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  • l-sanfilippo/bielefeld-ce-bi-tec-temp
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src/contents/Human Practices/HP svgs/understanding.tsx
View file @ 3fd292fd
......@@ -517,7 +517,7 @@ export const HPUnderstanding = () => (
fontSize: "21.96px",
fontFamily: "Arial",
/* writingMode: "lr-tb",*/
fill: "#ffffff",
fill: "var(--ourbeige)",
fillOpacity: 1,
fillRule: "nonzero",
stroke: "none",
......@@ -568,7 +568,7 @@ export const HPUnderstanding = () => (
fontSize: "21.96px",
fontFamily: "Arial",
/* writingMode: "lr-tb",*/
fill: "#fff6f3",
fill: "var(--ourbeige)",
fillOpacity: 1,
fillRule: "nonzero",
stroke: "none",
......@@ -639,7 +639,7 @@ export const HPUnderstanding = () => (
fontSize: "21.96px",
fontFamily: "Arial",
/* writingMode: "lr-tb",*/
fill: "#ffffff",
fill: "var(--ourbeige)",
fillOpacity: 1,
fillRule: "nonzero",
stroke: "none",
......@@ -681,7 +681,7 @@ export const HPUnderstanding = () => (
<path
id="path76"
style={{
fill: "#fff6f3",
fill: "var(--ourbeige)",
fillOpacity: 1,
fillRule: "evenodd",
stroke: "none",
......@@ -703,7 +703,7 @@ export const HPUnderstanding = () => (
<path
id="path80"
style={{
fill: "#fff6f3",
fill: "var(--ourbeige)",
fillOpacity: 1,
fillRule: "evenodd",
stroke: "none",
......
src/contents/Human Practices/HP-abstract.tsx
View file @ 3fd292fd
import { LoremMedium } from "../../components/Loremipsum"
import { Section } from "../../components/sections"
import { useTabNavigation } from "../../utils/TabNavigation";
export function HPAbstract(){
useTabNavigation();
return(
<Section title="Abstract" id="Abstract">
<LoremMedium/>
<p>As the iGEM Bielefeld-CeBiTec team, we adopted a human-centered approach and provided a comprehensive framework of <strong>seven reglementations</strong> for future iGEM teams, establishing <strong>seven frameworks</strong> that were validated through our project. Our methodology was shaped by over <strong>80 interviews with stakeholders and institutions</strong> , creating a comprehensive and multidimensional perspective on the complex challenge of gene therapy for Cystic Fibrosis (CF). Through <strong>33 key interviews</strong> , we navigated <strong>seven thematic areas</strong> simultaneously, incorporating <strong>five iterative feedback loops</strong> to ensure the project’s refinement at every stage.
</p>
<p> Our approach offers future iGEM teams and the synthetic biology community a valuable blueprint for responsible and ethical project development. By actively engaging with CF patients, clinicians, researchers, and regulatory bodies, we ensured that real-world needs and societal values were central to our project's evolution. Our efforts culminated in a thoughtful, scientifically sound solution, setting new standards for accessibility, patient focus, and regulatory compliance. Future teams can leverage our documented methodologies to further their own impact, ensuring that innovation remains aligned with the needs of those it serves.
</p>
</Section>
)
}
\ No newline at end of file
src/contents/Human Practices/IHP.tsx
View file @ 3fd292fd
import { ButtonOne } from "../../components/Buttons";
import { ButtonOne, ButtonOneWithScroll } from "../../components/Buttons";
import { H4, H5 } from "../../components/Headings";
import { HPTimeline } from "../../components/HP-timeline";
import { LoremMedium, LoremShort } from "../../components/Loremipsum";
import PreCyse from "../../components/precyse";
import { Section, Subesction } from "../../components/sections";
import { HPFeedback } from "./Feedback";
import { useNavigation } from "../../utils";
import { useTabNavigation } from "../../utils/TabNavigation";
import { HPconclusion } from "./Conclisuin";
import { HPFeedback } from "./Feedback";
import { HP3new } from "./HP svgs/hp3";
import {WholeMendelow} from "./HP svgs/mendelows";
import { StakeholderManagement } from "./HP svgs/stakeholder-management";
import { SWOTone } from "./HP svgs/swots";
import { HPUnderstanding } from "./HP svgs/understanding";
export function HPIntegrated(){
const { goToPageWithTabAndScroll} = useNavigation();
useTabNavigation();
return(
<Section title="Integrated Human Practices" id="Integrated Human Practices">
<Subesction title="Framework" id="Integrated Human Practices1">
<div className="row align-items-center" style={{marginTop: "5vh", marginBottom: "5vh"}}>
<p>Our team has defined a framework for managing interactions with stakeholders during each step of the project:
</p>
<ol>
<li> <b>Stakeholder Management Framework </b>- To identify stakeholders and define how potential stakeholders fit our project.</li>
<li><b>Power-Interest Matrix </b>- According to Mendelow to identify and evaluate the priorities of potential stakeholders.</li>
<li><b>SWOT analysis</b> - Assessment of the strengths, weaknesses, opportunities and threats of the various groups previously identified as potential stakeholders.</li>
<li><b>AREA Framework</b> - To structure and optimize project-related decisions.</li>
<li><b>Third-Party Feedback Templates </b>- To introduce the stakeholders, summarize the interactions and implement the output in our project.</li>
<li><b>Feedback-Cycle</b> - To structure interactions with stakeholders for efficient and informative design and maximization of potential.</li>
<li><b>Cycle of Reflection</b> - According to Gibbs demonstrate the response and implementation as a part of our feedback cycle.</li>
</ol>
<div className="row align-items-center">
<div className="col">
<ButtonOne text="Stakeholder Management" open="stakemamange" openclass="framecycletab"></ButtonOne>
<ButtonOneWithScroll text="Stakeholder Management" scrollId="stakeH" open="stakemamange" openclass="framecycletab"></ButtonOneWithScroll>
</div>
<div className="col">
<ButtonOne text="Mendelow Analysis" open="mendelow" openclass="framecycletab"></ButtonOne>
<ButtonOneWithScroll text="Mendelow Analysis" open="mendelow" scrollId="mendelowH" openclass="framecycletab"></ButtonOneWithScroll>
</div>
<div className="col">
<ButtonOne text="SWOT analysis" open="swot" openclass="framecycletab"></ButtonOne>
<ButtonOneWithScroll text="SWOT analysis" scrollId="swotH" open="swot" openclass="framecycletab"></ButtonOneWithScroll>
</div>
</div>
<div className="row align-items-center" style={{marginBottom: "5vh"}}>
<div className="row align-items-center">
<div className="col">
<ButtonOneWithScroll scrollId="areaH" text="AREA Framework" open="area" openclass="framecycletab"></ButtonOneWithScroll>
</div>
<div className="col">
<ButtonOne text="Feedback Cycle" open="feedcycle" openclass="framecycletab"></ButtonOne>
<ButtonOneWithScroll scrollId="thirdH" text="Third Party Feedback" open="thirdparty" openclass="framecycletab"></ButtonOneWithScroll>
</div>
<div className="col">
<ButtonOne text="Third Party Feedback" open="thirdparty" openclass="framecycletab"></ButtonOne>
<ButtonOneWithScroll scrollId="feedH" text="Feedback-Cycle" open="feedcycle" openclass="framecycletab"></ButtonOneWithScroll>
</div>
<div className="col">
<ButtonOne text="Cycle of Reflection" open="refcycle" openclass="framecycletab"></ButtonOne>
<ButtonOneWithScroll scrollId="gibsonH" text="Gibbs' Cycle" open="gibson" openclass="framecycletab"></ButtonOneWithScroll>
</div>
</div>
<div className="col framecycletab" id="stakemamange" style={{display: "block"}}>
<br/>
<div className="align-items-center">
<StakeholderManagement/>
</div>
<H4 text="Stakeholder Management Framework" id="stakeH"/>
<p>To clearly communicate to our stakeholders how they can support the <PreCyse/> project and the specific areas requiring
their input, we developed a stakeholder management framework highlighting four key areas. Starting at the bottom right of the
figure and moving clockwise, the first area needing feedback is <b>implementation</b>. It is crucial for our team to ensure that the project
has all necessary components for realistic application in healthcare settings, focusing on the gene therapy treatment for Cystic Fibrosis.
This includes both the technical design and the appropriate business model to ensure successful implementation.
</p>
<p>The second key area is <b>sustainability and social impact.</b> We seek to understand how our project aligns with current healthcare initiatives and facilities in Germany and Europe, but also in the globally, and how it interacts with social efforts related to CF awareness and gene therapy strategies.
</p>
<p>Next, we want to shape the <b>public perception</b> of our project, which is based on synthetic biology to tackle a major healthcare challenge. We ask stakeholders about their knowledge and opinions on our gene therapeutic approach and its potential application to improve CF treatments.
</p>
<p>The final area involves <b>regulatory and safety considerations</b>. Given the stringent regulations governing healthcare in Germany, which vary by municipality, it is essential to consult with stakeholders familiar with these regulations to ensure our project complies with all necessary safety standards.
</p>
</div>
<div className="col framecycletab" id="mendelow" style={{display: "none"}}>
<H4 text="Power-Interest Matrix according to Mendelow" id="mendelowH"/>
<p>Using the Mendelow Power-Interest Matrix, we classified the potential stakeholders associated with our project in terms of their power (the ability to provide important feedback on our project design and influence our project development) and interest in our project. The matrix consists of four quadrants arranged in descending order according to their priority level in the project:
</p>
<ul>
<li>High Power, High Interest (Key Players)</li>
<li>High Power, Low Interest (Keep Satisfied)</li>
<li>Low Power, High Interest (Keep Informed)</li>
<li>Low Power, Low Interest (Minimal Effort)</li>
</ul>
<p>This categorization helps to develop appropriate communication and management strategies for the different stakeholder groups and to ensure that their needs and influences are considered during the project.
</p>
<WholeMendelow/>
<div className="row">
<br/>
<div className="col-4">
<div className="" >
<HPUnderstanding/>
</div>
<div className="col">
<a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "mendelow-one", scrollToId: "keyplayersH" })}>
<img src="https://static.igem.wiki/teams/5247/scientific-figures/mendelow-1-aktuell.svg"/>
</a>
</div>
<div className="col">
<div className="col understandingtab" id="under-reflection" style={{display: "block"}}>
<div><LoremShort/></div>
</div>
<div className="col understandingtab" id="under-responsibility" style={{display: "none"}}>
<div><LoremShort/></div>
</div>
<div className="col understandingtab" id="under-responsive" style={{display: "none"}}>
<div><LoremShort/></div>
</div>
<a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "mendelow-two", scrollToId: "keepH" })}>
<img src="https://static.igem.wiki/teams/5247/scientific-figures/mendelow-2.svg"/>
</a>
</div>
</div>
</div>
<div className="col framecycletab" id="mendelow" style={{display: "none"}}>
<div className="col">
<a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "mendelow-three", scrollToId: "informedH" })}>
<img src="https://static.igem.wiki/teams/5247/scientific-figures/mendelow-3-1.svg"/>
</a>
</div>
<div className="col">
<a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "mendelow-four", scrollToId: "minimalH" })}>
<img src="https://static.igem.wiki/teams/5247/scientific-figures/mendelow-4.svg"/>
</a>
</div>
</div>
<p style={{ textAlign: "center", marginTop: "10px", fontStyle: "italic" }}>
Click on the images above to learn more about each category.
</p>
{/* TABS */}
<div className="mendelowtab" id="mendelow-one" style={{display: "none"}}>
<H4 text="Key Players" id="keyplayersH"/>
<H5 text="High Power, High Interest - Priority Level 4"/>
<p>
Close support and extensive feedback are necessary to successfully implement <PreCyse/>.
Target groups include experts and physicians with expertise in gene therapy and treatment
strategies for Cystic Fibrosis. The project has a reasonable, responsible, and future-oriented
significance for the world. Furthermore, scientific-technological knowledge and biosafety are
crucial. Collaboration with specialists in the field of gene therapy, Cystic Fibrosis, and different
treatment strategies ensures the quality and effectiveness of the solutions developed.
</p>
<p><b>Scientific community and research institutions</b></p>
<p>
This group includes researchers, scientists, and research institutions specializing in gene editing
and gene therapy. They have a high interest in the success of prime editing technology and have
the power to significantly influence the project, be it through collaborations, scientific support,
or critical reviews of the research results.
</p>
<p><b>Regulatory authorities (e.g. FDA, EMA)</b></p>
<p>
Regulatory agencies have both the power and the interest to ensure that gene therapy is safe and
effective. They are crucial for the approval and authorization of the therapy for clinical use.
</p>
</div>
<div className="mendelowtab" id="mendelow-two" style={{display: "none"}}>
<H4 text=" Keep Satisfied" id="keepH"/>
<H5 text="High Power, Low Interest - Priority Level 3"/>
<p>
In order to successfully implement <PreCyse/>, all concerns must be fully considered, even if they
may differ from the project interests. CF specialists, bioethics specialists, and regulatory experts
provide important information. Their input is beneficial to the implementation of our project.
These categories of experts help to ensure that the project is ethical and compliant with the law.
Their expertise ensures that all relevant aspects are covered.
</p>
<p><b>Government and public health authorities</b></p>
<p>
Although these stakeholders have some interest in new therapies that improve population health,
their day-to-day interest may not be specifically focused on the iGEM project. However, they have
the power to influence the project through funding, political support, or regulatory action.
</p>
<p><b>Pharmaceutical companies and industries</b></p>
<p>
Large pharmaceutical companies have the power to significantly support the project through
investment or strategic partnerships. However, their interest may be low for the time being until
the project has proven that it is commercially promising.
</p>
</div>
<div className="mendelowtab" id="mendelow-three" style={{display: "none"}}>
<H4 text="Keep Informed" id="informedH"/>
<H5 text="KEEP INFORMED - Low Power, High Interest - Priority Level 2"/>
<p> <b>Potential users of <PreCyse/></b> </p>
<p>
are important for understanding the needs, even if their expertise
may not be sufficient to solve all the problems that arise in the project. It is therefore important to
keep them informed. These users include stakeholders as their needs coincide with the project.
Their feedback can provide valuable insights. This collaboration helps to better tailor the project
to user needs.
</p>
<p><b>Patients and patient organizations</b></p>
<p>
Patients suffering from Cystic Fibrosis and the organizations that support them have a high interest in
new therapies that could improve their quality of life. However, their power to influence the project
is limited.
</p>
<p> <b>Academic community and students</b></p>
<p>
This group includes universities, professors, and students who are interested in the latest research
in gene therapy. Although they do not have the direct power to influence the project, they can
support and promote the project through research and academic publications.
</p>
</div>
<div className="mendelowtab" id="mendelow-four" style={{display: "none"}}>
<H4 text="Minimal Effort" id="minimalH"/>
<H5 text="Low Power, Low Interest - Priority Level 1" />
<p><b>Stakeholders who are regularly monitored</b></p>
<p>
may not be interested in the project and do not provide feedback. However, this can change over time.
Stakeholders in this category are involved in the right situation. For example, the public of Bielefeld is
essential for understanding the background information about the awareness of gene therapy or Cystic Fibrosis.
Their involvement occurs when it is relevant and other groups of people cannot meet their needs.
</p>
<p><b>General public of Bielefeld</b></p>
<p>
The public may have little interest and power in relation to the specific iGEM project. While a certain level
of education and awareness of the topic is important, intensive involvement of this group is generally not necessary.
</p>
<p><b>Media</b></p>
<p>
The media may have an interest in scientific breakthroughs and new technologies, but they tend to have little
power in terms of directly influencing the project. Nevertheless, strategic communication with the media can
be important to manage public perception.
</p>
</div>
</div>
<div className="col framecycletab" id="swot" style={{display: "none"}}>
<H4 text="SWOT Analyses" id="swotH"/>
<p>A <b>SWOT</b> analysis is a strategic tool used to evaluate the <b>S</b>trengths, <b>W</b>eaknesses, <b>O</b>pportunities, and <b>T</b>hreats of an
organization. In the context of our Human Practice approach, this analysis helps us identify internal strengths and weaknesses of our project, such as
technical capabilities and resource limitations, while also examining external opportunities, like potential collaborations, and threats, such as regulatory
challenges or public perception.
</p>
<SWOTone/>
<div className="row align-items-center" style={{marginTop: "5vh", marginBottom: "1vh"}}>
<div className="col">
<ButtonOne text="Patients" open="pats" openclass="subcycletab"></ButtonOne>
<div className="row align-items-center">
<div className="col ">
<ButtonOne text="Patients" open="swot&subTab=pats" openclass="subcycletab"></ButtonOne>
</div>
<div className="col">
<ButtonOne text="Industry" open="inds" openclass="subcycletab"></ButtonOne>
<div className="col ">
<ButtonOne text="Industry" open="swot&subTab=inds" openclass="subcycletab"></ButtonOne>
</div>
<div className="col">
<ButtonOne text="Academia" open="acs" openclass="subcycletab"></ButtonOne>
<div className="col ">
<ButtonOne text="Academia" open="swot&subTab=acs" openclass="subcycletab"></ButtonOne>
</div>
<div className="col">
<ButtonOne text="Healthcare" open="healths" openclass="subcycletab"></ButtonOne>
<div className="col ">
<ButtonOne text="Healthcare" open="swot&subTab=healths" openclass="subcycletab"></ButtonOne>
</div>
</div>
<div className="row align-items-center" style={{ marginBottom: "5vh"}}>
<div className="col">
<ButtonOne text="Government" open="govs" openclass="subcycletab"></ButtonOne>
<div className="row align-items-center">
< div className="col ">
<ButtonOne text="Government" open="swot&subTab=govs" openclass="subcycletab"></ButtonOne>
</div>
<div className="col">
<ButtonOne text="Community" open="comms" openclass="subcycletab"></ButtonOne>
<div className="col ">
<ButtonOne text="Community" open="swot&subTab=comms" openclass="subcycletab"></ButtonOne>
</div>
<div className="col">
<ButtonOne text="The Public" open="pubs" openclass="subcycletab"></ButtonOne>
<div className="col ">
<ButtonOne text="The Public" open="swot&subTab=pubs" openclass="subcycletab"></ButtonOne>
</div>
<div className="col">
<ButtonOne text="Business" open="busi" openclass="subcycletab"></ButtonOne>
<div className="col ">
<ButtonOne text="Business" open="swot&subTab=busi" openclass="subcycletab"></ButtonOne>
</div>
</div>
<div className="col subcycletab" id="pats" style={{display: "block"}}>
<H5 text="Patient Advocacy and Support Groups"/>
<table cellPadding={10} cellSpacing={0} >
<thead>
<tr>
<th>Strengths</th>
<th>Weaknesses</th>
<th>Opportunities</th>
<th>Threats</th>
</tr>
</thead>
<tbody>
<tr>
<td>Strong support network for patients </td>
<td>Limited funding and resources </td>
<td>Increased patient engagement and support for new therapies</td>
<td>Potential opposition from groups skeptical of new treatments</td>
</tr>
<tr>
<td>Influential in policy advocacy</td>
<td>Potential resistance to new technologies</td>
<td>Advocacy can drive policy changes</td>
<td>Misinformation or miscommunication about new therapies</td>
</tr>
<tr>
<td>Personal connection to patient communities</td>
<td>Dependence on donations and grants</td>
<td>Enhancing awareness and education</td>
<td>Competing priorities among advocacy groups</td>
</tr>
</tbody>
</table>
</div>
<div className="col subcycletab" id="inds" style={{display: "none"}}>
<H5 text="Pharmaceutical Companies" />
<table cellPadding={10} cellSpacing={0}>
<thead>
<tr>
<th>Strengths</th>
<th>Weaknesses</th>
<th>Opportunities</th>
<th>Threats</th>
</tr>
</thead>
<tbody>
<tr>
<td>Resources for large-scale production and distribution</td>
<td>Focus on profitability may conflict with patient affordability</td>
<td>Potential for developing new treatment modalities</td>
<td>High cost and risk associated with new therapy development</td>
</tr>
<tr>
<td>Experience with regulatory processes</td>
<td>Long development timelines</td>
<td>Market expansion and competitive advantage</td>
<td>Stringent regulatory and compliance requirements</td>
</tr>
<tr>
<td>Strong research and development capabilities</td>
<td>Potential for high pricing</td>
<td>Partnerships with research institutions</td>
<td>Potential public backlash over high treatment costs</td>
</tr>
</tbody>
</table>
<H5 text="Bioengineering and Biotechnology Departments "/>
<table cellPadding={10} cellSpacing={0}>
<thead>
<tr>
<th>Strengths</th>
<th>Weaknesses</th>
<th>Opportunities</th>
<th>Threats</th>
</tr>
</thead>
<tbody>
<tr>
<td>Agile and innovative, can quickly adapt to new technologies</td>
<td>Focused on niche markets and cutting-edge solutions</td>
<td>Opportunity for rapid growth and partnership with larger firms</td>
<td>High risk of failure and market volatility</td>
</tr>
<tr>
<td>Strong entrepreneurial spirit</td>
<td></td>
<td>Potential to disrupt existing markets with innovative solutions</td>
<td>Intense competition and fast-paced industry changes</td>
</tr>
<tr>
<td></td>
<td></td>
<td>Opportunities for collaboration and acquisition</td>
<td>Regulatory and market entry barriers</td>
</tr>
</tbody>
</table>
</div>
<div className="col subcycletab" id="acs" style={{display: "none"}}>
<H5 text="Genetic Research Institutions "/>
<table cellPadding={10} cellSpacing={0}>
<thead>
<tr>
<th>Strengths</th>
<th>Weaknesses</th>
<th>Opportunities</th>
<th>Threats</th>
</tr>
</thead>
<tbody>
<tr>
<td>Expertise in genetic disorders and advanced research capabilities</td>
<td>May have limited practical experience with therapy delivery</td>
<td>Collaboration can lead to breakthroughs in gene editing</td>
<td>High competition for funding and research grants</td>
</tr>
<tr>
<td>Access to state-of-the-art research facilities</td>
<td>Potential disconnect between research and clinical application</td>
<td>Potential for groundbreaking research and new discoveries</td>
<td>Rapid technological changes requiring constant adaptation</td>
</tr>
<tr>
<td>Strong track record of innovation</td>
<td>High operational costs</td>
<td>Collaborative research opportunities</td>
<td>Ethical and regulatory challenges</td>
</tr>
</tbody>
</table>
<H5 text="Universities and Academic Researchers "/>
<table cellPadding={10} cellSpacing={0}>
<thead>
<tr>
<th>Strengths</th>
<th>Weaknesses</th>
<th>Opportunities</th>
<th>Threats</th>
</tr>
</thead>
<tbody>
<tr>
<td>Strong research foundation and innovation capabilities</td>
<td>May have limited resources for commercialization</td>
<td>Established research infrastructure</td>
<td>Funding limitations and bureaucratic challenges</td>
</tr>
<tr>
<td>Access to a diverse pool of academic talent</td>
<td>Limited industry connections</td>
<td>High competition for academic grants</td>
<td>Intellectual property and technology transfer issues</td>
</tr>
<tr>
<td>Potential for groundbreaking research and new discoveries</td>
<td>Collaboration with industry for applied research</td>
<td>Opportunities for interdisciplinary research</td>
<td>Balancing academic research with practical applications</td>
</tr>
</tbody>
</table>
</div>
<div className="col subcycletab" id="healths" style={{display: "none"}}>
<H5 text="Pulmonologists and Respiratory Specialists"/>
<table cellPadding={10} cellSpacing={0}>
<thead>
<tr>
<th>Strengths</th>
<th>Weaknesses</th>
<th>Opportunities</th>
<th>Threats</th>
</tr>
</thead>
<tbody>
<tr>
<td>Deep understanding of CF and its complications</td>
<td>May lack expertise in gene editing and new delivery systems</td>
<td>Can provide valuable insights for therapy optimization</td>
<td>Resistance to adopt new and unproven therapies</td>
</tr>
<tr>
<td>Direct patient care experience</td>
<td>Limited time and resources for research involvement</td>
<td>Opportunities for clinical trials and patient feedback</td>
<td>Potential liability issues with new treatments</td>
</tr>
<tr>
<td>Established patient trust and rapport</td>
<td>Risk of slow adoption of new technologies</td>
<td>Enhanced treatment protocols through collaboration</td>
<td>Competition from alternative therapies</td>
</tr>
</tbody>
</table>
<H5 text="Specialized Clinics and Hospitals"/>
<table cellPadding={10} cellSpacing={0}>
<thead>
<tr>
<th>Strengths</th>
<th>Weaknesses</th>
<th>Opportunities</th>
<th>Threats</th>
</tr>
</thead>
<tbody>
<tr>
<td>Direct access to patient population</td>
<td>May lack expertise in gene therapy and new delivery systems</td>
<td>Clinical trials and real-world application of new therapies</td>
<td>High cost and complexity of integrating new therapies</td>
</tr>
<tr>
<td>Ability to conduct clinical trials</td>
<td>High operational costs</td>
<td>Opportunities for improving patient care and outcomes</td>
<td>Resistance from clinical staff to adopt new technologies</td>
</tr>
<tr>
<td>Experienced medical staff and infrastructure</td>
<td>Potential liability and ethical concerns</td>
<td>Collaboration with research institutions for therapy development</td>
<td>Competition for patients and clinical trial participation</td>
</tr>
</tbody>
</table>
<H5 text="Healthcare Networks"/>
<table cellPadding={10} cellSpacing={0}>
<thead>
<tr>
<th>Strengths</th>
<th>Weaknesses</th>
<th>Opportunities</th>
<th>Threats</th>
</tr>
</thead>
<tbody>
<tr>
<td>Broad reach and influence</td>
<td>Variability in network capabilities and resources</td>
<td>Established relationships with patient communities</td>
<td>Organizational resistance to change</td>
</tr>
<tr>
<td>Potential for large-scale impact on patient care</td>
<td>Ability to implement new therapies widely</td>
<td>Standardizing care across multiple facilities</td>
<td>Economic pressures and budget constraints</td>
</tr>
<tr>
<td>Leveraging network for large-scale clinical trials</td>
<td>Complex coordination required</td>
<td>Potential disparities in care quality</td>
<td>Variability in regulatory compliance across regions</td>
</tr>
</tbody>
</table>
</div>
<div className="col subcycletab" id="govs" style={{display: "none"}}>
<H5 text="Bioethics Committees "/>
<table cellPadding={10} cellSpacing={0}>
<thead>
<tr>
<th>Strengths</th>
<th>Weaknesses</th>
<th>Opportunities</th>
<th>Threats</th>
</tr>
</thead>
<tbody>
<tr>
<td>Ensure ethical standards are met</td>
<td>May be conservative and slow to approve new technologies</td>
<td>Establishing ethical frameworks for new therapies</td>
<td>Ethical dilemmas and public perception issues</td>
</tr>
<tr>
<td>Provide guidance on complex issues</td>
<td>Potential for conflicting opinions within the committee</td>
<td>Enhancing public trust through ethical oversight</td>
<td>Rapid advancements outpacing ethical guidelines</td>
</tr>
<tr>
<td>Enhance credibility and acceptance of new therapies</td>
<td>Limited authority to enforce decisions</td>
<td>Collaboration with regulatory agencies for comprehensive oversight</td>
<td>Misalignment with fast-paced technological advancements</td>
</tr>
</tbody>
</table>
<H5 text="Legal Experts"/>
<table cellPadding={10} cellSpacing={0}>
<thead>
<tr>
<th>Strengths</th>
<th>Weaknesses</th>
<th>Opportunities</th>
<th>Threats</th>
</tr>
</thead>
<tbody>
<tr>
<td>Navigate complex legal landscapes</td>
<td>High costs for legal services</td>
<td>Development of comprehensive legal frameworks for new therapies</td>
<td>Legal challenges and liability issues</td>
</tr>
<tr>
<td>Ensure compliance with regulations</td>
<td>Potential for lengthy legal processes</td>
<td>Protecting intellectual property and ensuring regulatory compliance</td>
<td>Rapidly changing legal environments and regulations</td>
</tr>
<tr>
<td>Expertise in healthcare law and genetic research regulations</td>
<td>Risk of stifling innovation with overly cautious advice</td>
<td>Advising on best practices for ethical and legal standards</td>
<td>Potential for litigation and legal disputes</td>
</tr>
</tbody>
</table>
</div>
<div className="col subcycletab" id="comms" style={{display: "none"}}>
<H5 text="Patient and Community Outreach Programs"/>
<table cellPadding={10} cellSpacing={0}>
<thead>
<tr>
<th>Strengths</th>
<th>Weaknesses</th>
<th>Opportunities</th>
<th>Threats</th>
</tr>
</thead>
<tbody>
<tr>
<td>Increase awareness and education</td>
<td>Limited reach and resources</td>
<td>Foster connections between patients, researchers, and healthcare providers</td>
<td>Public skepticism and resistance to new technologies</td>
</tr>
<tr>
<td>Build public support</td>
<td>Potential misinformation</td>
<td>Dependence on volunteer efforts</td>
<td>Competing messages and priorities among various community groups</td>
</tr>
<tr>
<td>Enhanced public engagement and support for new therapies</td>
<td>Opportunities for community-driven advocacy</td>
<td>Raising funds and resources through public engagement</td>
<td>Miscommunication leading to misinformation</td>
</tr>
</tbody>
</table>
</div>
<div className="col subcycletab" id="pubs" style={{display: "none"}}>
<H5 text="Educational Institutions"/>
<table cellPadding={10} cellSpacing={0}>
<thead>
<tr>
<th>Strengths</th>
<th>Weaknesses</th>
<th>Opportunities</th>
<th>Threats</th>
</tr>
</thead>
<tbody>
<tr>
<td>Educate future scientists and healthcare professionals</td>
<td>Limited funding and resources for large-scale projects</td>
<td>Opportunity to foster new talent and innovation</td>
<td>Funding constraints and academic pressures</td>
</tr>
<tr>
<td>Promote research and innovation</td>
<td>Potential for academic bureaucracy</td>
<td>Collaboration with industry and research institutions</td>
<td>Balancing academic objectives with practical applications</td>
</tr>
<tr>
<td>Established infrastructure for education and research</td>
<td>High competition for research grants</td>
<td>Opportunities for interdisciplinary education and research</td>
<td>Risk of brain drain to more lucrative industries</td>
</tr>
</tbody>
</table>
</div>
<div className="col subcycletab" id="busi" style={{display: "none"}}>
<H5 text="Funding Agencies and Venture Capitalists"/>
<table cellPadding={10} cellSpacing={0}>
<thead>
<tr>
<th>Strengths</th>
<th>Weaknesses</th>
<th>Opportunities</th>
<th>Threats</th>
</tr>
</thead>
<tbody>
<tr>
<td>Provide crucial financial support</td>
<td>Focus on ROI may limit funding for high-risk projects</td>
<td>Extensive networks and industry connections</td>
<td>Economic downturns affecting investment capacity</td>
</tr>
<tr>
<td>Drive innovation through investment</td>
<td>High competition for funding</td>
<td>Risk aversion may limit innovative but uncertain projects</td>
<td>Potential for high financial losses</td>
</tr>
<tr>
<td>Potential for high returns and impact on healthcare</td>
<td>Supporting transformative technologies</td>
<td>Identifying and nurturing breakthrough technologies</td>
<td>Changes in economic and policy environments</td>
</tr>
</tbody>
</table>
</div>
<div className="col subcycletab" id="pats" style={{display: "block"}}> pats </div>
<div className="col subcycletab" id="inds" style={{display: "none"}}>inds </div>
<div className="col subcycletab" id="acs" style={{display: "none"}}>acs </div>
<div className="col subcycletab" id="healths" style={{display: "none"}}> healths </div>
<div className="col subcycletab" id="govs" style={{display: "none"}}>govs </div>
<div className="col subcycletab" id="comms" style={{display: "none"}}>comms </div>
<div className="col subcycletab" id="pubs" style={{display: "none"}}>pubs </div>
<div className="col subcycletab" id="busi" style={{display: "none"}}>busi </div>
</div>
<div className="col framecycletab" id="thirdparty" style={{display: "none"}}>
none
<H4 text="The Third-Party Feedback Template" id="thirdH"/>
<p>To drive stakeholder interactions for the <PreCyse/> project, our team implemented the “Third-Party Feedback Template”. This template helps us introduce individuals to our stakeholders and demonstrate their significance to our project. There are three main sections in this template. First, we address the important question of who our stakeholders are, introducing their backgrounds and explaining why we reached out to them. Next, we summarize the conversations and knowledge exchanged during our interactions. Finally, we share our reflections from these stakeholder conversations and how these interactions have guided the next steps of our project. </p>
<div className="col-4">
<div className="" >
<HPUnderstanding/>
</div>
</div>
<div className="col">
<div className="col understandingtab" id="under-reflection" style={{display: "block"}}>
<div><p>The reflection component of the template is essential in evaluating how stakeholder interactions have shaped the <PreCyse/> project. After summarizing conversations with stakeholders, we analyze the feedback they provided. This reflection process allows our team to carefully consider the insights shared and assess their relevance to our project's goals. By reflecting on this input, we gain a clearer understanding of how external perspectives align or differ from our initial assumptions, fostering an environment of continuous learning and improvement. This stage ensures that we approach the next steps with a deeper awareness of both our project’s impact and the expectations of our stakeholders.</p></div>
</div>
<div className="col understandingtab" id="under-responsibility" style={{display: "none"}}>
<div><p>Responsibility is embedded in how we engage with stakeholders and respond to the trust they place in our team. By thoughtfully selecting who we reach out to, we demonstrate responsibility in choosing the most relevant and impactful individuals for our project. Additionally, it is our duty to honor the contributions of stakeholders by effectively integrating their feedback into our decision-making process. The template allows us to document these contributions, emphasizing our commitment to not only listen but also act responsibly based on their input. Our responsibility extends to ensuring that stakeholder interests are respected and that their input is actively contributing to the ethical and sustainable development of the <PreCyse/> project.</p></div>
</div>
<div className="col understandingtab" id="under-responsive" style={{display: "none"}}>
<div><p>Responsiveness is about how we adapt and move forward based on the knowledge and perspectives shared by our stakeholders. After gathering feedback, we assess how it aligns with our project’s objectives and adjust our strategies accordingly. This ensures that stakeholder contributions are not only heard but also acted upon in a timely and constructive manner. The final section of the template emphasizes how these interactions have guided the future steps of the project. Being responsive is crucial for maintaining dynamic and evolving relationships with our stakeholders, demonstrating that we value their input and are willing to incorporate it into our ongoing development.</p></div>
</div>
</div>
</div>
<div className="col framecycletab" id="feedcycle" style={{display: "none"}}>
<div className="col framecycletab" id="area" style={{display: "none"}}>
<H4 text="AREA Framework" id="areaH"/>
<p>The AREA Framework Analysis is a model that helps structure and optimize decision-making in complex projects, especially when multiple stakeholders are involved. Click on the cycle to find out more about each aspect.
</p>
<div id="hp3-wrapper">
<div className="col">
<HP3new/>
</div>
</div>
<H5 text="Agenda"/>
<p>
The central goal of the <PreCyse/> project is to develop an innovative gene therapy solution for Cystic Fibrosis (CF) that is not only technically effective but also socially acceptable and ethically justifiable. The primary questions include:
</p>
<ul>
<li>How can the project improve the lives of CF patients?</li>
<li>How can the project ensure that the proposed therapy meets ethical and regulatory standards?</li>
<li>How can the solution be integrated into current healthcare initiatives?</li>
<li>How does the project influence, and how is it influenced by, the perceptions of stakeholders, particularly patients, regulatory authorities, and the scientific community?</li>
</ul>
<p>
Here, the need is defined to develop a holistic understanding of the project’s impacts, considering both technological aspects and human perspectives.
</p>
<H5 text="Research"/>
<p>
To gain a comprehensive understanding of the needs and expectations, the <PreCyse/> team employed various methods to collect data from relevant stakeholders, including:
</p>
<p>
<b>Surveys of CF patients and their families:</b> These provided insights into the specific challenges and needs that CF patients face in daily life. Critical questions regarding safety, accessibility, and the long-term application of gene technology were addressed.
</p>
<p>
<b>Expert consultations with researchers and physicians:</b> Scientists and doctors working in gene therapy provided essential technical feedback and helped assess the feasibility and effectiveness of the proposed therapy.
</p>
<p>
<b>Regulatory authorities:</b> Feedback from agencies like the FDA and EMA played a central role in assessing safety requirements and regulatory challenges that need to be addressed before clinical application. This research phase was critical to ensuring that the technical solution aligned with patient needs and regulatory standards.
</p>
<H5 text="Evaluation"/>
<p>Based on the research phase results, a detailed assessment of the strengths, weaknesses, opportunities, and threats (SWOT analysis) was conducted (see above). The evaluation helped the team reflect on the gathered insights and focus on key challenges to ensure long-term feasibility and acceptance.
</p>
<H5 text="Agenda"/>
<p>
Based on the analysis, several measures were taken to ensure that the <PreCyse/> project is not only scientifically advanced but also socially and regulatorily acceptable:
</p>
<p>
<b>Integrated feedback loops:</b> Stakeholders were continuously involved, and their feedback was directly incorporated into the adaptation and improvement of the project design. An example of this is the application of Gibbs' Reflection Cycle to ensure that all feedback is thoroughly analyzed and incorporated into future decisions.
</p>
<p>
<b>Regulatory and ethical adjustments:</b> By working closely with regulatory authorities and ethics committees, measures were taken to ensure that the project complies with regulatory requirements and remains ethically justifiable.
</p>
<p>
<b>Safety considerations:</b> The safety of the therapy was a key concern in stakeholder interactions. Specific safety protocols were developed to minimize risks for patients.
</p>
<p>
<b>Public awareness:</b> To increase public awareness of the potential and safety of gene technologies, targeted communication measures were taken to address misunderstandings and improve acceptance of the technology.
</p>
</div>
<div className="col framecycletab" id="feedcycle" style={{display: "none"}}>
<H4 text="The Feedback Cycle of our IHP Approach" id="feedH"/>
<p>To foster productive conversations and ensure our team maximizes each stakeholder interaction for the <PreCyse/> project, we developed a feedback cycle that outlines a structured approach for our meetings. The first step in our cycle involves listening to each stakeholder's experiences, personal stories, or insights. Our team then follows up by asking relevant questions to dive deeper into the shared information or to introduce new topics or directions in the conversation. Towards the end of the meeting, we ask clarifying questions and reiterate key points to ensure our understanding is accurate and that the stakeholder has no additional input. Finally, we explore new directions and ideas inspired by the stakeholder interaction, encouraging our team to pursue innovative and novel concepts.
</p>
<div>
<img src="https://static.igem.wiki/teams/5247/photos/hp/cycle2.svg"/>
</div>
</div>
<div className="col framecycletab" id="gibson" style={{display: "none"}}>
<H4 text="Cycle of Reflections according to Gibbs" id="gibsonH"/>
<p>For the <PreCyse/> project to impact the world, it needs to work with all types of stakeholders. Therefore, we are optimizing Gibbs' Reflection Cycle to demonstrate our inclusive response to the challenges we encountered during our iGEM journey. The cycle includes the impetus for our engagement, the unbiased two-way communication with stakeholders, our thorough analysis of the feedback and our actions to implement the stakeholder inputs into our project.
</p>
<div style={{height: "20%"}}>
<img src="https://static.igem.wiki/teams/5247/scientific-figures/gibbsreflection.svg" style={{height: "20%"}}/>
</div>
<p>
<b>Impetus:</b> When developing a systematic approach to a recurring problem, it's easy to get distracted by multiple points of view. We need to prioritize quality over quantity. This section presents the successive impetus that drives the continuous implementation of Human Practices activities.
</p>
<p>
<b>Two-way communication:</b> For our solution to be human-centered, the two-way communication method is to communicate to stakeholders the values we are incorporating into our project design. We make sure they understand the project clearly so that they can articulate their concerns and suggestions precisely.
</p>
<p>
<b>Analysis:</b> The two-way communication with our stakeholders is carefully analyzed. In this section, stakeholder feedback is processed and turned into constructive guidance for project implementation, allowing us to consider what work should be prioritized to best address stakeholder concerns.
</p>
<p>
<b>Implementation:</b> Implementation shows our measures for evaluating and refining the project. These actions are fully integrated into the project designs and other parts of our project to ensure that our project and activities are good, responsible and engaging for the world.
</p>
<p>
In the areas of <b>Communication & Implementation</b>, <b>Necessity & Relevance</b>, <b>Science & Technology</b>, <b>Ethics & Regulation</b>, our Human Practice activities are conducted with X groups of stakeholders throughout our iGEM journey. In doing so, we explore the contexts that define projects, idealize solutions and evaluate outcomes for our human practice approach.
</p>
</div>
<div className="col framecycletab" id="refcycle" style={{display: "none"}}>refcycle </div>
</Subesction>
<Subesction title="Timeline" id="Integrated Human Practices2">
<HPTimeline/>
</Subesction>
<Subesction title="Implementation" id="Integrated Human Practices3">
<LoremMedium/>
</Subesction>
<Subesction title="Feedback" id="Integrated Human Practices4">
<Subesction title="Implementation & Feedback" id="Integrated Human Practices3">
<HPFeedback/>
</Subesction>
<Subesction title="Conclusion" id="Integrated Human Practices5">
<LoremMedium/>
<Subesction title="Conclusion" id="Integrated Human Practices4">
<HPconclusion/>
</Subesction>
</Section>
)
......
src/contents/Human Practices/Introduction.tsx
View file @ 3fd292fd
import { ButtonOne } from "../../components/Buttons";
import { LoremMedium } from "../../components/Loremipsum";
import { H5 } from "../../components/Headings";
import PreCyse from "../../components/precyse";
import { BlockQuoteB } from "../../components/Quotes";
import { Section } from "../../components/sections";
import { useNavigation } from "../../utils";
import { useTabNavigation } from "../../utils/TabNavigation";
export function HPIntroduction(){
const {goToPageAndScroll} = useNavigation();
useTabNavigation();
return(
<Section title="Introduction" id="Introduction">
<div className="row align-items-center" style={{marginTop: "5vh", marginBottom: "1vh"}}>
<div className="col">
<H5 text="- Connecting our project to real life -"/>
<BlockQuoteB text="Science and everyday life cannot and should not be separated." cite="Rosalind Franklin"/>
<div className="row align-items-center">
<div className="col ">
<ButtonOne openclass="intro-cycletab" text="Our Understanding of HP" open="understanding"></ButtonOne>
</div>
<div className="col">
<div className="col ">
<ButtonOne openclass="intro-cycletab" text="Our Mission & Vision" open="mission"></ButtonOne>
</div>
<div className="col">
<div className="col ">
<ButtonOne openclass="intro-cycletab" text="Our Approach" open="approach"></ButtonOne>
</div>
<div className="col ">
<ButtonOne openclass="intro-cycletab" text="Our Target Groups" open="targets"></ButtonOne>
</div>
</div>
<br/>
<div className="col intro-cycletab" id="understanding" style={{display: "block"}}> understanding <LoremMedium/> </div>
<div className="col intro-cycletab" id="mission" style={{display: "none"}}>mission <LoremMedium/> </div>
<div className="col intro-cycletab" id="targets" style={{display: "none"}}>targets <LoremMedium/> </div>
<div className="col intro-cycletab" id="understanding" style={{display: "block"}}>
<p>This year, we at iGEM Bielefeld-CeBiTec have consciously chosen a <b>human-centered project design</b>. At the heart of our iGEM project is
one key pillar: <b>Human Practice</b>. Our goal is to understand the impact of our project on society, the scientific community and the world
as whole. This is not just about the technical effectiveness of our parts, but also about how the solution is embraced in everyday
practice, and the potential long-term impact it could have for Cystic Fibrosis patients and their families all over the world.
</p>
<p>With our human-centered approach, we aim to address fundamental iGEM Human Practice questions and beyond:
</p>
<p style={{textAlign: "center"}}> <b>How does our project affect the world around us?</b></p>
<p style={{textAlign: "center"}}> <b>How does the world around us influence our project? </b></p>
<p>From the very beginning, it was our priority to identify various stakeholders and meet people affected by Cystic Fibrosis early on to <b>actively involve</b> them throughout the planning and development process. This collaborative approach allowed us to ensure that our project
addresses real needs and contributes to solutions for as many different people as possible. Without the critical advice, varied perspectives
and input from our stakeholders, it would have been impossible to identify and reflect on all aspects of our project. We made every
effort to <b>deeply understand</b> their values and backgrounds, allowing us to integrate their feedback into our solutions.
</p>
</div>
<div className="col intro-cycletab" id="mission" style={{display: "none"}}>
<p>We view Human Practice as an opportunity to <b>go beyond practical lab work and traditional science</b> and to learn about
the needs of people affected by Cystic Fibrosis. It’s a chance for us to creatively engage with different aspects of our project
while developing an awareness of the responsibilities that come with it.
</p>
<p>
As part of our <PreCyse/> project, we performed intensive brainstorming sessions and expert consultations. We conducted comprehensive
<a onClick={() => goToPageAndScroll("our-surveys-on--Cystic Fibrosis-and-gene-therapy", "/human-practices")}> surveys</a> among the public and people with Cystic Fibrosis and their relatives. We focused on critical aspects such as the <b>needs of our
target groups, safety, ethics, design, implementation, and business</b> — each guided by the core values of our team. Based on these interactions
and the recommendations of the Human Practice committee, we have developed an optimal strategy for our project, ensuring that our work is not
only innovative but also mindful of its broader impact on society.
</p>
</div>
<div className="col intro-cycletab" id="approach" style={{display: "none"}}>
<p>It was important to us as a team to not only offer technical solutions, but to show that our project can contribute to the larger context of ongoing initiatives and movements to optimize health care. We wanted to really understand the feedback and insights of the stakeholders to gain a better understanding of how our project fits into the overall picture of living with Cystic Fibrosis, the current state of research and how it can be used to reduce the health care gap.
</p>
<p><b>Our strategy includes:</b></p>
<ul>
<li>Identifying key target groups</li>
<li>Establishing meaningful and lasting communication with stakeholders</li>
<li>Effectively engaging with the diverse backgrounds of those involved</li>
<li>Understand the ethical, social and scientific values that inspired our project</li>
<li>Integrating feedback and adapting our approach to align with stakeholder goals</li>
<li>Designing and incorporating representative surveys</li>
<li>Reflect on how these values have been incorporated into our project</li>
</ul>
<p><b>With this approach and the support of our stakeholders, our ultimate goals are to:</b></p>
<ul>
<li>Improve care for Cystic Fibrosis patients</li>
<li>Optimize the availability of essential medications</li>
</ul>
</div>
<div className="col intro-cycletab" id="targets" style={{display: "none"}}>
<p>Our target groups are composed of <b>academic and clinical experts</b> to build a bridge between research and practical application.
In addition, engaging with CF <b>patients</b> across different age groups and countries and their relatives leads to an awareness of
the special needs and demands of our project and to an understanding of the limitations and challenges in everyday CF life. The
integration of regulatory ethics serves to evaluate how our project might be implemented in German and international <b>government </b>
regulations for gene therapeutics. The exchange with <b>companies</b> contributes to understanding the chances and challenges in business of
establishing a start-up and what it takes to get our idea on the market for real life application. The continuous exchange between the
different groups contributes significantly to the successful implementation of the project. First-hand information from our target groups
and the matching and merging of the information is therefore essential for defining and achieving our goals.
</p>
</div>
</Section>
)
}
\ No newline at end of file
src/contents/Human Practices/Overwiev.tsx
View file @ 3fd292fd
import { BlockQuoteB } from "../../components/Quotes";
import { useTabNavigation } from "../../utils/TabNavigation";
export function HPOverview(){
useTabNavigation();
return(
<div className="col">
<section id="OverviewH">
<h2 id="Overview"></h2>
<img src="https://static.igem.wiki/teams/5247/ihp-schaubild.webp" alt="Stakeholder Overview"></img>
<strong>Figure: Visualization of the Human-Centric Approach by iGEM Bielefeld-CeBiTec 2024</strong><p>Our project has been supported by over 80 individuals and institutions, enhancing our integrative human practices. We are deeply grateful for the invaluable feedback and the collective effort of everyone who has contributed to our vision of safe and precise gene therapy through PreCyse. Thank you for your support!
</p>
<span id="hp-quote"><BlockQuoteB
text="Human Practices is the study of how your work affects the world, and how the world affects your work."
cite="- Peter Carr, Director of Judging"
/> </span>
<img src="https://external-content.duckduckgo.com/iu/?u=https%3A%2F%2Ftse1.mm.bing.net%2Fth%3Fid%3DOIP.6mRPyWPFEIQzo-HP4kEukgHaEK%26pid%3DApi&f=1&ipt=ad1e62d3df6a343c1c163a8246d424a7b61015ac43a0cbe279976cf544be7aa7&ipo=images" alt="placeholder"></img>
<p>In the development of our project, we embraced a holistic and human-centric approach, putting people at the core of our efforts. Our goal was to foster interdisciplinary collaboration and open dialogue, ensuring that diverse perspectives shaped the trajectory of our work. Over the course of this journey, we have engaged with more than 80 individuals and institutions, who have significantly influenced our project.
The strength of our approach lies in its interdisciplinary nature. We have brought together representatives from a wide range of categories, each playing a vital role in shaping the direction and success of our initiative. These categories include: </p>
<ol>
<li>
<strong>Patients:</strong> At the heart of any healthcare-related project, patients provide invaluable insights. Their lived experiences, needs, and challenges have guided our efforts to develop solutions that are truly patient-centered.
</li>
<li>
<strong>Healthcare Institutions:</strong> Collaborating with hospitals, clinics, and other healthcare providers allowed us to understand the practical implications of our work and to ensure that our solutions are aligned with the realities of healthcare delivery.
</li>
<li>
<strong>Academia – Research and Science:</strong> By partnering with researchers and scientists from leading academic institutions, we were able to base our work on the latest scientific knowledge and methodologies. Their contributions ensured that our approach is both innovative and evidence-based.
</li>
<li>
<strong>Industry – Entrepreneurship and Business:</strong> Engaging with industry professionals and entrepreneurs enabled us to explore the commercial potential of our project. Their expertise in innovation, business models, and scalability helped us to envision how our solutions could be brought to market and widely implemented.
</li>
<li>
<strong>Government – Biosafety and Ethics:</strong> Regulatory bodies and ethics committees played a crucial role in ensuring that our project adheres to the highest standards of safety, ethics, and compliance. Their oversight has been instrumental in maintaining the integrity and social responsibility of our work.
</li>
<li>
<strong>Local and International Communities:</strong> Our outreach to both local and global communities allowed us to understand the broader societal impacts of our project. These interactions helped us to address diverse cultural, social, and economic factors, ensuring that our solutions are accessible and relevant to different populations.
</li>
<li>
<strong>Public Organizations:</strong> Partnering with non-governmental organizations and public institutions enabled us to align our project with public health goals and contribute to social good. Their mission-driven focus helped us to maintain a sense of purpose and responsibility in all our endeavors.
</li>
</ol>
<p>By engaging with these diverse stakeholders, we have built a project that is not only comprehensive but also adaptable to the multifaceted needs of society. The insights and feedback from patients, healthcare professionals, researchers, businesses, governments, communities, and public organizations have ensured that our human-centric approach remains dynamic, inclusive, and impactful.
In summary, our project reflects the power of collaboration across sectors, disciplines, and geographies. The collective input of more than 80 individuals and institutions has enabled us to create a well-rounded and robust framework that addresses the challenges we set out to solve. We are proud of the interdisciplinary nature of our work and are committed to continuing this inclusive approach as we move forward. </p>
</section>
</div>
......
src/contents/attributions.tsx
View file @ 3fd292fd
import { useEffect } from "react";
import { BackUp } from "../components/Buttons";
import { H2 } from "../components/Headings";
import { BlockQuoteB } from "../components/Quotes";
export function Attributions() {
const teamID = import.meta.env.VITE_TEAM_ID;
......@@ -26,8 +28,17 @@ export function Attributions() {
<>
<div className="row mt-4">
<div className="col">
<H2 text="One iGEM project - Million people behind"/>
<BlockQuoteB text="With a lot of responsibility comes a bigger workload and even more people working together" cite="former iGEMer"/>
<p>We would like to thank everyone who supported our team and provided assistance, resources or useful tips in order to fulfill our project.</p>
<p>The following overview lists all team members, who have been instrumental in our iGEM project. We are proud of our work and look forward to what awaits us after the iGEM competition.</p>
<p>We would like to thank our principal investigators, instructor and supervisors for guiding us along the way and helping to shape our project.</p>
<p>We contributed all external partners that have influenced the project below. We would like to thank all iGEM teams, former iGEM team members of the university of Bielefeld or external team members, contractors, technicians, laboratory students, post-docs and collaborators for their support in any form. </p>
<p>We would also like to thank our sponsors, who have supported us with their generous donations. First and foremost, Bielefeld University, and particularly Prof. Dr. Alfred Pühler, who revived iGEM and allows us to participate in this year's competition. Additionally, we are thankful for the support and feedback of the steering committee.</p>
<p>In addition to cooperation and collaborative work, time management focuses on efficient operation of every iGEM project. The following list of our project timeline serves as an orientation and gives an overview of the temporal staggering of the project. We would like to thank all participants for the coordination of our activities</p>
</div>
<br/>
</div>
<iframe
style={{ width: "100%" }}
......
src/contents/description.tsx
View file @ 3fd292fd
......@@ -2,524 +2,602 @@ import { InfoBox } from "../components/Boxes";
import { TabButtonRow } from "../components/Buttons";
import Collapsible from "../components/Collapsible";
import { SupScrollLink } from "../components/ScrollLink";
import { H2, H4} from "../components/Headings";
import { LoremMedium, LoremShort } from "../components/Loremipsum";
import { Circle } from "../components/Shapes";
import { Complex } from "../components/Svgs";
import { H4 } from "../components/Headings";
import { ButtonRowTabs } from "../components/Tabs";
import PieChart from "../components/Graph";
import PreCyse from "../components/precyse";
import { Section, Subesction } from "../components/sections";
import { symptomdata, SymptomDatensatz } from "../data/symptom-data";
import { drugdata, DrugDatensatz } from "../data/drug-data";
import { useTabNavigation } from "../utils/TabNavigation";
import { QuizQuestion } from "../components/Quiz";
import PrimeEditingComplex from "../components/Complex-svg";
import { useNavigation } from "../utils";
import DescSources from "../sources/description-sources";
import { OneFigure } from "../components/Figures";
export function Description() {
export function Description() {
useTabNavigation();
return (
<div className="row mt-4">
<div className="col">
<Section title="Abstract" id="Abstract">
<p id="obenindescription" >We are proud to introduce our next-generation prime editing technology <PreCyse/> . We aim to develop an innovative gene therapy against cystic fibrosis, tackling the most common mutation F508del of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. We optimize lipid nanoparticles (LNPs) for the efficient and cell-specific delivery of our therapeutic mRNA. Current treatment strategies are limited in terms of speed, precision and effectiveness, often failing to achieve long-lasting improvements. In addition, high costs and limited accessibility of pharmaceuticals contribute to adverse prognosis of many patients. We want to develop a monthly applied which represents a cure that is more advanced and user-friendly compared to other medications due to its longer lasting time, lowering the frequency of use. </p>
</Section>
<Section title="Our Motivation" id="Our Motivation">
<p>We chose to focus on CF and specifically the F508del mutation due to its prevalence and the severe impact it has on patients' lives. Additionally, our team includes members who have close friends affected by this condition, giving us a personal connection and a strong motivation to find a solution. By targeting the F508del mutation, we aim to develop a therapy that could potentially, not only benefit many CF patients and make a significant improvement in their lives, but also can serve as a template, which research groups can use to target other genetic diseases. </p>
const { goToPagesAndOpenTab, goToPageWithTabAndScroll } = useNavigation();
const { goToPageAndScroll, goToPageWithTabAndCollapsible } = useNavigation();
return (
<div className="row mt-4">
<div className="col">
<Section title="Abstract" id="Abstract">
<p id="obenindescription">We are proud to present <PreCyse />, a next-generation Prime Editing technology, as innovative gene therapy approach for Cystic Fibrosis (CF)
specifically targeting the most common mutation <b>F508del</b> of the CFTR gene. Cystic Fibrosis is a severe disorder that primarily affects the lungs
and for which only short-term symptomatic treatments exist. PreCyse aims to provide long-term relief by delivering a small genetic payload with speed
and precision. Our approach integrates <b>PrimeGuide</b>, a highly optimized Prime Editing system, with <b>AirBuddy</b>, a novel lipid nanoparticle
(LNP) delivery platform. The <b>SORT LNPs</b> used in AirBuddy are optimized for pulmonary delivery, offering precise organ targeting and structural
stability throughout the inhalation process. Small payload size is key for effective delivery and compatibility with viral vectors, which have a
limited capacity. PrimeGuide embodies this vision by utilizing a smaller, more efficient editing complex. Current treatments often require daily
administration whereas PreCyse is currently developed as a monthly applied therapy. In addition, Prime Editing holds the promise to offer a causal cure,
when mutations are corrected in stem cells. Our approach aims to reduce medication frequency while improving patient outcomes.
By lowering costs and improving accessibility, PreCyse aspires to offer an advanced and user-friendly cure for Cystic Fibrosis.</p>
</Section>
<Section title="Our Motivation" id="Our Motivation">
<div className="row align-items-center">
<div className="col" >
</div>
<p>Our project started with a personal story. Rather than being driven purely by academic curiosity, our motivation came from someone close to one of our team members — Max Beckmann, a friend who has lived with Cystic Fibrosis (CF) since his birth. Specifically, he carries the F508del mutation, the most common genetic cause of the disease. Seeing the impact of CF on his daily life—frequent treatments and physical strain—made us realize how much more can be done to improve the lives of those affected, which inspired us to pursue this project. </p>
<p>As we explored Cystic Fibrosis further, we were struck by how widespread it is, being the most common genetic disorder in Germany. Approximately 70% of those with CF are specifically affected by the F508del mutation<SupScrollLink label="1" /> . This mutation is the most prevalent and well-studied of the thousands of genetic variations that cause CF, making it an important focus of research and intervention. In fact, about 90% of Europeans and individuals of European descent with CF have at least one F508del allele<SupScrollLink label="2" />. This widespread prevalence highlighted the significance of our project—not just for our friend, but for the thousands of others affected by this mutation across Europe and beyond. </p>
</div>
<div className="col" >
<img className="img" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/other/max-bild.webp"
alt1=""
description="Picture from our interview with Max."
num="1 "
/>
</div> {/* */}
</div>
<p>Max</p>
</Section>
<Section title="Cystic Fibrosis" id="Cystic Fibrosis">
<Subesction title="Overview" id="Cystic Fibrosis1">
<div className="row align-items-center">
<div className="col">
<p data-aos="zoom-y-out" >Cystic fibrosis (CF) is the most common life-limiting genetic disorder in the Caucasian population. In Europe, CF affecting about 1 in 3,000 newborns
<SupScrollLink label="1"/>.</p>
<p> It is caused by mutations in the CFTR gene, which controls ions and water movement in cells. This leads to thick mucus, clogging airways, and frequent infections. The defective CFTR protein impacts the respiratory and digestive systems, causing chronic lung infections, breathing difficulties, and malnutrition. CF's severity varies, but it reduces life quality and expectancy. There are over 1,700 CFTR mutations; the F508del mutation is most common, present in 70% of cases. It prevents proper protein folding, affecting its function. </p>
<Collapsible id="fanzorcas-collapsible" title="Cas vs. Fanzor">
<p>The mutations can be divided into six classes [9]:</p>
<p>Class I mutations prevent the synthesis of CFTR proteins altogether, meaning no channels are produced.</p>
<p>Class II mutations, which include the common F508del mutation (responsible for about 85% of cases [10]), disrupt the maturation process of the protein. As a result, the defective channels are quickly degraded by the cell.</p>
<p>Class III mutations, known as “gating” mutations, reduce the likelihood that the CFTR channel will open correctly, impairing its function.</p>
<p>Class IV, V, and VI mutations are rare. These mutations result in the production of unstable or inefficient CFTR proteins, which do not function adequately and are produced in insufficient numbers.</p>
</Collapsible>
<p><LoremMedium/></p>
</div>
<div className="row-if-small col-2 ">
<Circle text="1:3000 newborns worldwide"/>
<Circle text="x:y newborns in Germany"/>
<Circle text="kosten"/>
<p>By focusing on the F508del mutation, we also hope to contribute valuable insights to the global Cystic Fibrosis community. Although this mutation is most common in European populations, it is also found in other regions around the world. Our research could thus help inform treatment strategies and health policies on an international scale. </p>
<p>With several team members focusing their studies on biomedical fields, we began by examining the current landscape of CF treatments. It quickly became clear that, despite recent progress, there is still no cure. Most therapies, such as CFTR modulators, focus on managing symptoms and improving lung function rather than addressing the underlying cause of the disease<SupScrollLink label="3" />{/* ehem6 */}. This realization led us to explore gene-editing technologies, thus leading us to Prime Editing—a next generation gene editing method—captured our attention. </p>
<p>While Prime Editing holds great promise, we found that its application for Cystic Fibrosis, particularly the F508del mutation, had not been fully explored. Recognizing this gap in the research inspired us to take on the challenge of optimizing Prime Editing for this specific mutation. Our mission became clear: we want to contribute to the development of a potential therapeutic approach for Cystic Fibrosis, specifically targeting the F508del mutation with prime editing, and bring us closer to a long-term solution for patients. </p>
</Section>
<Section title="Cystic Fibrosis" id="Cystic Fibrosis">
<Subesction title="Overview" id="Cystic Fibrosis1">
<p data-aos="zoom-y-out" >Cystic Fibrosis (CF) is a common life-limiting genetic disorder, particularly affecting the Caucasian population, with approximately <b>162,400 people worldwide</b> living with the condition<SupScrollLink label="4" />{/* ehem7 */}. Statistically, about <b>one in every 3,300</b> white newborns is born with CF<SupScrollLink label="5" />{/*ehem8*/}. And according to the German Cystic Fibrosis Registry, the average life expectancy for children born with CF in 2021 was around 57 years<SupScrollLink label="6" />{/*ehem9*/}, highlighting the severe and life-shortening nature of the disease. </p>
<p>The modern understanding of CF dates back to 1922 when Dr. Dorothy Andersen, a pediatric specialist, first described the disease and coined the term "Cystic Fibrosis"<SupScrollLink label="7" />{/*ehem10*/}. In Germany, it is commonly known as "Mukoviszidose," derived from the Latin words meaning "mucus" and "viscous"<SupScrollLink label="7" />{/*ehem10*/}, emphasizing the characteristic thick, sticky mucus that defines the condition<SupScrollLink label="8" />{/*ehem11*/}. </p>
<p>Genetic research has identified over 1,700 mutations in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene, with the F508del mutation being the most common, affecting about 70% of CF patients. This mutation prevents the proper folding of the CFTR protein, significantly impairing its function<SupScrollLink label="9" />{/*ehem13*/}. </p>
<p>The CFTR protein regulates the flow of chloride ions across the membranes of cells in the lungs, digestive system, and other organs. This ion flow is essential for drawing water into surrounding tissues, which helps maintain the proper hydration and consistency of mucus. In patients with CF, the disruption of this process prevents sufficient water from entering the mucus, making it abnormally thick and sticky. The accumulation of this mucus leads to an obstruction of airways and digestive ducts, resulting in chronic lung infections, inflammation, impaired digestion, and malnutrition<SupScrollLink label="10" />{/*ehem14*/}. </p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/project-description/lung-ephitel-biorender.png"
num={2}
bg="white"
description="Human lung epithelium with corrected CFTR expression (left) and Cystic Fibrosis (right)."
alt1="Lung ephitelium of human with correct CFTR expression (left) and Cystic Fibrosis (right)."
/>
<Collapsible id="classes-mutations-collapsible" title="Different classes of mutations">
<p>The mutations can be divided into <u>six classes</u><SupScrollLink label="11" />{/*ehem15*/} :</p>
<p><b>Class I</b> mutations prevent the synthesis of CFTR proteins altogether, meaning no channels are produced.</p>
<p><b>Class II</b> mutations, which include the common F508del mutation (responsible for about 85% of cases<SupScrollLink label="11" />{/*ehem16*/}, disrupt the maturation process of the protein. As a result, the defective channels are quickly degraded by the cell.</p>
<p><b>Class III</b> mutations, known as “gating” mutations, reduce the likelihood that the CFTR channel will open correctly, impairing its function.</p>
<p><b>Class IV, V</b> and <b>VI</b> mutations are rare. These mutations result in the production of unstable or inefficient CFTR proteins, which do not function adequately and are produced in insufficient numbers.</p>
</Collapsible>
<p>The prevalence of CF varies globally, with higher concentrations of cases in Europe, North America, and parts of Oceania. This geographic variation underscores the need for regionally tailored healthcare solutions. </p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/charts-maps/cfper10-000.png"
alt1=""
num="3"
bg="white"
description="Global distribution of cystic fibrosis cases: Countries are color-coded based on the number of reported cases, highlighting regional prevalence patterns."
/>
<p>CF is often diagnosed early through newborn screening programs, which detect elevated levels of immunoreactive trypsinogen (IRT). A positive result typically leads to a sweat test, the gold standard for diagnosing CF, which measures the concentration of chloride in sweat. </p>
<p>Although there is currently no cure for CF, patients must manage the disease throughout their lives, relying on treatments that alleviate symptoms but do not address the root cause. This lifelong management imposes significant financial burdens on affected families and healthcare systems, particularly in regions with a high prevalence of CF<SupScrollLink label="11" />{/*ehem15*/}. In recent years, <b>CFTR modulators</b>, which target the underlying genetic defect, have offered new hope for many patients. </p>
</Subesction>
<Subesction title="The CFTR Protein" id="Cystic Fibrosis2">
<p>The CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) protein is an ion channel that facilitates the movement of chloride ions across epithelial cell membranes<SupScrollLink label="12" />{/*ehem18*/}. This movement is essential for controlling the flow of water in tissues such as the lungs and intestines<SupScrollLink label="13" />{/*ehem19*/}. This increase in ion concentration in the extracellular space draws water out of the cells and into the surrounding mucus or fluid, ensuring it stays thin and mobile<SupScrollLink label="13" />{/*ehem20*/}.</p>
<p>The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is a specialized protein that plays a crucial role in maintaining the balance of ions and water on the surface of certain cells, particularly in the lungs, pancreas, and other organs. </p>
<H4 text="Structure of CFTR" id="structure-cftr" />
<p>CFTR is a large protein embedded in the cell membrane. It belongs to a family of proteins known as ABC transporters (ATP-Binding Cassette transporters), which typically move molecules across cell membranes<SupScrollLink label="14" />{/*ehem17*/}. CFTR, however, is unique because it functions as an ion channel rather than a transporter<SupScrollLink label="14" />{/*ehem18*/}. </p>
<p>The protein consists of several important regions<SupScrollLink label="15" />: </p>
<ul>
<li><b>Two transmembrane domains (TMDs)</b>: These span the cell membrane and create the channel through which ions can flow.</li>
<li><b>Two nucleotide-binding domains (NBDs)</b>: Located on the cytoplasmic side of the membrane, these domains bind and hydrolyze ATP (adenosine triphosphate). ATP binding and hydrolysis regulate the opening and closing of the chloride channel.</li>
<li><b>Regulatory (R) domain</b>: This domain is unique to CFTR and controls the activity of the protein. It requires phosphorylation by protein kinase A (PKA) to activate the ion channel.</li>
</ul>
<div className="row align-items-center">
<div className="col">
{/* This one this has tp stay a figure and not be a OneFigure */}
<div className="figure-wrapper">
<figure>
<div className="col gif-wrapper">
<img className="CFTR-gif" src="https://static.igem.wiki/teams/5247/fanzor/cftr-wt.gif"></img>
</div>
<figcaption> <b>Animation 1.</b> Model of a functional CFTR protein.</figcaption>
</figure>
</div>
<H4 text="Function of CFTR" id="function-cftr" />
<p>CFTR functions primarily as a chloride ion channel. It is responsible for transporting chloride ions (Cl⁻) across epithelial cell membranes. Here's how it works<SupScrollLink label="12" />{/*ehem18*/}:</p>
<ol>
<li><b>Regulation by phosphorylation</b>: The R domain must first be phosphorylated by PKA to allow channel activation. This phosphorylation is often triggered by cyclic AMP (cAMP), a signaling molecule.</li>
<li><b>Opening the channel</b>: Once the R domain is phosphorylated, ATP binds to the NBDs, causing conformational changes that open the chloride channel.</li>
<li><b>Chloride transport</b>: With the channel open, chloride ions move from inside the cell to the outside. This movement of chloride helps draw water out of the cell, thinning mucus and maintaining proper hydration of the epithelial surfaces.</li>
<li><b>Closing the channel</b>: Hydrolysis of ATP causes the channel to close after a certain period, tightly regulating chloride transport.</li>
</ol>
<p>CFTR plays a critical role in maintaining the fluid balance on the surfaces of tissues such as the airways, digestive tract and sweat glands. By allowing chloride ions to flow out of the cells, CFTR ensures that water follows, preventing the accumulation of thick, sticky mucus.</p>
<H4 text="CFTR in Cystic Fibrosis" id="CFTR-in-cftr" />
<p>In the lungs, this water movement is crucial for maintaining a thin, slippery layer of mucus that can trap and remove particles like dust and bacteria. The mucus is then moved out of the lungs by the action of cilia, tiny hair-like structures on the surface of epithelial cells. When the CFTR protein is defective, as in Cystic Fibrosis, chloride cannot properly exit the cells. This disrupts the osmotic gradient, preventing water from entering the mucus. As a result, the mucus becomes thick and sticky, making it difficult to clear and creating an ideal environment for bacterial infections, which leads to chronic inflammation and lung damage over time.</p>
<p>In the intestines, CFTR regulates fluid secretion into the digestive tract, which is vital for the normal movement of digestive contents. Without proper CFTR function, water movement is reduced, leading to thickened digestive fluids, blockages, and impaired nutrient absorption. This contributes to malnutrition and other digestive complications in Cystic Fibrosis patients. </p>
<p>By correcting the genetic mutations that lead to CFTR malfunction, such as the F508del mutation, we aim to restore the proper balance of chloride and water movement, which is crucial for preventing the buildup of mucus and ensuring normal function in both the respiratory and digestive systems.</p>
</div>
</div>
{/* <Linear
xAxis={[{ data: [1, 2, 3, 5, 8, 10] }]}
series={[
{
data: [2, 5.5, 2, 8.5, 1.5, 5],
},
]}
width={500}
height={300}
/> */}
</div>
<div className="col">
<img src="https://static.igem.wiki/teams/5247/charts-maps/cfper10-000.png"></img>
</div>
</Subesction>
<Subesction title="The CFTR Protein" id="Cystic Fibrosis2">
<div className="row align-items-center">
<figure>
</Subesction>
<Subesction title="F508del" id="Cystic Fibrosis3">
<p>More than 1,000 mutations in the CFTR gene are responsible for the development of Cystic Fibrosis. The most common variant is the F508del mutation, found in approximately 70% of affected individuals of Caucasian descent in Canada, Northern Europe, and the United States<SupScrollLink label="1" />{/* ehem1 */}. It is estimated that around 90% of people with Cystic Fibrosis in Europe and those of European heritage carry at least one F508del allele<SupScrollLink label="2" />{/* ehem23 */}<sup>,</sup><SupScrollLink label="9" />{/* ehem24 */}. Research suggests that this mutation originated in Western Europe at least 5,000 years ago<SupScrollLink label="2" />{/* ehem23 */}.</p>
<p>The F508del mutation involves the deletion of three nucleotides, "CTT," at position 508, which removes a phenylalanine
residue without causing a frameshift. This deletion impairs the kinetic and thermodynamic folding of the NBD1 domain
<SupScrollLink label="9" />{/* ehem24 */}. As a result, the CFTR protein not only misfolds but also experiences defects in trafficking
and premature degradation, leading to a reduction in its surface expression<SupScrollLink label="16" />{/* ehem25 */}. This specific mutation is particularly severe because it affects both the production and function of CFTR, resulting in a more aggressive disease course. Consequently, patients with the F508del mutation may respond better to CFTR modulators, which target these specific defects in protein folding and function.</p>
<Collapsible id="statistical-distribution-collapsible" title="Statistical distribution of F508del mutations">
<p>In 2023, a comprehensive analysis was conducted to assess the distribution of mutations in the CFTR gene associated with Cystic Fibrosis (CF) worldwide. Data was sourced from two reputable databases: the <a href="https://cftr.iurc.montp.inserm.fr/cgi-bin/variant_list.cgi" title="CFTR-database-1" >CFTR Mutation Database</a> and the <a href="https://cftr2.org/mutations_history" title="CFTR-database-2" >CFTR2 Database</a>. </p>
<p>The results indicate the following distribution of CFTR mutation types and their frequencies in percent: </p>
<div className="row align-items-center">
<div className="col" >
<ul>
<li><b>Insertions (ins)</b>: 0.00088%</li>
<li><b>Deletions (del)</b>: 72.64%</li>
<li><b>Substitutions (subs)</b>: 23.84%</li>
<li><b>Insertions/Deletions (indel)</b>: 0,00485%</li>
<li><b>Other mutations</b>: 0,00370%</li>
</ul>
</div>
<div className="col" >
<PieChart />{/* Render the PieChart component */}
</div>
</div>
</Collapsible>
<p>Overall, the statistical distribution of CFTR mutations reveals significant variations in mutation types and their frequencies worldwide, with deletions (72.64%) being the most common mutation type. This underscores the need for continued research and monitoring of these genetic variations to improve patient care and treatment strategies. CF not only affects the directly affected organs, but also many other areas of the body that are indirectly affected by the extent of the disease, e.g. through the condition of diseased organs. </p>
<div className="row">
<div className="col">
<img src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
<div className="col">
<img src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
<div className="col">
</div>
<div className="col"></div>
</div>
<figcaption><b>Figure x.</b> </figcaption>
</figure>
<div className="col">
<p>Text about CFTR <LoremMedium/></p>
</Subesction>
<Subesction title="Symptoms" id="Cystic Fibrosis4">
<p>Since the CFTR gene is expressed in nearly all tissues of the human body, Cystic Fibrosis affects as a metabolic disease a wide range of vital organs.</p>
<Collapsible id="symptoms-collapsible" title="How the symptoms affect different parts of the body" >
<TabButtonRow data={symptombuttonrowdata} opentype="meditabs" closing="" />
<ButtonRowTabs data={symptombuttonrowdata} cla="meditabs" />
</Collapsible>
</Subesction>
<Subesction title="Diagnosis" id="Cystic Fibrosis5">
<p>With Cystic Fibrosis being a hereditary disease, the diagnostic methods have evolved significantly. Early diagnosis is crucial, as it
allows for timely interventions that can improve the quality of life and longevity for CF patients<SupScrollLink label="37" />{/* ehem58 */}.
With advancements in screening and diagnostic tools, many individuals are diagnosed shortly after birth, enabling early management of
the disease.</p>
<p>Cystic Fibrosis can be diagnosed through a variety of methods, often starting in infancy or even before birth. The most common diagnostic test
is the newborn screening, which involves a blood test that checks for elevated levels of a protein called immunoreactive trypsinogen
(IRT). Elevated IRT levels can indicate potential CF, prompting further testing<SupScrollLink label="31" />{/* ehem61 */}. </p>
<Collapsible id="newborn-screening-collapsible" title="Newborn screening">
<p>Newborn screening for Cystic Fibrosis (CF) has been a major advancement in early detection and management, leading to significantly
improved patient outcomes. This practice, which started in the late 1960s, became more widespread in the 1970s. The screening
typically involves a blood test within the first few days of life, measuring immunoreactive trypsinogen (IRT), a marker that is
elevated in newborns with CF. Elevated IRT levels prompt further genetic testing to identify CFTR mutations<SupScrollLink label="39" />{/* ehem62 */}.
If mutations are found, a sweat chloride test is often conducted to confirm the diagnosis. </p>
<p>Many countries, including the United States, Canada, the United Kingdom, Australia, and several European nations, have implemented
newborn screening programs for CF. However, a survey of CF screening in Europe revealed that the implementation of such programs varies
widely, with some countries adopting more comprehensive protocols than others. Early diagnosis through
screening offers significant benefits, such as improved growth, better lung function, and overall enhanced health outcomes
<SupScrollLink label="40" />{/* ehem64 */}. The discovery of the CFTR gene has further refined diagnostic techniques and underscored the crucial role of newborn screening in the early detection and management of CF. </p>
<p>Technological advancements and improved medical procedures have greatly transformed the diagnosis of Cystic Fibrosis.
While newborn screening has revolutionized early detection and treatment, traditional methods such as the sweat test and
symptom observation continue to play a vital role, particularly in regions where screening programs are not yet widely
available.</p>
</Collapsible>
<p>Another widely used method is the sweat test, which measures the concentration of chloride in a person's sweat. CF patients
typically have higher-than-normal chloride levels due to defective CFTR protein function. While the sweat test is non-invasive and reliable for
indicating CF, it is limited in scope. For definitive diagnosis and to guide specific treatments, a genetic analysis is usually required
to identify the exact CFTR mutation, such as the F508del mutation<SupScrollLink label="41" />{/* ehem66 */}.</p>
<Collapsible id="sweat-test-collapsible" title="Sweat test">
<p>Traditionally, Cystic Fibrosis (CF) has been diagnosed using the sweat test, which measures chloride levels in sweat. A
chloride level below 40 mmol/L (millimoles of chloride per litre of sweat) is considered normal and unlikely to indicate CF.
Levels between 40 and 60 mmol/L require further investigation, while levels above 60 mmol/L strongly suggest the presence of CF<SupScrollLink label="41" />{/* ehem65 */}.</p>
<p>This quick and painless test has been the gold standard for CF diagnosis for many years. Despite its accuracy, the sweat test requires specialized lab personnel and can be difficult to perform on newborns. While diagnosing CF based on symptoms can be useful, it is not always reliable, particularly in mild or atypical cases.</p>
</Collapsible>
</Subesction>
<Subesction title="Treatment" id="Cystic Fibrosis6">
<p>Current Cystic Fibrosis treatments focus on managing symptoms, slowing disease progression, and improving quality of
life. Since there is still no cure for CF, treatment is typically lifelong and involves multiple approaches, including medications,
physical therapy, and dietary adjustments<SupScrollLink label="42" />{/* ehem66 */}. </p>
<p>The primary goal of CF treatment is to clear the thick mucus from the lungs to prevent infections and improve
breathing. Airway clearance techniques, such as chest physiotherapy, are often used alongside inhaled medications, like
bronchodilators and mucolytics, to thin the mucus and open the airways<SupScrollLink label="43" />{/* ehem69 */}<sup>,</sup><SupScrollLink label="44" />{/* ehem70 */}.
Antibiotics are frequently prescribed to treat or prevent lung infections caused by trapped bacteria in the
airways<SupScrollLink label="45" />{/* ehem71 */}.</p>
<p>One of the most significant advances in CF treatment has been the development of CFTR modulators, which target the underlying protein
dysfunction caused by mutations in the CFTR gene<SupScrollLink label="44" />{/* ehem70 */}<sup>,</sup><SupScrollLink label="46" />{/* ehem72 */}.
These drugs, such as ivacaftor, lumacaftor, and elexacaftor, work by improving the function of the defective CFTR protein, particularly
in patients with specific mutations like F508del<SupScrollLink label="46" />{/* ehem24 */}. While CFTR modulators can dramatically
improve lung function and overall health in many patients, they are not effective for all CFTR mutations and often are very
expensive<SupScrollLink label="47" />{/* ehem73 */}.</p>
<p>Digestive enzyme supplements are essential for CF patients who suffer from pancreatic insufficiency, helping them to absorb nutrients
from food<SupScrollLink label="22" />{/* ehem68 */}. Additionally, high-calorie diets and vitamins are recommended to support growth and maintain
body weight<SupScrollLink label="22" />{/* ehem68 */}.</p>
<p>Although current treatments can significantly improve quality of life and life expectancy, managing CF remains a daily challenge for
patients. Continued research into gene therapy and other innovative treatments offers hope for more
permanent solutions in the future.</p>
<Collapsible id="drugs-collapsible" title="Different types of drugs" >
<TabButtonRow data={medibuttonrowdata} opentype="symptabs" closing="" />
<ButtonRowTabs data={medibuttonrowdata} cla="symptabs" />
</Collapsible>
<H4 text="CF treatment with gene therapy"></H4>
<p>While mentioned medications have improved the quality of life for numerous CF patients, they only manage symptoms rather than cure the disease. Moreover, most of them are expensive and not world-wide accessible. Our research is focused on the development of a gene therapy that targets the underlying cause of CF by correcting the defective CFTR gene. <PreCyse /> aims to halt disease progression and reduce the treatment burden for patients.</p>
</Subesction>
</Section>
<Section title="Our Approach" id="Approach">
<Subesction title="Mechanism" id="Approach1">
<p>The development of an improved Prime Editing complex holds great promise for advancing gene editing technologies. Our enhanced system, Prime Guide, addresses key limitations of conventional Prime Editing by focusing on four main areas: editing efficiency, precision, size, and safety. Prime Guide has been designed to target the F508del mutation in Cystic Fibrosis with high accuracy, while minimizing off-target effects. By optimizing the pegRNA, reverse transcriptase, and nickase components, we aim to deliver precise and efficient genetic modifications.</p>
<InfoBox title="Prime Editing" id="prime-editing">
<details>
<summary>Prime editing is a new method of gene editing based on an RNA-Protein complex. It was developed by a group of
researchers revolving around Professor David Liu from Harvard University in 2019.<SupScrollLink label="55" />{/*ehem15*/} </summary>
<p></p>
<p><b>How does Prime Editing work?</b></p>
<p>Prime Editing builds on the well-known CRISPR technology, offering a more precise and controlled approach to DNA modification.
Traditional CRISPR-Cas9 methods typically involve creating double-strand breaks in DNA, which can be repaired by the cell in ways that
might introduce unintended mutations. Prime Editing, by contrast, circumvents this issue by using a more refined method that avoids
double strand breaks altogether<SupScrollLink label="55" />{/* ehem91 */}.</p>
<p>At the heart of Prime Editing is a fusion protein, which combines two key components: a modified Cas9 enzyme, known as a "nickase,"
and a
reverse transcriptase enzyme. The nickase is responsible for making a single strand cut in the DNA, unlike the traditional Cas9,
which cuts both strands. This single strand cut minimizes the risk of unintended mutations or large-scale DNA damage. The reverse
transcriptase attached to the nickase then modifies the DNA at the targeted site by incorporating new genetic information.</p>
<p>To guide this process, Prime Editing uses a specialized RNA molecule known as prime editing guide RNA (pegRNA). This pegRNA serves
two functions: it directs the Cas9 nickase to the specific location on the genome, and it carries a template for the desired DNA
modification. Now, let’s go through the process in more detail, referencing the image above.<SupScrollLink label="56"/></p>
<ol>
<li><b>DNA Nicking</b>: In the first step (top left in the image), the Cas9 nickase, guided by the pegRNA, binds to the target
genomic DNA and creates a single-strand break, or "nick," at the precise location. This is a key difference from standard CRISPR,
where both DNA strands are cut, increasing the risk of unwanted mutations.</li>
<li><b>Primer Binding and Reverse Transcription</b>: Once the DNA is nicked, the primer binding site (PBS) on the pegRNA hybridizes
with the exposed single-stranded DNA, as shown in the middle of the image. This alignment allows the reverse transcriptase (RT)
enzyme, also fused to the nickase, to begin copying the edit into the target DNA. The reverse transcriptase uses the template
encoded within the pegRNA to create a complementary DNA sequence (depicted as the new sequence in the image), ensuring the corrected
genetic sequence is accurately inserted into the genome.</li>
<li><b>Flap Formation and Equilibration</b>: The process continues as the reverse transcriptase copies the new genetic sequence into
the DNA strand, creating what is called a "3' flap" (as shown in the bottom part of the image). This flap contains the newly edited
sequence. At this point, there is an equilibration between the new flap (which encodes the intended edit) and the unedited 5' flap,
which still contains the original, unmodified DNA sequence. The cell's natural mechanisms typically degrade the unedited 5' flap,
favoring the integration of the 3' flap encoding the edit.</li>
<li><b>Flap Resolution and Final Editing</b>: In some cases, an additional nick (seen in the PE3/PE5 systems in the image) is
introduced in the non-target DNA strand to promote repair and favor the incorporation of the edit. This step increases the efficiency
of Prime Editing by ensuring that the newly edited strand is preferentially used during the cell's DNA repair process.
The mismatch repair (MMR) system of the cell also plays a role in determining whether the edit is retained or reverted to the
original sequence. For systems like PE4 and PE5, inhibition of the mismatch repair system (e.g., by MLH1dn) further promotes the
integration of the desired edit.</li>
<li><b>Final outcome</b>: Once the unedited flap is degraded and the new sequence is integrated, the cell completes the repair,
and the edit becomes permanently incorporated into the DNA. As shown in the diagram, the result is a successful genetic modification,
where the new, corrected sequence replaces the original faulty sequence.</li>
</ol>
{/* This figure has to stay this way */}
<figure>
<img className="gif-wrapper" src="https://static.igem.wiki/teams/5247/project-description/prime-editing-animation-10fps.gif" />
<figcaption>
<b>Animation 2. </b>
Illustration of the Prime Editing process and its possible outcomes.
</figcaption>
</figure>
<p>Overall, there are many different Prime Editing systems with a variety of components and complexity, starting from PE2 up to PE7. Possible edits could integrate substitutions, inserts and deletions in the range of one base up to hundreds of nucleotides, with gradually decreasing editing efficiency. Therefore Prime Editing technology allows targeted modifications of specific genes. </p>
</details>
</InfoBox>
<p>However, the Prime Editing complex is relatively large, posing challenges for therapeutic delivery<SupScrollLink label="60"/>{/* ehem3 */}. Additionally, Prime Editing has been shown to be relatively inefficient in terms of gene editing rates, which could limit its therapeutic utility<SupScrollLink label="62"/>{/* ehem4 */}. Our project aims to enhance the Prime Editing approach by miniaturizing its components and enhancing its efficiency, as well as precision. </p>
<p>As shown in the image, we developed two potential configurations for Prime Guide, each using a different nickase: one based on the Fanzor (nSpuFz1) nickase and the other on a CasX (nPlmCasX) nickase. Both configurations are designed to improve the precision and stability of the Prime Editing system. The pegRNA scaffold, reverse transcriptase (PE6c), and primer binding site (PBS/RTT) work together in both systems to introduce precise edits, with the La(1-193) enhancing stability and function.</p>
<div className="figure-wrapper">
<figure>
<div className="col gif-wrapper">
<img className="fanzor gif" src="https://static.igem.wiki/teams/5247/fanzor/cftr-wt.gif"></img>
<div className="row">
<div className="col">
<img className="img" src="https://static.igem.wiki/teams/5247/project-description/primeguide.png" />
</div>
<div className="col"><PrimeEditingComplex /></div>
</div>
<figcaption> <b>Figure 3.</b>Phase contrast image of HEK293T at 20x magnification</figcaption>
<figcaption>
<b>Figure 4. </b>
Illustration of our newly designed Prime Editors, "PrimeGuide".
</figcaption>
</figure>
</div>
</div>
</div>
</Subesction>
<Subesction title="F508del" id="Cystic Fibrosis3">
<p>A multitude of mutations in the CFTR gene, exceeding 1,000, are responsible for the development of cystic
fibrosis. The most prevalent variant is F508del, observed in approximately 70% of affected individuals of
Caucasian descent in Canada, Northern Europe, and the United States<SupScrollLink label="14"/>. It is estimated that around 90% of
the European population and people of European heritage with cystic fibrosis carry at least one F508del
variant <SupScrollLink label="15"/><sup>,</sup><SupScrollLink label="16"/>. Analyses have demonstrated that the F508del mutation originated in Western Europe at least
5,000 years ago <SupScrollLink label="15"/>. </p>
<p>It is a deletion of the three nucleotides "CTT" at position 508, which removes the phenylalanine residue
without causing a frameshift. This deletion leads to defects in the kinetic and thermodynamic folding
of the NBD1 domain <SupScrollLink label="16"/>. However, this not only leads to misfolding of CFTR but also to defects in
trafficking and premature degradation, resulting in reduced surface expression of CFTR <SupScrollLink label="17"/>. </p>
<div className="row">
<div className="col">
<img src="https://static.igem.wiki/teams/5247/charts-maps/cfper10-000.png"/>
</div>
<div className="col-4">
<QuizQuestion name="schreibweise" front="What do the codes F508del and F508del stand for?" back="they..."/>
</div>
</div>
</Subesction>
<Subesction title="Symptoms" id="Cystic Fibrosis4">
<p>Since the CFTR gene is expressed in nearly all tissues of the human body, cystic fibrosis affects as a metabolic disease a wide range of vital organs.</p>
<Collapsible id="symptoms-collapsible" title="How the symptoms affect different parts of the body" >
<TabButtonRow data={symptombuttonrowdata} opentype="meditabs" closing=""/>
<ButtonRowTabs data={symptombuttonrowdata} cla="meditabs"/>
</Collapsible>
</Subesction>
<Subesction title="Diagnosis" id="Cystic Fibrosis5">
<p>About the ways one can be diagnosed <LoremMedium/></p>
<div className="row align-items-center">
<div className="col" >
<img src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
<div className="col" >
How newbornscreening affected the numbers.
<LoremMedium/>
<p>To develop our innovative Prime Editing system, Prime Guide, we worked closely with several leading experts in the field. Among them were Mattijs
Bulcaen, Makato Saito, Dr. Hammer, Jan-Phillipp Gerhard and Prof. Kristian Müller, whose insights helped guide our decisions. Prime Guide
is a highly specialized Prime Editing complex, designed to target the F508del mutation in Cystic Fibrosis with precision and efficiency. </p>
<p>Our Prime Guide system consists of carefully selected components, each optimized for its role. For the nickase, we chose between SpuFz1 and CasX
nickases due to their smaller size and structural advantages, which suggest increased stability for the pegRNA within the Prime Editing
complex. Smaller nickases also provide benefits in terms of overall efficiency and ease of delivery, aligning with the compact design
we aimed for.</p>
<Collapsible id="fanzorcas-collapsible" title="Advantages of Fanzor/PlmCasX over Cas9">
<p>From the start of our project we have been examining the established Prime Editing complex, known for its effectiveness
but also for several limitations, including its relatively large size and structural vulnerabilities. A key component
of this complex is the Cas9 nickase, an enzyme that selectively cuts one of the two DNA strands at a precise location.
This nickase was originally engineered by introducing mutations into the Cas9 endonuclease, which typically cuts both DNA
strands. By disabling one of the two active sites, the Cas9 nickase was designed to nick only one strand, a function essential
to the success of the Prime Editing process<SupScrollLink label="55" />{/* ehem92 */}. </p>
<p>Our aim was to improve the Prime Editing complex, not only by reducing its size but also by enhancing its stability. To achieve
this, we sought alternative endonucleases that are smaller and possess other desirable properties. Our strategy involved
identifying endonucleases with suitable characteristics and then developing methods to mutate them into nickases, allowing
them to selectively cut a single DNA strand. CasX and Fanzor emerged as promising candidates, offering structural advantages
beyond their smaller size. </p>
<p>In Cas9-based systems, the spacer region of the guide RNA (gRNA)—the part that binds to the target DNA—is located at the 5' end
of the RNA-protein complex. However, in CasX and Fanzor, the spacer is positioned at the 3' end<SupScrollLink label="57" />{/* ehem93 */}<sup>,
</sup><SupScrollLink label="58" />{/* ehem94 */}. This reversal offers several benefits: the 3' end of the RNA is typically more
susceptible to degradation by RNases, which can compromise the stability and effectiveness of the Prime Editing complex. In CasX and
Fanzor, however, the 3’ terminus is positioned at the spacer enclosed by the protein, potentially protecting from RNase degradation. </p>
<p>Additionally, this reversed architecture alters the positioning of the reverse transcription template (RTT) and primer binding site (PBS)
on the pegRNA. In Cas9-based systems, the RTT is located at the 3' end of the pegRNA, which leaves it more exposed and increases the risk of
reverse transcription continuing past the intended stop point. This "scaffold read-through" effect can result in the synthesis of unintended
DNA sequences, leading to undesired mutations or genomic alterations at the target site<SupScrollLink label="55" />{/* ehem95 */}, potentially compromising the
safety of the Prime Editing process. In CasX and Fanzor systems, however, the RTT is positioned at the 5' end of the pegRNA, while the spacer is
located near the 3' end and is closely bound to the protein. This reversed layout helps ensure that reverse transcription stops precisely at
the end of the RTT sequence, significantly reducing the risk of unintended extensions and improving the precision and reliability of the editing process.</p>
<p>By incorporating these smaller, more stable nickases into the Prime Editing complex, we aim to reduce its overall size while maintaining
or even enhancing its functionality and reliability. </p>
</Collapsible>
<p>In terms of the pegRNA, we opted for a pegRNA, including a 16-base primer binding site (PBS) and a 30-base reverse transcription
template (RTT), with no silent edits and a structural motif, the tevopreQ1. After extensive screening using a reporter system, this
pegRNA demonstrated the highest performance, leading us to select it as the best candidate for further development. While other
pegRNAs also showed promise, pegRNA_PEAR_04 was ultimately chosen for its superior results in our testing.</p>
<Collapsible id="pegRNA-genau-collapsible" title="Optimization of the pegRNA">
<p><b>Stability improvement: tevopreQ1 extension</b></p>
<p>The pegRNA was specifically optimized to enhance its stability in the cellular environment. To achieve this, a structural motif
known as tevopreQ1 was added to the basic pegRNA structure. This motif was selected based on its known ability to improve RNA
stability by preventing degradation. By integrating tevopreQ1, the goal was to extend the half-life of the pegRNA, allowing it
to remain functional in cells for a longer duration, thus improving the likelihood of successful gene edits. This stabilizing
addition was particularly valuable in the context of CFTR gene editing, where higher RNA stability could lead to better editing
outcomes.</p>
<p><b>Precision enhancement: Spacer selection</b></p>
<p>A major focus during the optimization of the pegRNA was the careful design of the spacer sequence, which plays a crucial role in
guiding the editing complex to the correct genomic location. Multiple spacer sequences were designed and tested via a software,
with the aim of minimizing off-target effects that can lead to unintended genetic changes. Through expert consultations and
theoretical modeling, a rational design strategy was employed to select a spacer sequence that would enhance the precision of
the editing process. This precision is especially important for therapeutic applications, such as in CFTR gene editing, where
unintended edits could have harmful consequences.</p>
<p><b>Improving Editing Efficiency: PBS and RTT length adjustments with Silent Edits</b></p>
<p>To maximize editing efficiency, various combinations of primer binding site (PBS) and reverse transcriptase template (RTT) lengths
were evaluated. The RTT, which provides the template for the desired genetic change, was carefully optimized, including the
introduction of silent edits—changes in the RTT that do not alter the protein sequence but can improve the editing process.
Both shortened and extended versions of the PBS and RTT were tested in combinations with each other, with and without these
silent edits, to identify the optimal configuration that would result in the highest editing efficiency. This step-by-step
screening process allowed for the selection of the most efficient pegRNA for targeting the CFTR gene, ensuring that the system
could achieve high levels of successful edits with minimal unintended consequences.</p>
</Collapsible>
<p>For the reverse transcriptase, we selected the PE6c variant, which has shown to provide the best editing efficiency and a more compact
structure compared to alternatives. Its advanced development stage and ability to offer high precision and editing performance made it
the ideal choice for Prime Guide. </p>
<p>Together, these components form a highly optimized Prime Editing system that balances size, stability, and efficiency. Our aim with
Prime Guide is to create a robust and precise solution for correcting the F508del mutation in Cystic Fibrosis, building on the
guidance from our expert collaborators and extensive testing of each individual component.</p>
<H4 text="Our PreCyse cassette" id="PreCyse-cassette" />
<p>We have developed our PrimeGuide, an optimized version of the Prime Editing system, designed to enhance editing efficiency, precision,
and versatility. As part of our continued efforts to improve and streamline the Prime Editing workflow, we introduce to you the
PreCyse-Cassette—a universal plasmid backbone specifically tailored for any Prime Editing system.</p>
<p>The PreCyse-Cassette is engineered to provide maximum flexibility for the construction of the Prime Editing systems. It includes BsaI
und SapI cloning sites, allowing easy insertion and exchange of essential components like a nickase and reverse transcriptase,
fundamental for Prime Editing. Additionally, it incorporates a cloning site for the guide RNA, ensuring seamless integration and
adaptation to various target sequences.</p>
<p>Moreover, the PreCyse-Cassette contains several advanced features designed to enhance system performance. The architecture of this
cassette is based on a combination of the PE4 and PE7 systems, providing the presence of the LA motif and MLH1dn. Thus allowing an
increased functionality and editing efficiency, while the CMV and T7 promoters ensure high expression levels across different systems.
These features make the cassette universally applicable to a wide range of Prime Editing contexts, enabling users to effortlessly
clone their desired components—nickase, reverse transcriptase, and guide RNA—without the need for complex modifications.</p>
<p>With this PreCyse-Cassette, researchers can easily set up and test their Prime Editing systems, bypassing much of the laborious cloning
work traditionally associated with these setups. The cassette provides an efficient and versatile platform for experimenting with and
refining Prime Editing applications, forming the ideal backbone for PrimeGuide and beyond.</p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/thaw/precyse-plasmid.webp"
alt1=""
description="Schematic diagram of the designed plasmid containing our PreCyse cassette"
num="5"
/>
</Subesction>
<Subesction title="Delivery" id="Approach2">
<div className='row align-items-center'>
<div className='col'>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/sort-lnp-ohne-beschriftung.webp"
alt1=""
description="3D Figure of our optimized SORT LNP called AirBuddy."
num="6"
/>
</div>
<div className='col'>
<p>We optimized LNPs as a robust delivery system to transport larger therapeutic cargo, such as Prime Editing mRNA, to lung
epithelial cells via inhalation. LNPs were chosen over other delivery systems, like Adeno-associated viruses (AAVs), due to
their superior cargo capacity and reduced immunogenicity. Our goal was to create a spray-dried lung-specific LNP named</p>
<figure>
<img className="gif-wrapper" src="https://static.igem.wiki/teams/5247/delivery/airbuddy.webp" />
</figure>
<img src="" style={{ maxHeight: "80pt" }} />
<p>capable of efficiently delivering of our Prime Editing components, referred to as PrimeGuide, to lung tissues through
inhalation. This approach is designed to advance precision medicine by ensuring targeted delivery with minimal off-target
effects.</p>
</div>
</div>
</div>
</Subesction>
<Subesction title="Treatment" id="Cystic Fibrosis6">
<p>Cystic fibrosis therapy means inevitably a complex and customized treatment plan for each patient. It consists of a range of components. These include medication such as CFTR modulators and antibiotics as well as inhalation therapy and mucolytics, physiotherapy, nutritional therapy and sports therapy. It is therefore essential that CF patients receive treatment at a specialist centre [1].</p>
<Collapsible id="drugs-collapsible" title="Different types of drugs" >
<TabButtonRow data={medibuttonrowdata} opentype="symptabs" closing=""/>
<ButtonRowTabs data={medibuttonrowdata} cla="symptabs"/>
</Collapsible>
<H2 text="CF treatment with gene therapy"></H2>
<p>While mentioned medications have improved the quality of life for numerous CF patients, they only manage symptoms rather than cure the disease. Moreover, most of them are expensive and not world-wide accessible. Our research is focused on the development of a gene therapy that targets the underlying cause of CF by correcting the defective CFTR gene. <PreCyse/> aims to halt disease progression and reduce the treatment burden for patients.</p>
<img src="https://static.igem.wiki/teams/5247/charts-maps/cfper10-000.png"/>
</Subesction>
</Section>
<Section title="Approach" id="Approach">
<Subesction title="Mechanism" id="Approach1">
<p>To correct the mutation, we are utilizing Prime Editing technologies. Prime Editing is a genome editing technique that allows precise DNA modifications without causing double-strand breaks<SupScrollLink label="2"/>. Structurally, the Prime Editing complex consists of a Cas9 endonuclease fused to a reverse transcriptase (RT) and guided by a pegRNA, which directs the complex to the target site in the genome. </p>
<InfoBox title="Prime Editing" id="prime-editing">
<details>
<summary>Prime editing is a new method of gene editing based on an RNA-Protein complex. It was developed by a group of researchers revolving around Professor David Liu from Harvard University in 2019. <SupScrollLink label="9"/></summary>
<p>Details</p>
<LoremMedium/>
</details>
</InfoBox>
<div className="row">
<div className="col">
<p>However, the Prime Editing complex is relatively large, posing challenges for therapeutic delivery<SupScrollLink label="3"/>. Additionally, Prime Editing has been shown to be relatively inefficient in terms of gene editing rates, which could limit its therapeutic utility<SupScrollLink label="4"/>. Our project aims to enhance the Prime Editing approach by miniaturizing its components. Fanzor, a recently discovered eukaryotic endonuclease, performs functions similar to Cas9, a crucial part of the Prime Editing complex, but is significantly smaller. We aim to substitute Cas9 with Fanzor. </p>
<p>Additionally, we plan to replace the reverse transcriptase in the Prime Editing complex with a smaller RT variant. Furthermore, MCP proteins will be added to the Prime Editing complex to increase its stability<SupScrollLink label="5"/>. </p>
</div>
<div className="img-right img-half col"><Complex></Complex></div>
</div>
<Collapsible id="fanzorcas-collapsible" title="Cas vs. Fanzor"> child </Collapsible>
<p>The pegRNA is optimized via an extension by a stem loop, which stabilizes the RNA by protecting it from RNases and serves as a binding site for the MCP, which also supports the secondary RNA structure.
This represents a major biosafety feature in that the complex is switched off after successful DNA editing and the subsequent increased influx of chloride ions into the cell. The pegRNA is combined with an optimized sgRNA resulting in higher on-target effect. Overall, its optimization leads to a longer shelf life and an increase in the biosafety of the complex. </p>
</Subesction>
<Subesction title="Delivery" id="Approach2">
<img className="img-left img-half spin" src="https://static.igem.wiki/teams/5247/scientific-figures/lnp.png" height={"200vw"}/>
<div>
<p>We chose LNPs as the delivery system of our Next-Generation Prime Editing Technology. Because of their large capacity and less immunogenic side effects compared to other delivery systems like Adeno-associated Viruses (AVV)<SupScrollLink label="6"/>. Our aim is to optimize the LNP formulation to improve delivery to lung tissue via inhalation. Because of our collaborations, we are able to test and optimize different delivery systems to improve our organ specific therapeutic approach. Therefore, our LNP design focusses on stability and targeting. Stability is achieved by a polyethylene glycol (PEG) coating that protects the LNPs from degradation by the immune system<SupScrollLink label="7"/>. Moreover, we use capsaicin in combination with chitosan to improve the uptake of our construct through their mucus-adhesive properties<SupScrollLink label="8"/>. </p>
</div>
<div className="row align-items-center">
<div className="col">
Lagertemperatur der Parts <LoremShort/>
<Collapsible id="Col1" open={false} title="LNPs explained">
<H4 text="What are LNPs?" id="text" />
<p>Lipid nanoparticles, short LNPs, are small, spherical structures made of lipids that serve as delivery vehicles for therapeutic
molecules, such as RNA, DNA, or drugs. They protect their cargo from degradation, enhance cellular uptake, and are widely used in
mRNA vaccines and gene therapy due to their efficiency and biocompatibility.</p>
<H4 text="LNPs and their impact on modern medicine" id="text" />
<p>LNPs are an advanced delivery system designed to transport therapeutic molecules like RNA, DNA or proteins into the cells. These
nanoparticles are tiny spheres made of lipids that form a protective shell around the cargo. The size of LNPs typically ranges from
50 to 200 nm in diameter, making them incredibly small - about 1,000 times thinner than a human hair<SupScrollLink label="59" />{/* ehem1 */}. </p>
<p>Overall, LNPs represent a significant advancement in drug delivery technology. LNPs offer exceptionally high drug-loading capacities,
making them highly effective for delivering substantial amounts of therapeutic agents in a single dose. Their advanced design allows
for the encapsulation of a large payload, which enhances the efficacy of treatments and reduces the frequency of administration<SupScrollLink label="60"/>{/* ehem3 */}. By
encapsulating and protecting therapeutic agents like mRNA, LNPs enhance the stability, targeted delivery, and effectiveness of
treatments. Their ability to be tailored for specific delivery needs, such as targeting particular organs or overcoming physiological
barriers, makes them a powerful tool in modern medicine<SupScrollLink label="60" />{/*ehem3*/}.</p>
<H4 text="Protection of cargo" id="text" />
<p> The primary function of LNPs is to shield the therapeutic agents they carry, such as mRNA, from degradation and facilitate their
delivery into cells. mRNA is a critical component in many modern vaccines and therapies, but it is highly susceptible to breaking
down before it can reach its target within cells. LNPs address this challenge by encapsulating the mRNA, thus protecting it from
harmful enzymes, like RNases and environmental conditions<SupScrollLink label="61" />{/* ehem2 */}. </p>
<H4 text="Delivery assurance" id="text" />
<p>LNPs come in various types tailored for different therapeutic needs. Solid Lipid Nanoparticles (SLNs) and Nanostructured Lipid Carriers
(NLCs) enhance drug stability and solubility, while Liposomes, with their bilayer structure, are versatile for encapsulating both
hydrophilic and hydrophobic drugs. Cationic LNPs are ideal for gene delivery due to their positive charge, whereas anionic and neutral
LNPs offer reduced interaction and lower toxicity, respectively<SupScrollLink label="60"/>{/* ehem3 */}. </p>
<p>To enhance their effectiveness, LNPs are designed with specific components. For instance, the Nebulized Lung Delivery 1 (NLD1)
nanoparticle, a particular type of LNP, includes a combination of lipids and polymers that stabilize the mRNA and allow it to be
delivered efficiently. This formulation includes small lipid particles that encapsulate the mRNA and can maintain stability for
several days under proper storage conditions<SupScrollLink label="61"/>.</p>
<H4 text="Role of surface modifications in targeting" id="text" />
<div className='row align-items-center'>
<div className='col'>
<OneFigure
pic1="https://ars.els-cdn.com/content/image/1-s2.0-S1773224724002156-gr3_lrg.jpg"
alt1="Aufnahme LNP"
num={7}
description={<span> Endosomal escape vs degradation of LNP cargo at endocytosis.</span>}
/>
</div>
<div className='col'>
<p>LNPs are pivotal not only for shielding mRNA but also for ensuring its efficient delivery into target cells. They
facilitate cellular uptake through endocytosis, where the cell membrane engulfs the nanoparticle. LNPs are acclaimed
for their high drug-loading capacities, which greatly enhance their therapeutic effectiveness. However, the success of
this delivery hinges on effective endosomal escape. Ideally, LNPs release their mRNA payload into the cytoplasm after
escaping from endosomes. If this escape process is inefficient, the mRNA can be degraded by lysosomes, which poses a
significant challenge for mRNA vaccines and therapies<SupScrollLink label="62"/>{/* ehem4 */}.</p>
</div>
</div>
<p>A crucial advancement in LNP technology involves the use of pH-sensitive cationizable lipids. These lipids remain neutral
at physiological pH but become cationic in the acidic environment of endosomes. This shift in charge helps dissociate the nanoparticles and
disrupt the endosomal membrane, enhancing the likelihood of successful endosomal escape<SupScrollLink label="63"/>{/* ehem5 */}. </p>
<p>Moreover, the surface of LNPs can be customized to improve targeting. For instance, incorporating specific lipids or modifying the surface with
charged groups can direct the delivery of mRNA to targeted organs like the lungs or spleen<SupScrollLink label="64" />{/* ehem6 */}.
Additionally, LNPs can be engineered with targeting ligands or antibodies to precisely direct their payload to specific cell types, further
enhancing their therapeutic efficacy<SupScrollLink label="65" />{/* ehem7 */}. Another approach can be chitosan-based nanoparticles have been
explored for their ability to adhere to mucus and enhance drug delivery through the respiratory tract. These nanoparticles can penetrate through
the mucus layer to reach the lung tissues more effectively<SupScrollLink label="66" />{/*ehem8*/}. This versatility in design is essential for
optimizing the delivery and effectiveness of LNP-based therapies.</p>
</Collapsible>
<Collapsible id="Col2" open={false} title="Challenges of working with LNPs">
<p>Maintaining the stability of LNPs throughout formulation, storage, and delivery is critical, as factors like temperature changes, pH shifts, or
mechanical stress can affect their integrity<SupScrollLink label="67" />{/* ehem1 */}<sup>,</sup><SupScrollLink label="68" />{/* ehem2 */}. Equally
important is ensuring efficient encapsulation of the genetic material, as any inefficiency can lead to degradation of the therapeutic cargo or inadequate
delivery to the target cells. Once inside the body, LNPs face the challenge of cellular uptake and successful endosomal escape<SupScrollLink label="69"/>{/* ehem3 */}<sup>,</sup><SupScrollLink label="62"/>{/* ehem4 */}.
If they cannot escape the endosome after entering the cells, there is a risk that the genetic material will be degraded in the lysosomes, limiting the
efficacy of the treatment. In addition, the formulation must minimize immunogenicity and toxicity, particularly with repeated dosing, which is often
necessary for chronic diseases<SupScrollLink label="68" />{/* ehem2 */}<sup>,</sup><SupScrollLink label="69"/>{/* ehem3 */}. Achieving this sensitive
balance is crucial for maximizing the therapeutic potential of LNPs
in gene delivery.</p>
<p>While these are general difficulties in the use of LNPs for gene therapy, further challenges arise when administering the LNPs via inhalation into the lungs,
due to the unique environment and anatomy of the respiratory system.</p>
<H4 text="Challenges of inhalated lung-specific LNPs" id="chall2" />
<p>These challenges range from formulation and particle size to overcoming biological barriers and maintaining consistent dosing, all of which
impact the overall efficacy of the therapy. </p>
<p>When transforming LNP formulations into inhalable particles, even greater attention must be paid to stability than is already the case. During
processes like nebulization or spray-drying, LNPs are exposed to strong <strong>mechanical stress</strong> such as shear forces during aerosolization
that can damage the LNP and thus their ability to protect and deliver genetic material effectively<SupScrollLink label="70"/>{/* ehem5 */}. Ensuring that the
LNPs maintain their structure throughout this transformation while remaining suitable for aerosol delivery is critical to the success of the therapy.</p>
<p>The <strong>size</strong> of the nanoparticles is another important factor. For successful lung delivery, LNPs should be smaller than 2
µm<SupScrollLink label="71" />{/* ehem6 */}. If the particles are too large, there is a risk that they will get stuck in the upper airways not able to reach the target cells; if they are too small, they may be exhaled before reaching the deeper lung tissue. The right particle size is crucial for the LNPs to reach the alveoli, where they can provide the greatest therapeutic impact.</p>
<p>Another major challenge is overcoming the lungs' natural <strong>protective barriers</strong>. The airways are lined with mucus and surfactants, which
help to defend against pathogens, but also make it difficult for LNPs to be transported. In diseases such as Cystic Fibrosis, the thickened mucus presents
an even greater obstacle, making it more difficult for the LNPs to reach the target cells<SupScrollLink label="70"/>{/* ehem5 */}.
The development of LNPs that can penetrate these barriers is essential for the success of gene therapy. </p>
<p>Finally, inhaled administration leads to fluctuations in the consistency of the <strong>dosage</strong>. Unlike intravenous administration, where
dosing can be strictly controlled, the results of inhalation are influenced by factors such as the patient's breathing pattern, lung capacity and
inhalation technique. These variables can affect how much of the LNP formulation actually reaches the lungs, complicating efforts to maintain a consistent
therapeutic dose over time, which is a reasonable price to pay when you consider that inhalation is a non-invasive form of therapy compared to systemic
therapy via injections into the bloodstream</p>
<p>All these challenges complicate the work with LNPs and present scientists with a great challenge, which makes working with LNPs even more important to
find solutions.</p>
</Collapsible>
<br />
<div id="airbuddy-hook" className='row align-items-center'>
<p>To optimize AirBuddy for pulmonary delivery, we collaborated extensively with several experts, including <a onClick={() => goToPagesAndOpenTab('weber', '/human-practices')}>Prof. Weber,
Dr. Große-Onnebrink</a> and <a onClick={() => goToPagesAndOpenTab('kolonkofirst', '/human-practices')}>Dr. Kolonko</a> as medical experts,
<a onClick={() => goToPagesAndOpenTab('kristian', '/human-practices')}>Prof. Dr. Müller</a>, <a onClick={() => goToPagesAndOpenTab('radukic', '/human-practices')}>Dr. Radukic</a>,
<a onClick={() => goToPagesAndOpenTab('moorlach', '/human-practices')}>Benjamin Moorlach</a> and the <a onClick={() => goToPagesAndOpenTab('biophysik', '/human-practices')}>Physical and Biophysical Chemistry working group</a>
as academic experts from Bielefeld University and FH Bielefeld as well as <a onClick={() => goToPagesAndOpenTab('corden', '/human-practices')}>Corden Pharma</a> and <a onClick={() => goToPagesAndOpenTab('rnhale', '/human-practices')}>RNhale</a> as industrial experts.
Throughout the <a onClick={() => goToPageWithTabAndScroll({ tabId: 'tab-delivery', path: '/engineering', scrollToId: "delivery-header" })}>development process</a>, we tested two commercially available kits: the
<strong>Cayman Chemical LNP Exploration Kit (LNP-102)</strong> and the <strong>Corden Pharma LNP Starter Kit #2</strong>. While the Cayman kit
showed good non-lung-specific transfection efficiency, the Corden Pharma formulation also proved not to be the right approach. Building on this,
we integrated the <strong>SORT LNP</strong> method based on Wang's research<SupScrollLink label="71" />{/* ehem1 */}, making our nanoparticles lung-specific.
Additionally, we employed the <strong>chitosan-complexation of the therapeutic cargo</strong> to improve the stability of our LNP, ensuring that it
withstands the inhalation process without degradation. Moreover, further stabilization approaches including the employment of the <strong>spray-drying
technique</strong> in cooperation with RNhale<SupScrollLink label="72" />{/* ehem2 */} are in the pipeline. This improved stability is crucial for the efficient
delivery of mRNA into lung epithelial cells, where PrimeGuide can effectively perform genome editing.</p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/big-plan-inhalation-teil-del.webp"
alt1=""
description="Schematic representation our LNP-based drug delivery system."
num="8"
/>
</div>
<div className="col">
Trocknung <LoremShort/>
</div>
</div>
<br/>
<p>We are furthermore optimising the LNPs for pulmonary therapy and investigating delivery by nebulisation as a non-invasive method compared to systemic approaches to make the therapy more convenient for patients. For specific targeting, we are focussing on marker proteins of basal cells and ionocytes that produce particularly high levels of CFTR protein and which we want to target with appropriate antibodies<SupScrollLink label="9"/>. Our workflow includes testing our next generation Prime Editing Technology delivered by our optimized LNPs in cell culture lines but also in primary nasal epithelial cells of CF patients to evaluate our optimizations and further improvements in vitro. We can also provide the outlook on the adaptation of the delivery system enabling systemic applications as well. </p>
</Subesction>
</Section>
<Section title="Our Vision" id="Our Vision">
<p>We are envisioning a potential integration into a broader therapeutic framework involving customized gene editing tools for various genetic disorders, that present similar problems/difficulties to the F508del mutation, as well as other genetic diseases of different causes. This could include collaborations with pharmaceutical companies to develop new treatment modalities for genetic diseases beyond cystic fibrosis, utilizing advanced delivery systems and personalized medicine approaches. </p>
<H2 text="Editing Statistics"/>
<PieChart /> {/* Render the PieChart component */}
</Section>
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);
<p>To evaluate the <strong>delivery efficiency</strong>, we transfected HEK293 and CFBE41o- cells using fluorescent cargo and quantified the results
through flow cytometry analysis. We also ensured that AirBuddy meets the necessary standards for safety and efficacy since we conducted extensive
<a onClick={() => goToPageAndScroll('In-Depth Characterization of LNPsH', '/materials-methods')}> characterization of the LNPs </a>using physicochemical
techniques such as Zeta potential analysis, Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), and Cryogenic Electron Microscopy (cryo-EM).
These methods confirmed the stability and optimal size distribution of the nanoparticles. Furthermore, <strong>cytotoxicity assessments</strong>
including MTT and proliferation assays demonstrated that our LNPs are biocompatible despite the incorporation of <a onClick={() => goToPageWithTabAndCollapsible({ tabId: 'tab-delivery', path: '/engineering', collapseId: "Col1" })}>PEG</a>
and other ambivalent components. These findings reinforce AirBuddy's potential as a safe and effective tool for pulmonary delivery, with broad
implications for gene therapies targeting lung diseases.</p>
</Subesction>
</Section>
<Section title="Our Achievement" id="Our Achievement">
<p>We have successfully demonstrated a <b>proof of concept</b> for our gene therapy approach targeting Cystic Fibrosis. In initial experiments, HEK cells carrying a 3-base deletion analogous to the <i>F508del</i> mutation were transfected with our prime editing complex. The results met our expectations, confirming the viability of our approach for precise gene correction. Based on these findings, we optimized the prime editing complex, leading to the creation of <b>PrimeGuide</b>, a more compact and efficient editing tool. </p>
<p>Central to our <b>delivery system</b> is <b>AirBuddy</b>, a lung-specific lipid nanoparticle designed to stabilize and protect the prime editing complex during transport to lung epithelial cells. <b>AirBuddy</b> ensures that the protein complex is delivered specifically to lung cells, enhancing the efficiency of the gene-editing process. By modifying the lipid nanoparticle with protective features, we achieved increased stability, ensuring effective delivery to the target cells. </p>
<p>We further optimized the prime editing fusion protein, <b>PrimeGuide</b>, to streamline its components, resulting in a smaller and more efficient prime editing complex. This improvement significantly enhances the precision of the gene editing process, reducing off-target effects and increasing the overall success of mutation correction. </p>
<p>In subsequent experiments, <b>HEK293 and CFBE41o- cells</b> carrying the CFTR <i>F508del</i> mutation were successfully <b>transfected</b> with the optimized prime editing complex. Our results indicated successful correction of the mutation, confirming the potential of our approach for treating Cystic Fibrosis. </p>
<p>Additionally, we explored <b>downstream applications</b>. Primary cell cultures were treated with lipid nanoparticles to introduce a reporter RNA and Patch Clamp measurements were explored as a validation method.</p>
</Section>
<Section title="Our Vision" id="Our Vision">
<p>At <b>PreCyse</b>, we envision a future where gene therapy for Cystic Fibrosis (CF) is as simple and user-friendly as using an inhaler. Our goal is to develop a fully integrated Prime Editing system, <b>PrimeGuide</b>, delivered via a cutting-edge lipid nanoparticle (LNP) platform, <b>AirBuddy</b>. The therapy would allow patients to inhale the therapeutic complex, targeting the underlying genetic mutation that causes CF—specifically, the F508del mutation in the CFTR gene. </p>
<p>The core of our vision is to create a highly efficient and safe Prime Editing complex, referred to as Prime Guide, that is delivered directly into lung epithelial cells. This complex will be packaged as mRNA into LNPs, with an optimal ratio of the Prime Editing components and its guide RNA (pegRNA). Once inside the cell, the mRNA will be translated, forming the active Prime Editing complex, which then translocates into the nucleus using nuclear localization sequences. There, the complex will precisely edit the genome to correct the F508del mutation. </p>
<p>To ensure safety, we are working on developing a robust mechanism that regulates the Prime Editing complex at the mRNA level. One concept we are exploring is using the XBP1 intron<SupScrollLink label="73" />{/* ehem96 */}, which responds to cellular stress signals. Additionally, in the future, we aim to develop more mutation-specific control mechanisms, such as RNA riboswitches that activate the editing complex only in the presence of the target mutation, offering an even greater level of precision and safety. </p>
<p>The long-term vision for PreCyse is to provide a gene therapy that can be administered through inhalation, much like an asthma spray. The patient would simply inhale the LNPs, which then deliver the therapeutic mRNA to the lungs. This approach offers a user-friendly and minimally invasive treatment that could suppress the symptoms of CF for several months. By correcting the mutation in the top layers of lung epithelial cells, where mucus buildup is most problematic, we could offer relief from symptoms over an extended period. However, since these epithelial cells naturally regenerate over time, the therapy would need to be reapplied periodically, likely every few months, balancing long-lasting effects with the need for occasional re-administration. </p>
<p>Ultimately, our vision is to create a therapeutic approach that not only offers a cure that is safe and efficient but also maximizes convenience for the patient. With an easy-to-use inhaler, patients could administer their treatment with minimal disruption to their daily lives, inhaling the gene therapy in just a few breaths, leaving the rest of the process to the science we've built into PreCyse. By reducing the frequency of administration and simplifying the delivery method, we aim to make gene therapy for Cystic Fibrosis both accessible and practical for patients around the world. </p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/big-plan-inhalation-del-mech.webp"
num={9}
description="Illustration of our path from final product to prime editing in lung epithelial cells."
alt1="Illustration of our path from final product to prime editing in lung epithelial cells."
/>
</Section>
<Section title="References" id="References">
<DescSources />
</Section>
</div>
</div>
);
}
let medibuttonrowdata =[
let medibuttonrowdata = [
{
node: createDrugSteckbrief(drugdata[0]),
buttonname: "Modulators",
node: createDrugSteckbrief(drugdata[0]),
buttonname: "Modulators",
cssname: "Med-First",
main: true
},
{
{
node: createDrugSteckbrief(drugdata[1]),
buttonname: "Mucolytics",
buttonname: "Mucolytics",
cssname: "Mucolytics"
},
{
node: createDrugSteckbrief(drugdata[2]),
buttonname: "Antibiotics",
buttonname: "Antibiotics",
cssname: "Antibiotics"
},
{
node: createDrugSteckbrief(drugdata[3]),
buttonname: "Enzymes",
buttonname: "Enzymes",
cssname: "Enzymes"
},
]
......@@ -528,49 +606,49 @@ export function Description() {
let symptombuttonrowdata = [
{
node: createSymptomSteckbrief(symptomdata[0]),
buttonname: "Pancreas",
node: createSymptomSteckbrief(symptomdata[0]),
buttonname: "Pancreas",
cssname: "Symp-First",
main: true
},
{
node: createSymptomSteckbrief(symptomdata[1]),
buttonname: "Intestines",
{
node: createSymptomSteckbrief(symptomdata[1]),
buttonname: "Intestines",
cssname: "intestines"
},
{
node: createSymptomSteckbrief(symptomdata[2]),
buttonname: "Liver",
node: createSymptomSteckbrief(symptomdata[2]),
buttonname: "Liver",
cssname: "liver"
},
{
node: createSymptomSteckbrief(symptomdata[3]),
buttonname: "Sexual glands",
node: createSymptomSteckbrief(symptomdata[3]),
buttonname: "Sexual glands",
cssname: "Sexual glands"
},
{
node: createSymptomSteckbrief(symptomdata[4]),
buttonname: "Lungs",
node: createSymptomSteckbrief(symptomdata[4]),
buttonname: "Lungs",
cssname: "lungs"
},
{
node: createSymptomSteckbrief(symptomdata[5]),
buttonname: "Skeletal System",
node: createSymptomSteckbrief(symptomdata[5]),
buttonname: "Skeletal System",
cssname: "Skeletal System"
},
{
node: createSymptomSteckbrief(symptomdata[6]),
buttonname: "Skin",
node: createSymptomSteckbrief(symptomdata[6]),
buttonname: "Skin",
cssname: "skin"
},
{
node: createSymptomSteckbrief(symptomdata[7]),
buttonname: "Nasal mucosa",
node: createSymptomSteckbrief(symptomdata[7]),
buttonname: "Nasal mucosa",
cssname: "Nasal mucosa"
},
{
node: createSymptomSteckbrief(symptomdata[8]),
buttonname: "Brain",
node: createSymptomSteckbrief(symptomdata[8]),
buttonname: "Brain",
cssname: "brain"
},
......@@ -579,59 +657,58 @@ let symptombuttonrowdata = [
function createSymptomSteckbrief(data: SymptomDatensatz){
let examplelist = [];
function createSymptomSteckbrief(data: SymptomDatensatz) {
let examplelist: JSX.Element[] = [];
for (let index = 0; index < data.introduction.length; index++) {
examplelist.push(
<li>{data.introduction[index]}</li>
)
examplelist.push(
<li key={index}>{data.introduction[index]}</li>
)
}
return(
return (
<div>
<H4 id={`${data.name}-btn`} text={data.name}/>
<H4 id={`${data.name}-btn`} text={data.name} />
<div className="row">
<div className="col-2">
<div className="symptom-img-wrapper">
<img src={data.picture} className="symptom-img"/>
<img src={data.picture} className="symptom-img" />
</div>
</div>
<div className="col">
<ul>{examplelist}</ul>
</div>
</div>
</div>
)
}
function createDrugSteckbrief(data: DrugDatensatz){
let examplelist = [];
function createDrugSteckbrief(data: DrugDatensatz) {
let examplelist: JSX.Element[] = [];
for (let index = 0; index < data.examples.length; index++) {
let absaetze = []
let absaetze: JSX.Element[] = []
for (let i = 0; i < data.examples[index].text.length; i++) {
absaetze.push(
<li>{data.examples[index].text[i]}</li>
<li key={i}>{data.examples[index].text[i]}</li>
)
}
examplelist.push(
<div className="drug">
<H4 text={data.examples[index].title}/>
<ul>{absaetze}</ul>
<div key={index + 500} className="drug">
<H4 text={data.examples[index].title} />
<ul key={index}>{absaetze}</ul>
</div>
)
}
return(
return (
<div>
<H4 id={`${data.name}-btn`} text={data.name}/>
<H4 id={`${data.name}-btn`} text={data.name} />
<div className="row">
<div className="col-2">
<div className="symptom-img-wrapper">
<img src={data.picture} className="symptom-img"/>
<img src={data.picture} className="symptom-img" />
</div>
</div>
<div className="col">
......@@ -639,7 +716,7 @@ function createDrugSteckbrief(data: DrugDatensatz){
</div>
</div>
<div className="col">
{examplelist}
{examplelist}
</div>
</div>
)
......
src/contents/engineering.tsx
View file @ 3fd292fd
import { ButtonOneEngineering } from "../components/Buttons";
import { LoremShort } from "../components/Loremipsum";
import { openElement } from "../utils/openElement";
import { H3, H4 } from "../components/Headings";
import { H2, H3, H4, H5, PhilipH3 } from "../components/Headings";
import { useTabNavigation } from "../utils/TabNavigation";
import { Collapsible } from "../components/Collapsible";
import { useNavigation } from "../utils";
import { TabScrollLink } from "../components/Link";
import { InfoBox } from "../components/Boxes";
import { DownloadLink } from "../components/Buttons";
import { Section } from "../components/sections";
import EngTrfsources from "../sources/eng-trf-sources";
import EngRepsources from "../sources/eng-reporter-sources";
import EngPEsystems from "../sources/eng-pe-sources";
import EngPegsources from "../sources/eng-peg-sources";
import EngNicksources from "../sources/eng-nickases-sources";
import EngDelsources from "../sources/eng-delivery-sources";
import { TwoLinePDF, PDF } from "../components/Pdfs";
import { OneFigure, TwoFigureRow } from "../components/Figures";
export function Engineering() {
useTabNavigation();
const {goToPagesAndOpenTab} = useNavigation ();
const {goToPageAndScroll} = useNavigation();
const {goToPageWithTabAndScroll} = useNavigation();
return (
<>
<div className="row mt-4">
<div className="col">
<br/> <br/> <br/>
<div id="tab-our-cycle" className="enginneeringtab" style={{display: "block"}}>
<br/> <br/>
<div id="tab-our-cycle" className="enginneeringtab" style={{display: "none"}}>
<section > <br id="obenengineering"/>
<div className="eng-box box" >
<H3 text="Our cycle" id="ourcycle"></H3>
<p>Hallo Prime Editing diesdas</p>
<H2 text="Our cycle" id="our-cycle-header"></H2>
<p>
In the course of our project, innovative thoughts were taken up, rejected, elaborated, discussed and tested. On this page we present a selection of ideas that occupied us the most in the last month. Our trains of thought are represented in form of iterations of engineering cycles. Each engineering cycle is comprised of four steps:
</p>
<ul>
<li>
<b>Design</b>: In this step the motivation or problem, that the individual iteration addresses, is mentioned. A theoretical approach addressing the motivation is explained.
</li>
<li>
<b>Build</b>: The practical examination of the theoretical plan described in the design section is executed, e. g. by planning an experiment, by creating a construct in silico, by cloning.
</li>
<li>
<b>Test</b>: The initial design is tested, either by conducting an experiment, testing a design in silico, e. g. by modeling, refuting ideas based on new input or background research or discussing approaches with an expert.
</li>
<li>
<b>Learn</b>: In this last section, test results are discussed and insights gained in testing the design are formulated.
</li>
</ul>
</div>
<br/>
......@@ -28,328 +58,1084 @@ export function Engineering() {
</div>
<div className="col button-left">
<div className="right"><ButtonOneEngineering label="Next" open="proof-of-concept" scrollToId="Proof of Concept"/></div>
<div className="right"><ButtonOneEngineering label="Next" open="reporter" scrollToId="reporter-header"/></div>
</div>
</div>
</section>
</div>
<div id="tab-proof-of-concept" className="enginneeringtab" style={{display: "none"}}>
<div className="enginneeringtab" id="tab-reporter" style={{display: "none"}}>
<section id="reporter sec" >
<div className="eng-box box" >
<H2 id="reporter-header" text="Prime Editing Reporter"></H2>
<p>Prime editing is a is a very precise and safe method. However, depending on the genomic locus targeted, the editing efficiency can be very low. The Cystic Fibrosis causing CFTR F508del mutation is, as <a onClick={() => goToPagesAndOpenTab('mattijsinv', '/human-practices')}> Mattijs Bulcaen </a> stated in our interview, one of, if not the most obvious application of prime editing, considering the large amount of people affected. The lack of publications addressing CFTR target implied, that the mutation might be particularly hard to edit. At low editing efficiency, successful edits are hard, if not impossible to distinguish from the background noise using conventional methods like sanger sequencing or qPCR. As a basis to effectively test our approach and screen for working pegRNAs, we needed a highly sensitive method of detection with as little noise as possible to optimize our prime editing approach for genomic CFTR targeting.</p>
</div>
<div className="box" >
<p id="rep1">
<H3 text="A Fluorescence Reporter" id="rep1head"/>
<H4 text="Design" id="design-head"/>
<p>
We reasoned that the easiest way of detecting DNA changes in a cell would be fluorescence. Our initial idea was to create pegRNAs targeting the coding sequence of a fluorescent protein, that would introduce a mutation resulting in a different emission, giving easily detectable feedback of correct editing. The original Aequorea victoria GFP protein differs from avGFP(Y66W), emitting light in a wavelength of around 509 nm (cyan), and avGFP(Y66H), emitting light in a wavelength of around 448 nm (blue) by only one amino acid substitution each.<TabScrollLink tab="tab-reporter" num="1" scrollId="desc-1"/> Prime editing could therefore be visualized by facilitating these substitutions with a prime editor.
</p>
<H4 text="Build" id="build-head"/>
<p>
To this end, the wild-type and edited versions of the avGFP were put in contrast and we started searching for potential pegRNAs for editing one into the other.
</p>
<figure>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/rep-it1.svg" alt="Illustration of fluorescence wavelength change reporter"/>
<figcaption><b>Figure 1: Illustration of a reporter system based on the introduction of a single amino acid substitution into GFP plasmids transformed into HEK293 cells changing the emission spectrum in a detectable way.</b> </figcaption>
</figure>
<H4 text="Test" id="test-head"/>
<p>
When trying to find protospacers for Cas9 and other possible <a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'nickase-header', path: '/engineering', tabId: 'tab-nickase'})}>nickases</a>, we noticed, that the locus of the mutations is too far away from any SpuFz1 TAM sequences. Additionally, the applicability of insights gained through pegRNA optimization in this locus to CFTR editing would also be very limited due to the vast differences in the sequence of protospacer and surrounding genomic region. Additionally, we learned from our interview with <a onClick={() => goToPagesAndOpenTab('mattijsinv', '/human-practices')}>Mattijs Bulcaen</a> that the type of edit (insertion, substitution or deletion) significantly impacts editing efficiency. A mutation changing GFP to BFP would have to be a substitution instead of the three-nucleotide insertion needed to correct CFTR F508del.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
From our observations we learned that a reporter system is only of use, if it can really mimic the genomic target of choice. The adjustments to be made to create a pegRNA targeting the genomic target from a pegRNA targeting the reporter should be as minor as possible. This includes a similar spacer and a similar edit to be made.
</p>
</p>
</div>
<div className="box" >
<p id="rep2">
<H3 text="Proof of Concept for PEAR" id="rep2head"/>
<H4 text="Design" id="design-head"/>
<p>
After extensive research we came across the prime editor activity reporter (PEAR) created by Simon et al. (2022)<TabScrollLink tab="tab-reporter" num="2" scrollId="desc-2"/>, which is the template our modified reporter plasmid is based on. The PEAR plasmid contains an eGFP coding sequence with an intron derived from the mouse Vim gene. If the intron is removed during RNA splicing, the two exons form a continuous open reading frame. By mutating the 5’ splicing signal, a target is created which, upon correct editing, leads to a gain-of-function. The resulting fluorescence can be imaged using confocal microscopy or quantified by means of flow cytometry. Notably, the area downstream of the 5’ splice signal is intronic, and thus can be edited without any impact on the coding sequence. Additionally, Simon et al. showed, that “efficiency of prime editing to modify PEAR plasmids is governed by the same factors as prime editing in genomic context”. We reasoned that this system might be flexible, and sensitive enough to build our optimizations strategies upon.
</p>
<H4 text="Build" id="build-head"/>
<p>
Since none of us had any experience in prime editing before our project, we wanted to test whether we can facilitate prime editing in the first place. To do this and also assess the functionality of the PEAR system, we set up a proof of concept using the PEAR 2in1 system. This plasmid includes not only the eGFP with and intron and disrupted 5’ splice site, but also a pegRNA expression cassette. The pegRNA is designed in a way that, in combination with a prime editing protein complex, corrects the disrupted splicing signal.
</p>
<H4 text="Test" id="test-head"/>
<p>
In the experiment, we transfected HEK293 cells (as recommended by <a onClick={() => goToPagesAndOpenTab('mattijsinv', '/human-practices')}>Mattijs Bulcaen</a>) with the <a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'current-pe-systems', path: '/engineering', tabId: 'tab-pe-systems' })}>pCMV-PE2 prime editor</a> plasmid and the pDAS12489_PEAR-GFP_2in1_2.0 mentioned above. Our first proof of concept succeeded as we could see fluorescent cells 72 h after transfection. In contrast, negative controls with only one of the plasmids transfected did not show any fluorescence. However, the transfection efficiency in our initial test runs was quite low, as indicated by a technical positive control.
</p>
<figure>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/rep-it2.svg" alt="Illustration of the proof of concept using the PEAR2in1 system"/>
<figcaption><b>Figure 2:</b> Illustration of the proof of concept experiment. HEK293 cells transiently transformed with the pDas12189_PEAR-GFP_2in1_2.0 plasmid on the left side show fluorescence after transformation with a prime editor expression plasmid.</figcaption>
</figure>
<H4 text="Learn" id="learn-head"/>
<p>
This proved, that not only we were able to use prime editing in our model, but also that the PEAR reporter system can report successful prime editing. Though this was a very promising start, further steps had to be taken to enable context specific testing of prime editing. Firstly, the transfection efficiency had to be improved (see <a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'transfection-header', path: '/engineering', tabId: 'transfection' })}> Transfection Optimization</a>). Secondly, the reporter had to be modified in a way that resembles the genomic CFTR target.
</p>
</p>
</div>
<div className="box" >
<p id="rep3">
<H3 text="Contextualization of PEAR" id="rep3head"/>
<H4 text="Design" id="design-head"/>
<p>
The original PEAR plasmid pDAS12124_PEAR-GFP-preedited that we bought from AddGene represents, as the name suggests, how the reporter should look like after successful editing and can thus be used as a positive control and for normalization. To alter the PEAR plasmid so that it mimics the mutated genomic CFTR target, we first analyzed the region surrounding CFTR F508del mutation. As the mutation is a three base pair deletion, we introduced the very same at the 5’ splicing signal. For this modification to reliably disrupt intron splicing and thus eGFP expression, we effectively removed the GT bases of the intronic 5’ splice donor site as well as the preceding, exonic G base of the 5’ flanking sequence. Secondly, we replaced the intronic region downstream of the four base pair 3’ flanking region with the respective sequence from the CFTR locus. This 27 bp substitute included a PAM sequence, an entire spacer as well as four additional base pairs in between present in the original gene sequence. Lastly, we introduced silent mutations upstream of the 5’ flanking sequence that lowered the GC content. This was to mimic the AT-rich region preceding the F508del mutation in the CFTR gene. This reveals one of the necessary shortcomings of this reporter: Edits upstream of the 5’ donor site are heavily restricted by the eGFP coding sequence.
</p>
<figure>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/rep-strategy-it3.svg" alt="Modification strategy for creation of the pPEAR_CFTR plasmid"/>
<figcaption><b>Figure 3:</b> Illustration of the modification strategy for the pPEAR_CFTR reporter. The pDAS12124_PEAR-GFP-preedited plasmid was modified by introducing a 3 bp deletion resembling the F508del mutation, inserting a 27 bp sequence from the genomic CFTR target including PAM and protospacer sequence as well as making silent edits to account for the AT-rich region upstream of the mutation.</figcaption>
</figure>
<H4 text="Build" id="build-head"/>
<p>
We constructed the reporter system by first analyzing the original plasmid to identify appropriate restriction sites. We then digested the plasmid backbone and cloned in a gene synthesis fragment ordered at IDT containing the edits via Gibson Assembly cloning. The correct cloning was validated first by colony PCR and then by sequencing the regions of the plasmid containing the cloning sites and our modifications.
</p>
<H4 text="Test" id="test-head"/>
<p>
We evaluated the functionality of our reporter system by co-transfecting our reporter construct with a pCMV-PE2 prime editor plasmid as well as a plasmid expressing pegRNA that targeted our reporter (see <a onClick={() => goToPagesAndOpenTab('pegrna', '/engineering')}> pegRNA engineering cycle</a>) into HEK293 cells. After 72 h we saw a significant number of cells showing fluorescence.
</p>
<p>
Additionally, for positive controls we transfected a technical control plasmid as well the unmodified pDAS12124_PEAR-GFP-preedited plasmid, which could be used to determine the transfection efficiency as well as normalize the editing efficiency. As negative controls, our modified plasmid, pCMV-PE2 and the pegRNA plasmid were transfected. The positive controls showed fluorescence, while the negative control did not.
</p>
<figure>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/rep-it3.svg" alt="Illustration of pegRNA testing using the pPEAR_CFTR system"/>
<figcaption><b>Figure 4:</b> Illustration of the pegRNA testing using the pPEAR_CFTR system. HEK293 cells transiently transformed with the pPEAR_CFTR plasmid (in the middle) show fluorescence after transformation with a prime editor and a reporter-specific pegRNA expression plasmid. The pDAS12124_PEAR-GFP-preedited is used as an internal positive control and for normalization.</figcaption>
</figure>
<H4 text="Learn" id="learn-head"/>
<p>
Our results demonstrate three things: Firstly, the original pDAS12124_PEAR-GFP-preedited plasmid leads to undisrupted expression of eGFP in the transfected cells. Secondly, the modifications that we made to create our own, context specific PEAR plasmid prevented proper expression of eGFP in transfected, unedited cells as planned and notably with no apparent noise. The last and most important insight gained was, that editing of the reporter plasmid using respective pegRNAs successfully restores eGFP expression, proving that our reporter works as intended.
</p>
<p>
<b>This achievement formed a convenient basis for the following optimization of prime editing in the CFTR F508del locus for us as well as other research groups.</b>
</p>
</p>
</div>
<div className="box" >
<p id="rep4">
<H3 text="Application in epithelial Cells" id="rep4head"/>
<H4 text="Design" id="design-head"/>
<p>
Although we could show that our PEAR reporter plasmid works in a HEK cell model, according to <a onClick={() => goToPagesAndOpenTab('ignatova', '/human-practices')}> Prof.Dr. Zoya Ignatova </a> insights gained here might still not entirely transfer to cells actively expressing CFTR. As recommended, we applied our reporter to a system closer to a therapeutic target <a onClick={() => goToPageAndScroll ('Cell Culture2H', '/materials-methods')}>CFBE41o-</a>. The cells are derived from bronchial epithelial cells of a Cystic Fibrosis patient and are homozygous for CFTR F508del.
</p>
<H4 text="Build" id="build-head"/>
<p>
For experimenting in CFBE41o- cells, the same reporter construct was used as for the HEK293 test. However, we used a different prime editor (pCMV-PE6c, see prime editing systems engineering cycle<a onClick={() => goToPagesAndOpenTab('pe-systems', '/engineering')}> prime editing systems circle </a>), and only pegRNAs were used, that proved the most efficient in preceding experiments (see <a onClick={() => goToPagesAndOpenTab('pegrna', '/engineering')}> pegRNA engineering cycle </a>).
</p>
<H4 text="Test" id="test-head"/>
<p>
Similar to the previous cycle, we evaluated the functionality of our reporter system by co-transfecting our reporter construct with a pCMV-PE6c prime editor plasmid as well as a plasmid expressing pegRNA that targeted our reporter this time into CFBE41o- cells. After 72 h we saw a significant number of cells showing fluorescence.
</p>
<p>
Like with the experiments in HEK cells, we transfected a technical control plasmid as well the unmodified pDAS12124_PEAR-GFP-preedited plasmid as positive controls and our modified plasmid, pCMV-PE6c and the pegRNA plasmid individually as negative controls. Again, the positive controls showed solid fluorescence, while the negative control did not.
</p>
<figure>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/rep-it4.svg" alt="Illustration of applying the pPEAR_CFTR system to lung epithelial cell lines"/>
<figcaption><b>Figure 5:</b> Illustration of the pegRNA testing using the pPEAR_CFTR system. CFBE41o- cells transiently transformed with the pPEAR_CFTR plasmid (in the middle) show fluorescence after transformation with a prime editor and a reporter-specific pegRNA expression plasmid. The pDAS12124_PEAR-GFP-preedited is used as an internal positive control and for normalization.</figcaption>
</figure>
<H4 text="Learn" id="learn-head"/>
<p>
This experiment confirms that our reporter can not only be used in cell lines distantly related to patient cells of interest, in our case HEK203 cells, but also works in cells actively expressing CFTR and carrying the mutation. The reporter still showed no noise.
</p>
</p>
</div>
<div className="box" >
<p id="rep5">
<H3 text="Application in Primary Cells" id="rep5head"/>
<H4 text="Design" id="design-head"/>
<p>
The model closest to application in actual patient cells are human derived primary cells. For our last test of our modified PEAR reporter, we thus chose to use <a onClick={() => goToPageAndScroll ('Cell Culture3H', '/materials-methods')}>human nasal epithelial cells</a> derived from members of our team.
</p>
<H4 text="Build" id="build-head"/>
<p>
For testing our reporter in the human nasal epithelial cells, the same constructs have been used as in the previous iteration with CFBE41o- cells.
</p>
<H4 text="Test" id="test-head"/>
<p>
The experimental setup for this experiment was a scaled down version of the previous cycle with the only altered variable being the cells transfected. In this case, we did not observe any fluorescence, neither in the tested cells, nor the technical or pDAS12124_PEAR-GFP-preedited positive controls.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
In this last experiment, the negative technical positive control implies a failed transfection of the cells. Thus, this attempt did not allow to draw any conclusion regarding the function of our reporter in primary cells. The experiment is to be repeated in the future.
</p>
</p>
</div>
<div className="box" >
<p id="rep6">
<H3 text="Outlook" id="rep6head"/>
<p>
Our CFTR contextualized PEAR reporter proved to consistently allow detection of prime editing without notable noise, laying the foundation for optimization of existing and testing of new prime editing systems. Although very versatile in the context of targeting CFTR F508del with the <a onClick={() => goToPagesAndOpenTab('pegRNA-genau-collapsible', '/description')}>spacer of our choice</a>, a wider applicability to other genomic targets and other possible prime editor variants working differently than Cas9-based systems would be favorable. In the original PEAR plasmid however, modification of variable region is quite impractical. Also, as a part the eGFP is RCF[1000] but not RCF[10] BioBrick standard conform and hardly compatible with other parts like our <a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'pe3', path: '/engineering', tabId: 'pe-systems'})}>PreCyse cassette</a>.
</p>
<H4 text="Design" id="design-head"/>
<p>
This is why, as an outlook and contribution for future iGEM teams, we aim to create a more modular and compatible part similar to our PreCyse Casette. For this we made use of the experience gained when cloning pegRNAs. An oligonucleotide-based golden gate cloning site in the region of interest surrounding the 5’ splice donor site allows for cheap and convenient modification of the sequence. The area between the TypeIIS restriction sites is designed as a dropout cassette coding for a fluorescence marker expressed in E. coli, that enables rapid screening for transformants containing correctly digested plasmid backbones.
</p>
{/* <H4 text="Build" id="build-head"/>
<p>
</p>
<H4 text="Test" id="test-head"/>
<p>
</p>
<H4 text="Learn" id="learn-head"/>
<p>
</p> */}
</p>
</div>
<Section title="References" id="references">
<EngRepsources/>
</Section>
<br/>
<div className="row ">
<div className="col">
<div className="left"><ButtonOneEngineering label="Previous" open="our-cycle" scrollToId="our-cycle-header"/></div>
</div>
<div className="col button-left">
<div className="right"><ButtonOneEngineering label="Next" open="transfection" scrollToId="transfection-header"/></div>
</div>
</div>
</section>
</div>
<div id="tab-transfection" className="enginneeringtab" style={{display: "none"}}>
<section >
<div className="eng-box box" >
<H3 id="Proof of Concept" text="Proof of Concept"></H3>
<p>To test prime editors, a reliable model system is required. HEK293 cells are a human derived cell line and widely used in a variety of fields in biology[1]. Apart from easy handling and comparatively easy transfection, they have, as we found out in our exchange with Mattijs Bulcaen, one advantage over other models: They are naturally impaired in DNA repair mechanisms and therefore easier to edit. To properly compare editing efficiencies, a high transfection efficiency is of utmost importance. This engineering cycle focuses on our work in simulating prime editing using the PEAR reporter system[2] and optimizing transfection protocols.</p>
<H2 id="transfection-header" text="Optimization of Transfection"></H2>
<p>
To test prime editors, a reliable model system is required. HEK293 cells are a human derived cell line and widely used in a variety of fields in biology<TabScrollLink tab="transfection" num="3" scrollId="desc-3"/>. Apart from easy handling and comparatively easy transfection, they have, as we found out in our exchange with <a onClick={() => goToPagesAndOpenTab('mattijsinv', '/human-practices')}>Mattijs Bulcaen</a>, one advantage over other models: They are naturally impaired in DNA repair mechanisms and therefore easier to edit. To properly compare editing efficiencies, a high transfection efficiency is of utmost importance. This engineering cycle focuses on our work in simulating prime editing using the PEAR reporter system<TabScrollLink tab="transfection" num="4" scrollId="desc-4"/> and optimizing transfection protocols.
</p>
</div>
<div className="box" >
<p id="cyc1">
<H3 text="Test of Lipofectamine 2000" id="text"/>
<H4 text="Test" id="text"/>
<p>While conducting research on transfection methods for HEK cells, particular attention was devoted to the delivery of the Prime Editing complex into the cells. In the literature, Lipofectamine is described as a common transfection agent. <i>Anzalone et al. 2019</i>[3] describe a transfection of prime-editing complexes with Lipofectamine 2000. The aim of this study is to introduce our prime-editing complex into HEK cells.</p>
<p>Transfection with Lipofectamine 2000 was performed in accordance with the Anzalone protocol. However, the result was characterized by insufficient transfection efficiency.</p>
<H4 text="Learn" id="text"/>
<p>The low efficiency of Lipofectamine 2000 indicated that the product is not optimally suited to the specific conditions under consideration. In contrast, Lipofectamine 3000 is described in the literature as potentially more efficient.</p>
<p id="trf1">
<H3 text="Test of Lipofectamine 2000" id="trf1head"/>
<H4 text="Design" id="text"/>
<p>In light of the aforementioned findings, the decision was taken to test Lipofectamine 3000, given its reputation for greater efficiency. A new test design was devised, utilizing Lipofectamine 3000 with an equivalent quantity of DNA and modified transfection conditions.</p>
<p>
Before testing any of our mechanistic approaches, we had to examine whether we can facilitate and detect prime editing in the first place. During our research we eventually stumbled upon the PEAR reporter system (see <a onClick={() => goToPagesAndOpenTab('pegrna', '/engineering')}> pegRNA engineering cycle </a>). The PEAR 2in1 plasmid reporter includes a GFP that is to be edited for sensitive prime editing detection, and a pegRNA expression cassette with a pegRNA targeting the plasmid itself. Having found a system capable of detecting even small-scale prime editing, the next step was to find transfection conditions that would work. In the literature, Lipofectamine is described as a common transfection agent.
</p>
<p>
Transfection with Lipofectamine 2000 was performed in accordance with the Anzalone protocol. However, the result was characterized by insufficient transfection efficiency.
</p>
<H4 text="Build" id="text"/>
<p>In accordance with the established protocol, the recommended ratio of 1 µg DNA to 2 µl Lipofectamine 3000, as provided by ThermoFisher, is to be employed.</p>
<p>
Anzalone et al. 2019<TabScrollLink tab="transfection" num="5" scrollId="desc-5"/> describe a transfection of prime-editing complexes with Lipofectamine 2000.
</p>
<H4 text="Test" id="text"/>
<p>
Transfection with Lipofectamine 2000 was performed in accordance with the Anzalone protocol. However, the result was characterized by insufficient transfection efficiency.
</p>
<H4 text="Learn" id="text"/>
<p>
The low efficiency of Lipofectamine 2000 indicates that the product is not optimally suited to the specific conditions under consideration. In contrast, Lipofectamine 3000 is described in the literature as potentially more efficient.
</p>
</p>
</div>
<div className="box" >
<p id="cyc2">
<H3 text="Initial Test with Lipofectamine 3000" id="text"/>
<H4 text="Test" id="text"/>
<p>Considering the favorable assessment of Lipofectamine 3000 in the scientific literature, the proof of concept was conducted once more.</p>
<p>The objective of the experiment was to enhance the transfection efficiency of Lipofectamine 3000. The transfection protocol was conducted in accordance with the manufacturer's instructions (1 µg DNA, 2 µl Lipofectamine 3000 reagent).</p>
<p>The outcome revealed that despite the modification, the transfection efficiency remained inadequate, although a marginal improvement was discernible.</p>
<H4 text="Learn" id="text"/>
<p>Although a switch to Lipofectamine 3000 resulted in a marginal improvement, the efficiency fell short of expectations. This indicates that further optimization is required in terms of the amount of Lipofectamine and DNA, as well as the medium used.</p>
<p id="trf2">
<H3 text="Initial Test with Lipofectamine 3000" id="trf2head"/>
<H4 text="Design" id="text"/>
<p>In order to optimize the transfection process, a new optimization test was designed, which incorporates a variable design with regard to the quantity of Lipofectamine 3000 and DNA.</p>
<p>
In light of the aforementioned findings, the decision was taken to test Lipofectamine 3000, given its reputation for greater efficiency. A new test design was devised, utilizing Lipofectamine 3000 with an equivalent quantity of DNA and modified transfection conditions.
</p>
<H4 text="Build" id="text"/>
<p>The protocol entails the utilization of varying concentrations of Lipofectamine 3000, specifically 1 µl and 1.5 µl, with a DNA quantity of 1 µg or 0.5 µg.</p>
<p>
In accordance with the established protocol, the recommended ratio of 1 µg DNA to 2 µl Lipofectamine 3000, as provided by ThermoFisher, was to be employed.
</p>
<H4 text="Test" id="text"/>
<p>
The objective of the experiment was to enhance the transfection efficiency of Lipofectamine 3000. The transfection protocol was conducted in accordance with the manufacturer's instructions (1 µg DNA, 2 µl Lipofectamine 3000 reagent).
</p>
<p>
The outcome revealed that despite the modification, the transfection efficiency remained inadequate, although a marginal improvement was discernible.
</p>
<H4 text="Learn" id="text"/>
<p>
Although a switch to Lipofectamine 3000 resulted in a marginal improvement, the efficiency fell short of expectations. This indicates that further optimization is required in terms of the amount of Lipofectamine and DNA, as well as the medium used.
</p>
</p>
</div>
<div className="box" >
<p id="cyc3">
<H3 text="Optimization of DNA and Lipofectamine Volumes" id="text"/>
<H4 text="Test" id="text"/>
<p>To enhance transfection efficiency, optimization tests were conducted, in which the quantities of Lipofectamine and DNA were varied. The objective of this iteration was to find the optimal ratio of Lipofectamine 3000 to DNA. To this end, 1 µl and 1.5 µl of Lipofectamine 3000 at a DNA concentration of either 1 µg or 0.5 µg were compared with each other.</p>
<H4 text="Learn" id="text"/>
<p>The experiment demonstrated that a quantity of 1 µl Lipofectamine 3000 is sufficient for successful transfection, and that increasing the quantity does not result in a notable difference. Additionally, the findings indicated that an amount of 1 µg DNA exhibited a higher efficiency than an amount of 0.5 µg DNA. It can be reasoned that additional factors may have contributed to the previously observed decline in transfection efficiency. One potential explanation is that the cells may have been in an excessively high passage level.</p>
<p>It can be reasonably deduced that the aforementioned factors may have contributed to the observed decline in transfection efficiency.</p>
<p id="trf3">
<H3 text="Optimization of DNA and Lipofectamine Volumes" id="trf3head"/>
<H4 text="Design" id="text"/>
<p>The results obtained were used to develop an optimized protocol that takes into account both the concentration of Lipofectamine and the amount of DNA.</p>
<p>
In order to optimize the transfection process, a new optimization test was designed, which incorporated a variable design with regard to the quantity of Lipofectamine 3000 and DNA.
</p>
<H4 text="Build" id="text"/>
<p>In the following experiments, a DNA quantity of 1 µg and a defined quantity of 1 µl Lipofectamine 3000 was used.</p>
<p>
The protocol entailed the utilization of varying concentrations of Lipofectamine 3000, specifically 1 µl and 1.5 µl, with a DNA quantity of 1 µg or 0.5 µg. In this phase, we developed the transfection method with calcium chloride (CaCl<sub>2</sub>) as an alternative to conventional lipofectamine transfection. The aim was to test whether this more cost-effective method offers comparable transfection efficiency. Three different DNA concentrations were used to investigate the effect on transfection efficiency.
</p>
<H4 text="Test" id="text"/>
<p>
To enhance transfection efficiency, optimization tests were conducted, in which the quantities of Lipofectamine and DNA were varied. The objective of this iteration was to find the optimal ratio of Lipofectamine 3000 to DNA. To this end, 1 µl and 1.5 µl of Lipofectamine 3000 at a DNA concentration of either 1 µg or 0.5 µg were compared with each other. In the next step, the tests were carried out with the different DNA concentrations using the CaCl<sub>2</sub> transfection method. The transfection efficiencies were compared with those from the Lipofectamine transfection to determine whether the new method represents an improvement.
</p>
<H4 text="Learn" id="text"/>
<p>
The experiment demonstrated that a quantity of 1 µl Lipofectamine 3000 was sufficient for successful transfection, and that increasing the quantity does not result in a notable difference. Additionally, the findings indicated that an amount of 1 µg DNA exhibited a higher efficiency than an amount of 0.5 µg DNA. It can be reasoned that additional factors may have contributed to the previously observed decline in transfection efficiency. One potential explanation is that the cells may have been in an excessively high passage level. It became clear from the tests that CaCl<sub>2</sub> transfection did not deliver better results than Lipofectamine transfection. On the contrary, the efficiency was significantly lower, although the method is less expensive. This led to the realisation that the CaCl<sub>2</sub> technique in this form was not a suitable alternative for our specific requirements.
</p>
<p>
It can be reasonably deduced that the aforementioned factors may have contributed to the observed decline in transfection efficiency.
</p>
</p>
</div>
<div className="box" >
<p id="cyc4">
<H3 text="Validation of optimized Protocol" id="text"/>
<p id="trf4">
<H3 text="Validation of optimized Protocol" id="trf4head"/>
<H4 text="Design" id="text"/>
<p>
The results obtained were used to develop an optimized protocol that takes into account both the concentration of Lipofectamine and the amount of DNA.
</p>
<H4 text="Build" id="text"/>
<p>
In subsequent research, a DNA quantity of 1 µg and a defined quantity of 1 µl of Lipofectamine 3000 will be utilized.
</p>
<H4 text="Test" id="text"/>
<p>Following the series of optimizations, the proof of concept was conducted once more to confirm the efficacy of the optimized protocol. This protocol involved the utilization of 1 µl Lipofectamine 3000, 1 µg DNA, 2 µl Reagent 3000 and Opti-MEM as a medium. The outcomes were encouraging, as the transfection efficiency was markedly enhanced.</p>
<p>
Following a series of optimizations, the proof of concept was conducted once more to confirm the efficacy of the optimized protocol. The objective was to perform the transfection with the final, optimized protocol. This protocol involved the utilization of 1 µl Lipofectamine 3000, 1 µg DNA, 2 µl Reagent 3000 and Opti-MEM as a medium. The outcomes were encouraging, as the transfection efficiency was markedly enhanced.
</p>
<H4 text="Learn" id="text"/>
<p>The utilization of an optimized quantity of 1 µl Lipofectamine 3000, a defined quantity of DNA and the suitable Opti-MEM medium resulted in a notable enhancement in transfection efficiency. This substantiates the assertion that the aforementioned conditions constitute an optimal foundation for the transfection of HEK cells with the prime editing complex.</p>
<H4 text="Design" id="text"/>
<p>This protocol establishes the standard procedure for the transfection of HEK cells with the Prime Editing Complex. The transfection reagent Lipofectamine 3000 is diluted in Opti-MEM to a final volume of 1 µl, and the DNA to be transfected is diluted to a final concentration of 1 µl.</p>
<p>
The utilization of an optimized quantity of 1 µl Lipofectamine 3000, a defined quantity of DNA and the suitable Opti-MEM medium resulted in a notable enhancement in transfection efficiency. This substantiates the assertion that the aforementioned conditions constitute an optimal foundation for the transfection of HEK cells with the prime editing complex.
</p>
</p>
</div>
<Section title="References" id="references">
<EngTrfsources/>
</Section>
<br/>
<div className="row ">
<div className="col">
<div className="left"><ButtonOneEngineering label="Previous" open="our-cycle" scrollToId="ourcycle"/></div>
<div className="left"><ButtonOneEngineering label="Previous" open="reporter" scrollToId="reporter-header"/></div>
</div>
<div className="col button-left">
<div className="right"><ButtonOneEngineering label="Next" open="pe-systems" scrollToId="PE Systems"/></div>
<div className="right"><ButtonOneEngineering label="Next" open="pe-systems" scrollToId="pe-systems-header"/></div>
</div>
</div>
</section>
</div>
<div className="enginneeringtab" id="tab-pe-systems" style={{display: "none"}}>
<div id="tab-pe-systems" className="enginneeringtab" style={{display: "none"}}>
<section id="PE Systems sec" >
<div className="eng-box box" >
<H3 id="PE Systems" text="PE Systems"></H3>
<p><LoremShort></LoremShort></p>
<H2 id="pe-systems-header" text="Prime Editing Systems"></H2>
<p>Different versions of the original prime editing system have been developed since its initial introduction.
Deciding on what system to use for the application in therapeutic human gene editing, especially concerning the correction of
F508del, was the goal of this engineering cycle.</p>
<p>
Since we aim to develop a therapy delivered to the human body, we wanted to obtain high editing efficiency while risking as
little off-targets as possible and also reducing the size for improved packability.
</p>
<InfoBox title="Existing Prime Editing Systems" id="current-pe-systems">
<details>
<summary>
</summary>
<div className='row align-items-center'>
<div className='col'>
<p>
<b>PE1</b><TabScrollLink tab="tab-pe-systems" num="6" scrollId="desc-6"/>{/* ehem 1 */}, the first version of the Prime Editor features a Cas9(H840A), a Streptococcus pyogenes Cas9 (SpCas9, hereafter just referred to as Cas9) mutant that only cuts the non-target strand of the DNA template<TabScrollLink tab="tab-pe-systems" num="7" scrollId="desc-7"/>{/* ehem 2 */}, and a wildtype reverse transcriptase from the Moloney Murine Leukaemia Virus (M-MLV RT) connected by a serine and glycine rich flexible linker.
</p>
<p>
<b>PE2</b><TabScrollLink tab="tab-pe-systems" num="6" scrollId="desc-6"/>{/* ehem 1 */} improves on this concept by incorporating an improved RT with five mutations improving affinity to the template RNA, enzyme processivity and thermostability (D200N/L603W/T330P/T306K/W313F). This version of the prime editor showed varying improvement of editing efficiency over all tested loci and edits with no apparent downsides. Building on the PE2 system, a smaller version of the M-MLV RT was introduced by Gao et al. (2022)<TabScrollLink tab="tab-pe-systems" num="8" scrollId="desc-8"/>{/* ehem3 */}. The RT was truncated by 621 bp through deletion of the RNaseH domain, which originally degrades the RNA template, but is not needed for prime editing. The codon optimized version of this truncated RT prime editor (in literature usually called PE2∆RNaseH) was named <b>PE<sup>CO</sup>-Mini</b> in the paper and will be addressed as such here.
</p>
</div>
<div className='col-4'>
<figure>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/pe2-open.svg" alt="PE2 Prime Editor"/>
<figcaption><b>Figure 1: Illustration of PE2 Prime Editor</b> </figcaption>
</figure>
</div>
</div>
<div className='row align-items-center'>
<div className='col'>
<p>
The <b>PE3</b><TabScrollLink tab="tab-pe-systems" num="6" scrollId="desc-6"/>{/* ehem 1 */} system, described in the same paper as PE1 and PE2, introduces the use of a second single guide RNA besides the pegRNA which leads to a nick in the strand opposite to the edited strand. This is supposed to improve integration of edits by directing cellular DNA repair systems to use the edited strand as a template for resolving base mismatches. Nicks positioned 3‘ of the edit about 40–90 base pairs from the pegRNA-induced nick were able to further increase editing efficiencies about threefold when compared to PE2, but with a higher range of on-target indels , meaning random Insertions and/or Deletions that appear after faulty repair of double strand breaks in the DNA. PE3b, where the protospacer for the nicking sgRNA lies within the edited regions, decreased the indel rate greatly compared to PE3.
</p>
</div>
<div className='col-4'>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/pe3-open.svg" alt="PE3 Prime Editor"/>
</div>
</div>
<div className='row align-items-center'>
<div className='col'>
<p>
<b>PE4</b> and <b>PE5</b><TabScrollLink tab="tab-pe-systems" num="8" scrollId="desc-8"/> {/* ehem 4 */}expand the PE2 and PE3 systems, respectively, by co-expressing a dominant negative MLH1 protein (MLH1(Δ754–756), hereafter referred to as MLH1dn). The MLH1 protein plays a crucial role in the mismatch repair (MMR) mechanism of the human cell<TabScrollLink tab="tab-pe-systems" num="9" scrollId="desc-9"/>{/* ehem 5 */} by recruiting other repair proteins and facilitating catalytic function. The mutant still recruits other factors but is impaired in its endonuclease function, disrupting function of the entire repair mechanism. This leads to an average 7.7-fold and 2.0-fold increase in editing efficiency, respectively, compared to PE2 and PE3. This is possibly due to slower repair of mismatches and thus more time for the proteins encoded by LIG1 and FEN1 genes to excise the non-edited 5’ flap and ligate the nick in the edited strand. Additionally, MLH1dn co-expression slightly reduced on-target indels as well as unintended editing outcomes in PE3 systems and did not lead to higher off-target indel rates or overall mutation rates.
</p>
<p>
With <b>PEmax</b><TabScrollLink tab="tab-pe-systems" num="8" scrollId="desc-8"/>{/* ehem 4 */}, the structure of PE2 is further enhanced by using human codon-optimized RT, a new linker containing a bipartite SV40 nuclear localization sequence (NLS)<TabScrollLink tab="tab-pe-systems" num="11" scrollId="desc-11"/>, an additional C-terminal c-Myc NLS<TabScrollLink tab="tab-pe-systems" num="12" scrollId="desc-12"/> and R221K N394K mutations in SpCas9 previously shown to improve Cas9 nuclease activity<TabScrollLink tab="tab-pe-systems" num="13" scrollId="desc-13"/>. These changes led to moderate improvements in editing efficiency compared to previous editor architectures.
</p>
</div>
<div className='col-4'>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/pe4-open-new.svg" alt="PE4 Prime Editor"/>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/pe5-open-new.svg" alt="PE5 Prime Editor"/>
</div>
</div>
<div className='row align-items-center'>
<div className='col'>
<p>
<b>PE6</b><TabScrollLink tab="tab-pe-systems" num="14" scrollId="desc-14"/> was made by improving the reverse transcriptase domain of the prime editor using Phage-Assisted Continuous Evolution (PACE). Multiple RT mutants (PE6a-d), derived from RTs of Escherichia coli Ec48 retron, Schizosaccharomyces pombe Tf1 retrotransposon and Moloney Murine Leukaemia Virus, were identified to increase editing efficiency over and/or were smaller than the M-MLV RT used in previous PE systems. Especially <b>PE6c</b> (evolved Tf1 RT) and <b>PE6d</b> (evolved M-MLV RT) showed significantly higher editing efficiencies than even PEmax depending on the targeted loci, with PE6d showing benefits especially in loci forming more complex secondary structures. Recent advancements in prime editing targeting the CFTR F508Δ mutation showed that PE6c was the most promising for editing in this loci<TabScrollLink tab="tab-pe-systems" num="15" scrollId="desc-15"/>. Improvements of nCas9 on the other hand (PE6e-g) were only marginal and highly site specific. All PE6 systems use nicking gRNAs (PE3) by default, but do not co-express MLH1dn.
</p>
</div>
<div className='col-4'>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/pe6c-open.svg" alt="PE6c Prime Editor"/>
</div>
</div>
<div className='row align-items-center'>
<div className='col'>
<p>
<b>PE7</b><TabScrollLink tab="tab-pe-systems" num="16" scrollId="desc-16"/> adds an additional RNA binding domain to the Prime Editor. The domain is derived from the La Protein (La(1-194)), an endogenous eukaryotic protein involved RNA metabolism and known for its role in binding polyuridine (polyU) tails at the 3’ ends of nascent transcripts, thus protecting them from exonuclease activity. PE7 showed considerable improvements over PEmax at different loci and different types of edits when used with the PE2 strategy (no nicking gRNAs, no MLH1dn co-expression). Notably, PE7 did perform worse when used with engineered pegRNAs than with regular ones (see pegRNA design).
</p>
</div>
<div className='col-4'>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/pe7-open.svg" alt="PE7 Prime Editor"/>
</div>
</div>
</details>
</InfoBox>
</div>
<div className="box" >
<p id="pe1">
<H3 text="" id="text"/>
<H4 text="Test" id="text"/>
<p></p>
<H4 text="Learn" id="text"/>
<p></p>
<PhilipH3 id="pe1head"><span>PE2 and PE<sup>CO</sup>-Mini</span></PhilipH3>
<H4 text="Design" id="text"/>
<p></p>
<p>
For our initial approach, we wanted to start from the beginning and use the PE2 prime editing system. Since our goal of stripping the size of the prime editor was a big factor from the beginning, we did a researched into that direction and found a truncated version of M-MLV RT, PE<sup>CO</sup>-Mini. We then ordered the plasmids for both PE2 and PE<sup>CO</sup>-Mini. Since the PE<sup>CO</sup>-Mini plasmid had a different promotor than pCMV-PE2, we decided to clone the PE<sup>CO</sup>-Mini RT into the pCMV-PE2 vector to allow for direct comparison.
</p>
<H4 text="Build" id="text"/>
<p></p>
<p>
We designed primers for the amplification of PE<sup>CO</sup>-Mini RT and cloned it into pCMV-PE2 via double digestion and Gibson assembly.
</p>
<H4 text="Test" id="text"/>
<p>
To compare the prime editing performances of M-MLV RT (PE2) and PE<sup>CO</sup>-Mini RT, both were tested using a 2in1 prime editing reporter plasmid system<TabScrollLink tab="tab-pe-systems" num="17" scrollId="desc-17"/> (see <a onClick={() => goToPageWithTabAndScroll({scrollToId: 'Proof of Concept', path: '/engineering', tabId: 'tab-transfection' })}>Proof of Concept</a>) in HEK293 cells. Contrary to the findings of Gao et al., here the PE<sup>CO</sup>-Mini prime editor performed a lot worse than the PE2 prime editor.
</p>
<H4 text="Learn" id="text"/>
<p>
Since we knew, that for a successful therapy targeting the F508del mutation a very high prime editing efficiency was of utmost importance, we decided against using PE<sup>CO</sup>-Mini as the basis for our approach and that we have to look for other alternatives.
</p>
</p>
</div>
<div className="box" >
<p id="pe2">
<H3 text="" id="text"/>
<H4 text="Test" id="text"/>
<p></p>
<H4 text="Learn" id="text"/>
<p></p>
<H3 text="PE6c" id="pe2head"/>
<H4 text="Design" id="text"/>
<p></p>
<p>
During our initial talk with <a onClick={() => goToPagesAndOpenTab('mattijsinv', '/human-practices')}>Mattijs Bulcaen</a>, he recommended a talk of <a onClick={() => goToPagesAndOpenTab('liu', '/human-practices')}>David Liu</a> at an online conference, where he presented unpublished data about his laboratory working on prime editing for F508del correction. We investigated it and through this came across the PE6 generation of prime editors. Seeing that the Liu Laboratory eventually decided on using the PE6c system, we adopted the findings.
</p>
<H4 text="Build" id="text"/>
<p></p>
<p>
We got the plasmid carrying the PE6c prime editor. Except for the RT and a few improving mutations in the Cas9 enzyme, it has the same architecture as PE2, which made comparison quite easy.
</p>
<H4 text="Test" id="text"/>
<p>
We tested PE6c against PE2 using the same reporter system as mentioned above for PE<sup>CO</sup>-Mini. PE6c, as expected from the literature, proved way more efficient in prime editing.
</p>
<H4 text="Learn" id="text"/>
<p>
The data from literature as well as our own experiments confirmed that PE6c architecture is superior to PE2 even without using nicking gRNAs that help suppress mismatch repair. This led us to the decision to use the PE6c reverse transcriptase and parts of the overall architecture for our subsequent tests.
</p>
</p>
</div>
<div className="box" >
<p id="pe3">
<H3 text="" id="text"/>
<H4 text="Test" id="text"/>
<p></p>
<H4 text="Learn" id="text"/>
<p></p>
<H3 text="PreCyse Casette" id="pe3head"/>
<H4 text="Design" id="text"/>
<p></p>
<p>
In the later stages of our project, the Liu laboratory published their own findings regarding CFTR F508del targeting with prime editing<TabScrollLink tab="tab-pe-systems" num="15" scrollId="desc-15"/>. The data showed that the editing efficiency of PE2 based systems, even when using PE6c reverse transcriptase, might not be sufficient for application in a therapy. Also, the plasmids of current prime editors did not include restriction sites that would have allowed replacing components like the nickase to test alternatives. This is why, in a cherry-picking manner, we combined the PE6c architecture prime editor with the most promising aspects of other prime editors, creating the PreCyse cassette.
</p>
<p>
Our decision on what components of existing prime editors we wanted to use was mainly driven by two factors: efficiency and precision. In prime editing, these two are often opposing forces, which means advancements improving efficiency often also increase the risk of off-targets mutations and on-target undesired editing. For this reason, we decided against using nicking gRNAs. Although they have been proven to reliably improve editing efficiency, they increase the risk and possible scope of off-target cleavage and mutations. Additionally, if <b>PE3b</b> is not applicable, there is a chance for double strand breaks to occur, which diminishes the safety advantage of prime editing over other common CRISPR-based methods. Co-expression of MLH1dn can improve editing efficiency in the same way as nicking gRNAs do, by helping to evade of the cellular mismatch repair mechanisms. The use of MLH1dn is especially impactful, when nicking gRNAs are not used, which is perfect in our case. Recently, the La poly(U)-binding motif has been shown to enhance prime editing efficiency, presumably through protection of the 3’ poly(U) tail of the pegRNA from RNases. The motif is also comparatively small, which aligns with the overall goal to create a compact prime editing tool. This is why PreCyse Casettes have been designed to include the La RNA binding motif fusion and the dominant negative MLH1 protein.
</p>
<H4 text="Build" id="text"/>
<p></p>
<p>
The PreCyse cassette comes in three versions: PreCyseA, the most basic version, comprises of a T7 promoter and an open reading frame, which includes NLS and one typeIIS restriction enzyme cloning site for a nickase and a reverse transcriptase each. For possible future additions like e. g. selection markers, a BamHI restriction site at the end of the coding sequence allows for easy in-frame Gibson cloning. Building on this basis, PreCyseB expands PreCyseA by the La Poly(U)-binding motif. PreCyseC additionally introduces the co-expressed MLH1dn. The cassettes were ordered in three individual parts to be put together with a pCMV-PE6c backbone via Gibson Cloning in different configurations to create the three variants. In the plasmid the cassette is expressed under a CMV promoter and followed by a polyadenylation signal. The PreCyse Casettes themselves can be used as a BioBrick RFC<TabScrollLink tab="tab-pe-systems" num="16" scrollId="desc-16"/> standard compatible composite part can thus be freely combined with other parts. The nickase and RT slots can be used for inserting any basic or composite part compatible with the Type IIS RCF[1000] standard for fusion proteins. The PreCyse Casette is meant to be a contribution to the iGEM community and a base for other teams to join us and researchers around the world to innovate in the exciting field of prime editing.
</p>
<div className="casettecontainer">
<div className="casettebox">
<H5 text="PreCyseA" id="PCA"/>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/precysea-casette.svg" alt="image 1" />
</div>
<div className="casettebox">
<H5 text="PreCyseB" id="PCB"/>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/precyseb-casette.svg" alt="image 2" />
</div>
<div className="casettebox">
<H5 text="PreCyseC" id="PCC"/>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/precysec-casette.svg" alt="image 3" />
</div>
</div>
{/* <div className="row align-items-center">
< div className='col align-items-center'>
<H5 text="PreCyseA" id="PreCyseA"/>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/precysea-casette.svg" alt="PreCyseA modular PE casette" style={{height: "80pt", width: "auto"}}/>
</div>
<div className='col align-items-center'>
<H5 text="PreCyseB" id="PreCyseB"/>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/precyseb-casette.svg" alt="PreCyseB modular PE casetter" style={{height: "80pt", width: "auto"}}/>
</div>
</div>
<div className='row align-items-center'>
<H5 text="PreCyseC" id="PreCyseC"/>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/precysec-casette.svg" alt="PreCyseC modular PE casette" style={{height: "80pt", width: "auto"}}/>
</div> */}
</p>
</div>
<Section title="References" id="references">
<EngPEsystems/>
</Section>
<br/>
<div className="row ">
<div className="col">
<div className="left"><ButtonOneEngineering label="Previous" open="proof-of-concept" scrollToId="Proof of Concept"/></div>
<div className="left"><ButtonOneEngineering label="Previous" open="transfection" scrollToId="transfection-header"/></div>
</div>
<div className="col button-left">
<div className="right"><ButtonOneEngineering label="Next" open="nikase" scrollToId="Nikase"/></div>
<div className="right"><ButtonOneEngineering label="Next" open="pegrna" scrollToId="pegrna-header"/></div>
</div>
</div>
</section>
</div>
<div className="enginneeringtab" id="tab-nikase" style={{display: "none"}}>
<section id="Nikase sec" >
<div className="enginneeringtab" id="tab-pegrna" style={{display: "none"}}>
<section id="pegRNA sec" >
<div className="eng-box box" >
<H3 id="Nikase" text="Nikase"></H3>
<p><LoremShort></LoremShort></p>
<img src="https://static.igem.wiki/teams/5247/fanzor/movie4-ezgif-com-video-to-gif-converter.gif"></img>
<img src="https://static.igem.wiki/teams/5247/fanzor/movie5-ezgif-com-video-to-gif-converter-2.gif"></img>
</div>
<div className="box" >
<p id="nik1">
<h3>nik1</h3>
<LoremShort></LoremShort>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
</p>
<H2 id="pegrna-header" text="pegRNA"></H2>
<p>The <a onClick={() => goToPagesAndOpenTab('pegrna', '/engineering')}> pegRNA </a> is of paramount importance for function and efficiency of prime editors, as it plays a role in every step of the prime editing mechanism. It is therefore equally important to optimize the pegRNA than it is to have an optimized prime editor. Hence this engineering cycle explains our process of optimizing the pegRNAs for our genomic target, CFTR F508del. Given that different areas of the pegRNA have different functionalities, the following iteration cycles will demonstrate how improvements and optimizations have been made to these various functional domains in relation to the CFTR context. This was achieved through research, the correspondence with of experts and experiments.</p>
<div className="casettecontainer">
<div className="pegrnabox" style={{height: 'auto', width: '50%'}}>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/pegrna-overview.svg" alt="Illustration of the key components in our pegRNAs, including spacer, gRNA scaffold, primer binding sequence, reverse transcriptase template, tevopreQ1 3' stem loop motif and templates for main and silent edits."/>
<figcaption><b>Figure 1:</b> Illustration of the key components in our pegRNAs, including spacer, gRNA scaffold, primer binding sequence
(PBS), reverse transcriptase template (RTT), tevopreQ1 3' stem loop motif and templates for main and silent edits. </figcaption>
</div>
</div>
</div>
<div className="box" >
<p id="nik2">
<h3>nik2</h3>
<LoremShort></LoremShort>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
<p id="peg1">
<H3 text="Initial pegRNA Design and Silent Edits" id="peg1head"/>
<p>
The first iteration of our engineering cycle, we designed our first set of pegRNAs targeting the
<a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'reporter-header', path: '/engineering',
tabId: 'reporter' })}>modified pPEAR_CFTR reporter</a>. We also focused on the incorporation of
silent edits.
</p>
<H4 text="Design" id="design-head"/>
<p>
Following an interview with <a onClick={() => goToPagesAndOpenTab('JPpegRNA', '/human-practices')}>
Jan-Phillipp Gerhard</a>, we came across the concept of silent edits. Silent edits refer to single-base
alterations of the nucleotide sequence that do not change the encoded amino acid. Jan-Phillipp pointed out
that introducing silent edits in addition to the intended edit offers two major advantages.
</p>
<p>
Firstly, silent edits can increase the likelihood of flap incorporation during the prime editing process,
especially in the context of MMR (Mismatch Repair) in the cell. Without silent edits, the cell is more likely
to detect the mismatches that only occur at the desired mutation site, leading to a higher chance of the
wild-type flap being reinserted. By introducing silent edits, multiple mismatches are present which this
increases the probability of the synthesized flap being incorporated.
</p>
<p>
Secondly, silent edits can prevent re-binding of the prime editing complex to the target region after
successful editing. This is be achieved by introducing silent edits to the regions making up PAM sequence
and/or protospacer. PAM or protospacer disruption make the editing process more secure. This is because it
reduces the likelihood of editing the target region repeatedly, which would increase the probability of
on-target undesired editing outcomes. He suggested that swapping cytosine or guanine bases for these silent
edits can be particularly effective in improving prime editing efficiency.
</p>
<H4 text="Build" id="build-head"/>
<p>
We designed several pegRNAs, both with and without silent edits. To assist with this, we used the pegFinder
software<TabScrollLink tab="tab-pegrna" num="18" scrollId="desc-18"/>, which generated possible variations of
pegRNAs based on the sequence of the reporter plasmid. We selected the optimal pegRNA as suggested by the
software, and then tested it in two forms: one unmodified and one with silent edits. For the unmodified
variant, we included a single silent edit that introduced a PAM disrupt in terms of our biosafety measures.
For the modified variant, we introduced three silent edits in total, adding two more to the initial edit.
</p>
<p>
Once we had designed these variants, we ordered them in their individual components and cloned them into a
pU6-peg-GG-acceptor backbone using Golden Gate cloning according to the protocol from Anzalone et al.
2019<TabScrollLink tab="tab-pegrna" num="19" scrollId="desc-19"/>. We then screened the assembled pegRNAs to ensure that the individual components had the correct orientation and then cloned them into the pU6-GG-pegRNA-acceptor plasmid so that they were ready to be tested.
</p>
<H4 text="Test" id="test-head"/>
<p>
These two variants were then tested against each other using our <a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'rep3', path: '/engineering', tabId: 'reporter' })}>pPEAR_CFTR reporter plasmid system</a> and a <a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'scroll target id', path: '/page', tabId: 'tabid' })}>PE2 prime editor</a>. The test of the pegRNAs was conducted by co-transfecting the reporter system, the pegRNA plasmids and the PE2 plasmids into HEK293 cells.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
The results showed that the editing efficiency of the variant without silent edits was superior to the variant with silent edits, which considering our input was not expected. But as we have learned in the interview with Jan-Phillipp Gerhard, these silent edits are especially effective in avoiding mismatch repair (MMR) inside human cells. Form <a onClick={() => goToPagesAndOpenTab('mattijsinv', '/human-practices')}> Mattijs Bulcaen </a> we learned, that HEK293 cells are deficient in this very mechanism. From this we deduced that we had to test the silent edits in lung ephital cells to get a valid result.
</p>
</p>
</div>
<div className="box" >
<p id="nik3">
<h3>nik3</h3>
<LoremShort></LoremShort>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
<p id="peg2">
<H3 text="Screening of pegRNA variants" id="peg2head"/>
<p>
In this second iteration, we focused on further optimizing our pegRNA by incorporating a stem loop and experimenting with different lengths of the PBS (Primer Binding Site) and RTT (Reverse Transcriptase Template). These modifications were inspired by a combination of literature research and expert interviews. After evaluating the performance of the pegRNAs using flow cytometry, we selected the three most effective candidates.
</p>
<H4 text="Design" id="design-head"/>
<p>
Based on literature reviews and our interview with <a onClick={() => goToPagesAndOpenTab('mattijsinv', '/human-practices')}> Mattijs Bulcaen </a>, we decided to modify our pegRNA by adding a stem loop to enhance its stability. Specifically, Mattijs recommended using the tevopreQ1 stem loop, a small structural motif that increases the pegRNA's resistance to RNases. This stem loop was added to the 3' end of the pegRNA, positioned after the PBS.
</p>
<p>
Additionally, during a webinar with B. Sc. Jordan Doman<TabScrollLink tab="tab-pegrna" num="20" scrollId="desc-20"/>, we learned that it is important to test various lengths of PBS and RTT, as there is no universally optimal length for all applications. Instead, the ideal lengths are application specific. Following this advice, we designed six different pegRNA variants with combinations of two different PBS lengths (16 and 17 nucleotides) and three different RTT lengths (27, 30, and 33 nucleotides).
</p>
<p>
We chose the PBS lengths of 16 and 17 nucleotides based on an earlier recommendation from Jan-Phillipp Gerhard, who emphasised that the annealing temperature of the PBS should match the environmental conditions relevant to the intended application. In our case, since we are exploring a potential therapeutic approach, it is important that the annealing temperature of the PBS is close to the body temperature of 37 °C, which is the case for these lengths. The RTT lengths were selected based on suggestions from the pegFinder software. As with our previous insights, we designed all six variants both with and without silent edits for a wider comparison of the silent edits, making it 12 variants in total.
</p>
<H4 text="Build" id="build-head"/>
<p>
Once we had designed these variants, we ordered them in their individual components and cloned them together using Golden Gate cloning. This was a much more resource-efficient and sustainable option, as only the PBS and/or RTT lengths differed. Thus, there was a constant pegRNA part, consisting of spacer and scaffold, and a variable part, consisting of PBS, RTT and stem loop. We then cloned these variants into the pU6-GG-pegRNA-acceptor plasmid and confirmed the correct orientation and successful cloning of all constructs through screening.
</p>
<H4 text="Test" id="test-head"/>
<p>
We tested these twelve pegRNA variants against each other and the two previous variants without the trevopreQ1 stem loop, again within the PE2 system, using our reporter system, to assess their editing efficiency. The experimental setup was similar to the cycle before.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
From this round of testing, we found out that our engineered pegRNA variants pegRNA04, 05, 07 and 08 exhibited the highest levels of efficiency and stability, while the pegRNA12 showed the lowest level of editing efficiency. Therefore, we reasoned to go with these four pegRNA variants as well as pegRNA12 as a negative example for follow-up experiments.
</p>
</p>
</div>
<div className="box" >
<p id="nik4">
<h3>nik4</h3>
<LoremShort></LoremShort>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
<p id="peg3">
<H3 text="Application lung epithelial cell lines" id="peg3head"/>
<p>
HEK cells are an easy to handle and easy to edit cell model. However, they are not particularly similar to the cells that would actually be useful targets for a gene therapy. In our context, two key differences are especially grave: HEK cells, as mentioned above, are impaired in mismatch repair, making them easier to edit, and they do not naturally express CFTR.
</p>
<H4 text="Design" id="design-head"/>
<p>
In this third iteration, we wanted to investigatee the applicability of a pegRNA optimized in a model closer to therapeutic application. In our case we used in <a onClick={() => goToPageAndScroll ('Cell Culture2H', '/materials-methods')}>CFBE41o- epithelial cells lines</a> homozygous for the CFTR F508del mutation.
</p>
<H4 text="Build" id="build-head"/>
<p>
For this test, we used one of the pegRNAs (pegRNA04) that showed the highest efficiencies in previous optimization steps. Since we expected only low editing efficiencies compared to HEK cells for reasons mentioned above, we used the <a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'pe2', path: '/engineering', tabId: 'pe-systems' })}>PE6c prime editor</a>. It had proven to be most effective in HEK cells in our <a onClick={() => goToPagesAndOpenTab('pe-systems', '/engineering')}> pe systems engineering cycle </a> and should ensure detectability of possible editing.
</p>
<H4 text="Test" id="test-head"/>
<p>
We co-transfected the CFBE41o- with our modified <a onClick={() => goToPagesAndOpenTab('reporter', '/engineering')}> reporter system </a>, the plasmid expressing pegRNA04 as well as pCMV-PE6c. As a result, we observed fluorescence, indicating successful editing of the reporter plasmid. The negative controls transfected with only one of the plasmids each showed no fluorescence, routing out other factors.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
Thanks to this experiment we knew, that our pegRNAs work not only in HEK, but also in epithelial cells that express CFTR F508del.
</p>
</p>
</div>
<div className="box" >
<p id="nik5">
<h3>nik5</h3>
<LoremShort></LoremShort>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
<p id="peg4">
<H3 text="Application to genomic CFTR targeting" id="peg4head"/>
<p>
In this fourth iteration, we aimed to transfer our findings in optimizing the pegRNAs, generated in previous iterations, to the genomic CFTR context. To this end we modified our pegRNAs to be used in the CFTR gene editing process.
</p>
<H4 text="Design" id="design-head"/>
<p>
Using the pegFinder software and our acquired expertise in creating pegRNAs, we designed the new variants specifically tailored to the genomic CFTR region. These pegRNAs included the same combinations of PBS and RTT lengths as the ones we created for our reporter plasmid. Notably, scaffold, spacer, PBS and a part of the RTT did not have to be changed from the reporter targeting to genome targeting pegRNAs. Of the created pegRNAs, we wanted to focus on testing the most effective four variants found in the previous cycles and also a variant designated comparatively ineffective to test for consistency of our reporter system.
</p>
<H4 text="Build" id="build-head"/>
<p>
The newly designed pegRNAs were ordered as separate components, identical to the process used for the pegRNAs targeting the reporter system. Each RNA had both a constant and variable region, which we assembled using Golden Gate cloning. Afterwards we confirmed the correctness and completeness of the cloning into the pU6-peg-GG-acceptor plasmid through colony PCR screening. Unfortunately to this point, we were not able to produce positive clones.
</p>
<H4 text="Test" id="test-head"/>
<p>
The next step is to test the correction of CFTR F508del using these pegRNAs in the CFBE41o- epithelial cells. Additionally, we also want to test the pegRNAs in primary cells derived from friend of the team and Cystic Fibrosis patient <a onClick={() => goToPagesAndOpenTab('maxfirst', '/human-practices')}> Max </a>, testing whether our approaches are applicable not only in model systems, but also work in patient cells. To validate the editing efficiency of our designed pegRNAs were going to co-transfect a plasmid carrying an eYFP variant which is sensitive to chloride and iodide ion concentrations<TabScrollLink tab="tab-pegrna" num="21" scrollId="desc-21"/><TabScrollLink tab="tab-pegrna" num="22" scrollId="desc-22"/>. The intensity of the fluorescence correlates with these ion concentrations, which in turn reflects the functionality of the CFTR channel. This enables us to evaluate the editing efficiency of the different pegRNA variants on a phenotypic level. After 72 hours, we are going to perform a final analysis using flow cytometry to quantify the results and determine the editing efficiency of each pegRNA. Secondly, we wanted to detect the editing on a genomic level by facilitating a qPCR with a primer specific only to the corrected F508del locus.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
With this experiment we hope to achieve two things: Firstly, we want to examine whether optimizations of pegRNAs designed for our reporter system actually transfer to the genomic CFTR targeting. Secondly and most importantly, we want to find out whether we actually created an effective gene editing strategy for the genomic context of CFTR, thereby providing a foundation for a future gene therapy with high efficiency and precision when used with the right prime editor.
</p>
</p>
</div>
<div className="box" >
<p id="nik6">
<h3>nik6</h3>
<LoremShort></LoremShort>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
<p id="peg5">
<H3 text="Outlook" id="peg5head"/>
<p>
In this final iteration, we focus on the outlook for future modifications and optimizations of our pegRNA design. These concepts are meant to further improve both the stability and editing efficiency through additional research and the implementation of new design strategies.
</p>
<H4 text="Design" id="design-head"/>
<p>
As we continued to refine our approach, further literature research was conducted, and new design ideas considered. The overarching goal remained to enhance both the stability and editing efficiency of the pegRNAs. One concept we are already exploring involves the incorporation of 3’ and 5’ UTRs (Untranslated Regions)<TabScrollLink tab="tab-pegrna" num="23" scrollId="desc-23"/>. These elements, typically found in mRNA, could be added to the pegRNA to increase its stability.
</p>
<p>
Another promising idea is the use of circular RNA (circRNA)<TabScrollLink tab="tab-pegrna" num="24" scrollId="desc-24"/>, which could provide additional stability by maintaining the closed-loop structure of the pegRNA. This would prevent degradation and increase the longevity of the pegRNA in the cell.
</p>
<p>
Additionally, further nucleotide modifications could be explored, such as experimenting with alternative silent edits to see if this leads to improved editing efficiency. We also nucleotide substitutions in the scaffold region to enhance RNA-binding affinity to the protein complex could be of use.
</p>
<H4 text="Build" id="build-head"/>
<p>
To implement these new design features, the individual components, such as UTRs, would need to be cloned into the existing pegRNAs. If we pursue alternative silent edits, the pegRNA sequences would need to be redesigned, ordered, and re-cloned. The circular RNA would also require a new assembly method to achieve the desired structure. However, the fundamental workflow would remain consistent with the processes used in previous iterations.
</p>
<H4 text="Test" id="test-head"/>
<p>
To maintain consistency and comparability, the same testing protocols used for the previous pegRNA screening would be applied. This includes co-transfection in the appropriate cell lines, fluorescence-based readouts for editing efficiency, and flow cytometry analysis. By keeping the experimental conditions the same, we can ensure that the effects of the new modifications can be accurately assessed and compared to previous results.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
From these tests, we would aim to derive new insights not only specific to our particular context but also for pegRNA design as a whole. These future modifications could also yield valuable information on how to further improve the overall efficiency and stability of pegRNAs, contributing to the broader field of gene editing.
</p>
</p>
</div>
<Section title="References" id="references">
<EngPegsources/>
</Section>
<br/>
<div className="row ">
<div className="col">
<div className="left"><ButtonOneEngineering label="Previous" open="pe-systems" scrollToId="PE Systems"/></div>
<div className="left"><ButtonOneEngineering label="Previous" open="pe-systems" scrollToId="pe-systems-header"/></div>
</div>
<div className="col button-left">
<div className="right"><ButtonOneEngineering label="Next" open="pegrna" scrollToId="pegRNA"/></div>
<div className="right"><ButtonOneEngineering label="Next" open="nickase" scrollToId="nickase-header"/></div>
</div>
</div>
</section>
</div>
<div className="enginneeringtab" id="tab-pegrna" style={{display: "none"}}>
<section id="pegRNA sec" >
<div className="enginneeringtab" id="tab-nickase" style={{display: "none"}}>
<section id="Nickase sec" >
<div className="eng-box box" >
<H3 id="pegRNA" text="pegRNA"></H3>
<p><LoremShort></LoremShort></p>
<H2 id="nickase-header" text="Alternative Nickases"></H2>
<p>The Cas9 nickase is the key component of most current prime editing system. It is needed for localizing the genomic target and cutting a single DNA strand. The complex's size and RNA stability issues limit its efficiency. To overcome these challenges, we explored smaller endonucleases like CasX and Fanzor, which not only reduce the size of the complex but also offer structural advantages such as a reversed guide RNA architecture. We theorize that this unique configuration protects the RNA from degradation and improves editing precision by reducing the risk of unwanted genomic alterations by scaffold readthrough, making CasX and SpuFz1 promising alternatives to Cas9-based systems for prime editing.</p>
</div>
<div className="box" >
<p id="peg1">
<h3>peg1</h3>
<LoremShort></LoremShort>
<p id="nic1">
<H3 text="SpuFz1 Zink Finger Mutation" id="nic1head"/>
<H4 text="Design" id="text"/>
<p>
In our quest to identify smaller endonucleases suitable for creating nickases, we focused on a newly characterized family of eukaryotic endonucleases known as Fanzor proteins first described in June 2023<TabScrollLink tab="tab-nickase" num="25" scrollId="desc-25"/>, with SpuFz1 (Fig. 1) being a standout candidate due to its smaller size compared to Cas9 (SpuFz1 consists of 638 amino acids<TabScrollLink tab="tab-nickase" num="25" scrollId="desc-25"/>, whereas Cas9 has a size of 1368 amino acids<TabScrollLink tab="tab-nickase" num="26" scrollId="desc-26"/>). We selected SpuFz1 not only because of its smaller size, but also due to structural advantages, such as the reversed positioning of the spacer, which provides better protection from RNase degradation and improves editing precision.
</p>
<p>
The Cas9 endonuclease contains two active domains, each responsible for cutting one of the two DNA strands. Cas9 uses the RuvC and HNH domains, with each domain making a cut on a different strand of the target DNA<TabScrollLink tab="tab-nickase" num="27" scrollId="desc-27"/>. To create a nickase from Cas9, scientists deactivate one of these active domains, typically the HNH domain, so that the enzyme only cuts one strand instead of both, producing a single-strand break rather than a double-strand break<TabScrollLink tab="tab-nickase" num="27" scrollId="desc-27"/>.
</p>
<p>
Based on the function of this prototypical Cas9 nickase, we assumed that SpuFz1 would operate similarly, with two active centers—RuvC and TNB—each cutting one DNA strand. Following this logic, we hypothesized that by deactivating the TNB domain, which contains a zinc finger motif (Fig. 2) crucial for DNA coordination, we could convert SpuFz1 into a nickase. To test this, we aimed to replace the cysteine residues involved in zinc ion coordination within the TNB domain with alanine, thereby impairing its DNA-binding ability and producing a SpuFz1 nickase that cuts only one strand. At that time, we believed both domains in SpuFz1 were directly responsible for DNA cleavage, and our strategy was based on this assumption.
</p>
<TwoFigureRow
pic1="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-3d-model.webp"
description="Schematic illustration of SpuFz1 (PDB code: 8GKH) visualized in ChimeraX"
alt1="Schematic illustration of SpuFz1 (PDB code: 8GKH) visualized in ChimeraX"
num={1}
num2={2}
description2="Close up of the zinc finger motif of SpuFz1 (PDB code: 8GKH) visualized in ChimeraX - in the middle of the image the zinc ion of the motif can be seen, which is coordinated by 4 surrounding cysteine residues"
pic2="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-3d-model-zinc-finger.webp"
/>
<H4 text="Build" id="text"/>
<p>
Using the protein visualization software ChimeraX, we carefully examined the structure of SpuFz1 to identify the key cysteine residues responsible for coordinating the zinc ion. With this insight, we designed our nickase candidates by modifying the wild-type sequence, specifically substituting these cysteines with alanine, to disrupt the zinc ion coordination and potentially alter the protein's function.
</p>
<H4 text="Test" id="text"/>
<p>
First, we discussed our approach with <a onClick={() => goToPagesAndOpenTab('hammerkai', '/human-practices')}> Kai Schülke </a>, a PhD student from the Hammer Group at Bielefeld University, which specializes in enzyme engineering. He confirmed that our plan to focus on specific mutation candidates was appropriate given the constraints of our project. He emphasized that we lacked the time and resources to conduct large-scale, quantitative studies on a wide range of mutations. Instead, he supported our decision to target specific candidates that could be thoroughly tested within the scope of our project.
</p>
<p>
Additionally, we carefully considered the potential effectiveness of our modified SpuFz1 nickase in a Prime Editing scenario, specifically targeting the F508del mutation in Cystic Fibrosis. During this detailed analysis, we identified a critical challenge: the TAM sequence required for SpuFz1 binding was located too far from the target mutation site. This distance could limit the efficiency of the Prime Editor, raising concerns about its overall effectiveness for this particular mutation.
</p>
<InfoBox title="TAM sequences" id="current-pe-systems">
<details>
<summary>
A TAM sequence is the equivalent to a PAM sequence for OMEGA systems.
</summary>
<p>
A <b>TAM sequence</b> (Targeted Activity Modification sequence) is a short DNA sequence, typically only a few bases long, that provides a binding site for the nickase within the Prime Editing complex. This sequence is crucial because it allows the nickase to bind to the DNA and make a precise single-strand cut. For the Prime Editing complex to correct a mutation at a specific location guided by the pegRNA, a TAM sequence must be located near that target site. While the pegRNA directs the editing machinery to the region where the correction will occur, the TAM sequence enables the nickase to physically interact with the DNA and initiate the cut. Therefore, both the pegRNA and the TAM sequence are essential for efficient and accurate editing: the pegRNA specifies the site of the correction, and the TAM sequence facilitates the nickase's binding and action. For instance, SpuFz1 recognizes the TAM sequence <b>5'-CATA-3'</b>, and CasX binds to <b>5'-TTCN-3'</b>.
</p>
</details>
</InfoBox>
<H4 text="Learn" id="text"/>
<p>
Through this iteration, we learned that targeted mutagenesis is a promising approach for generating our mutant nickases. We also recognized the importance of carefully selecting the appropriate PAM or TAM sequences for our chosen endonucleases. Specifically, we realized that the TAM sequence for SpuFz1 might be too far from our target mutation, prompting us to explore other endonucleases within the Fanzor family that could serve as better candidates for nickase development. Additionally, this process highlighted the critical role of expert consultation in refining our strategies and ensuring the feasibility of our approach.
</p>
</p>
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</div>
<div className="box" >
<p id="peg2">
<h3>peg2</h3>
<LoremShort></LoremShort>
<p id="nic2">
<H3 text="Fusion Protein from GtFz1 & SpuFz1" id="nic2head"/>
<H4 text="Design" id="design-head"/>
<p>
In our ongoing exploration of Fanzor proteins, we identified another potential candidate, GtFz1, which had a suitable TAM sequence for our target application of correcting the F508del mutation in Cystic Fibrosis. However, GtFz1 showed low cutting efficiency in the tests reported in the literature<TabScrollLink tab="tab-nickase" num="25" scrollId="desc-25"/>. To address this, we devised a strategy to combine the favorable TAM-binding properties of GtFz1 with the higher cutting efficiency of SpuFz1. Specifically, we planned to engineer a fusion protein by replacing the TAM-binding domain of SpuFz1 with that of GtFz1. This approach aims to create an endonuclease that retains the strong TAM-binding ability of GtFz1 while utilizing the robust cutting efficiency of SpuFz1, optimizing it for our Prime Editing application.
</p>
<p>
Given that we were swapping entire domains rather than just single amino acids, we realized that the fusion protein might not retain the ideal TAM-binding efficiency or cutting efficiency of the original proteins. Our strategy was to create a fusion protein that could bind to the TAM site and perform DNA cutting to a certain extent, albeit weakly. We planned to use directed evolution techniques, such as Phage Assisted Continuous Evolution (PACE), to enhance these functionalities over time. This approach relies on having a starting point with some degree of the desired activity, which can then be incrementally improved through evolution.
</p>
<H4 text="Build" id="build-head"/>
<p>
The build phase involved designing this fusion protein by integrating the TAM-binding region from GtFz1 into the SpuFz1 protein structure. We engineered the sequence to include this hybrid configuration, intending to test its functionality as a nickase after introducing the zinc finger mutation, which we had hypothesized would inactivate one of the DNA-cutting domains.
</p>
<H4 text="Test" id="test-head"/>
<p>
To validate our approach, we conducted two key interviews. First, we consulted with <a onClick={() => goToPagesAndOpenTab('hammer', '/human-practices')}> Prof. Dr. Hammer </a> from Bielefeld University, who highlighted the possibility that the zinc finger domain might be structurally significant and cautioned that mutating it could destabilize the protein. He recommended that we explore whether there were any known enzymes with similar mechanisms where analogous mutations had successfully converted endonucleases into nickases. This approach, he suggested, might offer a more reliable pathway.
</p>
<p>
Next, we spoke with <a onClick={() => goToPagesAndOpenTab('svenja', '/human-practices')}> Svenja Finke </a>, a Postdoctoral Fellow at the Harvard Institute and an expert in directed enzyme evolution, including PACE. We reached out to her specifically because we anticipated that our fusion protein might require optimization to achieve strong TAM-binding and cutting efficiency. Svenja informed us that while PACE could theoretically optimize our fusion protein, the process was too complex and time-consuming for the scope of our project. As a result, we decided to reconsider this method and look for simpler, more feasible alternatives.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
From this iteration, we learned several important lessons. First, we decided to abandon the fusion protein approach. Feedback from Svenja’s interview highlighted that this strategy was too complex, time-consuming, and involved significant uncertainty regarding its effectiveness. Given the long testing times and the inherent risks, we concluded that this approach was not viable within our project’s constraints. Initially, we considered moving away from SpuFz1 due to the TAM sequence being too far from the ΔF508 mutation. However, with ongoing improvements in reverse transcriptases within Prime Editing systems, which allow for greater distances between the mutation site and the TAM sequence, we refocused our efforts on SpuFz1, considering it a viable candidate for further development.
</p>
<p>
Secondly, we realized the importance of verifying whether the zinc finger mutation we proposed is structurally feasible and whether it might compromise protein stability. This insight further emphasized the need to carefully assess our design choices before proceeding to extensive testing.
</p>
</p>
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</div>
<div className="box" >
<p id="peg3">
<h3>peg3</h3>
<LoremShort></LoremShort>
<p id="Modeling of Mutant Structural Integrity">
<H3 text="nic3" id="nic3head"/>
<H4 text="Design" id="design-head"/>
<p>
In the previous iteration, we consulted <a onClick={() => goToPagesAndOpenTab('hammer', '/human-practices')}> Prof. Dr. Hammer </a>, who suggested that the zinc finger domain in the SpuFz1 protein might play a critical structural role. Based on this feedback, the goal of this iteration was to investigate whether mutating the zinc finger would destabilize the protein and compromise its function. Specifically, we aimed to determine if altering this domain would still be a viable strategy for generating a SpuFz1-based nickase without losing structural integrity.
</p>
<H4 text="Build" id="build-head"/>
<p>
Before, we identified the specific amino acids responsible for coordinating the zinc ion within the zinc finger domain. Using the software Geneious, we proceeded to design DNA sequences by substituting these key amino acids with ones that would impair their ability to coordinate the zinc ion. These designed sequences corresponded to our potential mutation candidates, which we prepared for further structural analysis.
</p>
<H4 text="Test" id="test-head"/>
<p>
We used AlphaFold to model the 3D structures of our zinc finger mutation candidates. After generating these models, we used ChimeraX to perform a structural overlay comparison between the native SpuFz1 protein and the mutated versions (Fig. 3). This comparison revealed significant differences, particularly in the TNB domain, indicating that the zinc finger plays a crucial structural role (Fig. 4).
</p>
<TwoFigureRow
pic1="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-vs-zf-nikase.webp"
pic2="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-vs-zf-nikase-zinc-finger.webp"
num={3}
num2={4}
alt1="Structural overlay of wildtype SpuFz1 (color: Lilac) (PDB code: 8GKH) and modeled zinc-finger mutation candidate (color: orange) visualized in ChimeraX – the yellow circle shows the location of the zinc-finger. A structural deviation of both proteins locally is evident"
description="Structural overlay of wildtype SpuFz1 (color: Lilac) (PDB code: 8GKH) and modeled zinc-finger mutation candidate (color: orange) visualized in ChimeraX – the yellow circle shows the location of the zinc-finger. A structural deviation of both proteins locally is evident"
description2="Close-up of the zinc finger motif of the structural overlay - the zinc finger appears to be structurally significant: there are strong structural differences locally"
/>
<H4 text="Learn" id="learn-head"/>
<p>
From this analysis, we concluded that the zinc finger mutation is not a suitable candidate for generating a nickase, as altering this domain would likely compromise the structural integrity of SpuFz1. Prof. Hammer suggested that instead of focusing on SpuFz1, we explore other endonucleases with similar mechanisms. His recommendation was to identify endonucleases that are structurally comparable to SpuFz1 and analyze the strategies used to convert these into nickases. We would then apply these same strategies to our selected endonucleases, adapting them for our purposes.
</p>
</p>
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</div>
<div className="box" >
<p id="peg4">
<h3>peg4</h3>
<LoremShort></LoremShort>
<p id="nic4">
<H3 text="nCas12 analog Mutations" id="nic4head"/>
<H4 text="Design" id="design-head"/>
<p>
After concluding that the zinc finger mutation approach was not suitable for converting SpuFz1 into a nickase, we revisited our understanding of its cutting mechanism. Initially, we believed that SpuFz1, similar to Cas9, contained two active centers that each cut one of the DNA strands, and that by deactivating one of these centers, we could generate a nickase that only cuts one strand. However, through further research, we discovered that this assumption was incorrect. SpuFz1 actually functions with a different cutting mechanism: the RuvC domain is responsible for cutting the non-target strand, while the TNB domain does not directly cut the DNA. Instead, it assists the process by guiding the target strand into the RuvC domain for sequential cleavage<TabScrollLink tab="tab-nickase" num="29" scrollId="desc-29"/>. This discovery shifted our focus from simply deactivating an active site to better understanding how the sequential cleavage works in order to inform future mutation strategies.
</p>
<p>
In addition to these insights, we noticed a significant phylogenetic relationship between Fanzor endonucleases, like SpuFz1, and Cas12 endonucleases<TabScrollLink tab="tab-nickase" num="25" scrollId="desc-25"/>. This connection was crucial, as Cas12 proteins have a similar cutting mechanism to Fanzor proteins, utilizing a single active site for cleavage while coordinating both DNA strands. More importantly, we identified a precedent in the literature where a Cas12a endonuclease was successfully converted into a nickase by substituting a single amino acid in the TNB domain<TabScrollLink tab="tab-nickase" num="30" scrollId="desc-30"/> (Fig. 5 and 6). This provided us with a clear model strategy to follow, as this targeted mutation allowed the endonuclease to selectively cut only one DNA strand, effectively converting it into a nickase.
</p>
<TwoFigureRow
pic1="https://static.igem.wiki/teams/5247/engineering-cycle/cas12-nikase.webp"
pic2="https://static.igem.wiki/teams/5247/engineering-cycle/cas12-nikase-close-up.webp"
num={5}
num2={6}
alt1="Schematic representation of Cas12a (PDB code: 8SFH) visualized in ChimeraX - the yellow circle highlights the position of arginine (R) (1226th amino acid in the primary structure) which, when replaced by an alanine (A), converts the Cas12a endonuclease into an nCas12a nickase"
description="Schematic representation of Cas12a (PDB code: 8SFH) visualized in ChimeraX - the yellow circle highlights the position of arginine (R) (1226th amino acid in the primary structure) which, when replaced by an alanine (A), converts the Cas12a endonuclease into an nCas12a nickase"
description2="Close-up of Cas12a (PDB code: 8SFH) - arginine (R) (1226th amino acid in the primary structure) is colored purple"
/>
<p>
Based on these findings, two key decisions emerged. First, recognizing the structural and mechanistic similarities between Fanzor and Cas12 endonucleases, we decided to explore CasX—a smaller Cas12-related endonuclease—as an additional candidate in our project. CasX shares many of the advantages of SpuFz1, such as a more compact structure compared to Cas9, making it ideal for applications requiring smaller editing systems. Secondly, we resolved to adapt the mutation strategy used to convert Cas12a into a nickase for both CasX and SpuFz1. By applying these learnings, we aimed to generate effective nickases from these endonucleases to further optimize the Prime Editing complex.
</p>
<InfoBox title="The rationale behind designing SpuFz1 and CasX Nickases" id="how-to-create-nickases-online-fast">
<details>
<summary>
The mutation strategy aimed to convert the endonucleases SpuFz1 and CasX into nickases by targeting specific positively charged amino acids, similar to R1226 in mutated to create a AsCas12a nickase, to disrupt their double-strand cleavage function while retaining single-strand cutting capability.
</summary>
<p>
In our project, we set out to engineer the endonucleases SpuFz1 and CasX into nickases, a process that required a more targeted approach than random mutagenesis due to the time and financial constraints of our project. Random mutagenesis, while a possible strategy, would have required an extensive scope, making it difficult to achieve meaningful results within our timeframe. As a result, we aimed to identify specific mutational candidates that would allow for a more focused and efficient approach.
</p>
<p>
One strategy we explored was finding an endonuclease with structural and mechanistic similarities to SpuFz1 and CasX, for which a successful precedent existed in converting an endonuclease into a nickase. After studying the phylogenetic relationships of SpuFz1 and CasX, we identified AsCas12a, an endonuclease with a similar sequential DNA cleavage mechanism. Importantly, there was already a known example where AsCas12a had been engineered into a nickase through a single mutation—specifically, the mutation of arginine 1226. This provided a strong foundation for us to develop a similar strategy for SpuFz1 and CasX.
</p>
<p>
We hypothesized that the role of arginine 1226 in the sequential cleavage mechanism of AsCas12a was to coordinate the DNA strands during the cutting process. AsCas12a performs a sequential cut, where the RuvC domain first cleaves the non-target strand, and the TNB (NUC) domain helps guide the target strand into the RuvC domain for cleavage (Fig. 7). We suspected that arginine 1226 could play a key role in this process by coordinating the DNA due to its long, positively charged side chain. If removing or mutating this arginine disrupts the sequential cut, it would suggest that the arginine helps guide the second strand into the RuvC domain.
</p>
<p>
Structurally, we observed that arginine 1226 protrudes from the NUC domain of AsCas12a and is oriented toward the RuvC domain (Fig. 8). This positioning led us to hypothesize that the arginine helps coordinate the DNA strand as it moves into the RuvC domain for cutting. Based on this observation, we speculated that the mutation of arginine 1226 disrupts this coordination, preventing the full double-strand cut and effectively converting AsCas12a into a nickase.
</p>
<TwoFigureRow
pic1="https://static.igem.wiki/teams/5247/engineering-cycle/ascas12a-nuc-domain.webp"
pic2="https://static.igem.wiki/teams/5247/engineering-cycle/ascas12a-nuc-domain-close-up.webp"
num={7}
num2={8}
description2="Close-up of NUC domain (colored purple) of AsCas12a(PDB code: 8SFH) - the arginines (R) (1226th amino acid in the primary structure) is colored yellow. Its positively charged side chain is oriented towards the RuvC domain, as well as the DNA strand fixated in the RuvC domain"
description="AsCas12a (PDB code: 8SFH) visualized in ChimeraX. The NUC domain (TNB) is colored purple and is attached to the RuvC domain. The DNA strand is colored orange"
alt1="AsCas12a (PDB code: 8SFH) visualized in ChimeraX. The NUC domain (TNB) is colored purple and is attached to the RuvC domain. The DNA strand is colored orange"
/>
<p>
We then applied this structural insight to SpuFz1 and CasX, searching for positively charged amino acids with long side chains, similar to arginine 1226, that were positioned at the interface between the NUC and RuvC domains. We specifically looked for amino acids that protruded from the NUC domain and oriented toward the RuvC domain, mirroring the structural role of arginine 1226 in AsCas12a. These amino acids became our mutational targets, allowing us to design a strategy to convert SpuFz1 and CasX into nickases by disrupting their ability to make double-strand cuts, while preserving their functionality for single-strand cuts. The amino acids we identified in SpuFz1 are the 564th and the 568th arginine located in its NUC domain. For CasX we identified the 904th arginine as a promising candidate.
</p>
</details>
</InfoBox>
<H4 text="Build" id="build-head"/>
<p>
We structurally analyzed CasX and SpuFz1, as well as the known AsCas12a nickase, using Chimera. Our objective was to understand why the specific amino acid substitution converted AsCas12a into a nickase. We then identified analogous amino acids in SpuFz1 (Fig. 7 and Fig. 8) and CasX (Fig. 9 and Fig. 10) that might play a similar role, allowing us to design new mutation candidates for our project. After designing these new mutation candidates, we modeled them using AlphaFold to predict their 3D structures and assess their potential effectiveness.
</p>
<TwoFigureRow
pic1="https://static.igem.wiki/teams/5247/engineering-cycle/casx-nikase.webp"
pic2="https://static.igem.wiki/teams/5247/engineering-cycle/casx-nikase-close-up.webp"
num={9}
num2={10}
description2="Close-up of PlmCasX (PDB code: 7WAZ) - arginine (R) (904th amino acid in the primary structure) and glutamine (Q) (907th amino acid in the primary structure) are purple in color"
alt1="Schematic representation of PlmCasX (PDB code: 7WAZ) in ChimeraX - the yellow circle highlights the position of arginine (R) (904th amino acid in the primary structure) and glutamine (Q) (907th amino acid in the primary structure), which, when replaced by an alanine (A), convert the endonuclease into a nickase, according to our hypothesis"
description="Schematic representation of PlmCasX (PDB code: 7WAZ) in ChimeraX - the yellow circle highlights the position of arginine (R) (904th amino acid in the primary structure) and glutamine (Q) (907th amino acid in the primary structure), which, when replaced by an alanine (A), convert the endonuclease into a nickase, according to our hypothesis"
/>
<TwoFigureRow
pic1="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-nikase.webp"
pic2="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-nikase-close-up.webp"
num={11}
num2={12}
description2="Close-up of SpuFz1 (PDB code: 8GKH) - the two arginines (R) (564th and 568th amino acid in the primary structure) are purple in color"
alt1="Schematic representation of SpuFz1 (PDB code: 8GKH) in ChimeraX - the yellow circle highlights the position of the two arginines (R) (564th and 568th amino acid in the primary structure), which, when replaced by an alanine (A), transform the endonuclease into a nickase according to our hypothesis"
description="Schematic representation of SpuFz1 (PDB code: 8GKH) in ChimeraX - the yellow circle highlights the position of the two arginines (R) (564th and 568th amino acid in the primary structure), which, when replaced by an alanine (A), transform the endonuclease into a nickase according to our hypothesis"
/>
<H4 text="Test" id="test-head"/>
<p>
To validate our approach, we conducted an interview with <a onClick={() => goToPagesAndOpenTab('saito', '/human-practices')}> Makoto Saito </a>, the lead author of the main Fanzor paper. Given his expertise, there was no better person to consult on this topic. We presented our project and our strategy for creating nickases, and he found our approach to be very plausible. He confirmed that the zinc finger mutation is likely structurally critical and agreed that our new strategy, based on the precedent with AsCas12a, was more promising. This conversation gave us confidence that we were on a good track.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
From this iteration, we gained several key insights. First, our initial understanding of the cutting mechanism used by SpuFz1—based on the assumption that it contained two active centers, like Cas9, each cutting a DNA strand—was incorrect. We discovered that SpuFz1 operates differently, with the RuvC domain cutting the non-target strand and the TNB domain assisting by guiding the target strand into the RuvC domain for sequential cleavage. This shift in understanding allowed us to refine our approach, moving away from deactivating an active site to focusing on the sequential cutting mechanism.
</p>
<p>
Additionally, we found that Fanzor endonucleases, like SpuFz1, share a significant phylogenetic relationship with Cas12 endonucleases, which have a similar single-site cutting mechanism. This connection, along with the precedent of converting Cas12a into a nickase through the substitution of a single amino acid in the TNB domain, provided us with a clear strategy for converting SpuFz1 and CasX into nickases. The similarity in cutting mechanisms between Fanzor and Cas12 proteins reinforced the viability of this approach.
</p>
<p>
This iteration led us to incorporate CasX, a smaller Cas12-related endonuclease, into our project. CasX offers the same advantages as SpuFz1, such as a compact structure, making it ideal for applications that require smaller editing systems. Additionally, we adapted the mutation strategy used to convert Cas12a into a nickase for both CasX and SpuFz1, guiding our future work in optimizing the Prime Editing complex.
</p>
</p>
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</div>
<div className="box" >
<p id="peg5">
<h3>peg5</h3>
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<p id="nic5">
<H3 text="Ongoing: In Vitro Cleavage Assays" id="nic5head"/>
<H4 text="Design" id="design-head"/>
<p>
In this iteration, our focus shifted to testing whether our mutation candidates had successfully converted the endonucleases into functional nickases. To do this, we adapted an existing assay that had been originally designed to determine whether mutated endonucleases exhibited nickase activity<TabScrollLink tab="tab-nickase" num="25" scrollId="desc-25"/>. We tailored this assay to fit our specific needs, allowing us to accurately assess the properties of our mutated proteins in the lab. The key question was whether the mutations had rendered the proteins dysfunctional, left them as endonucleases, or successfully converted them into nickases.
</p>
<H4 text="Build" id="build-head"/>
<p>
We started off by amplification of our nickase candidates, ordered as gene syntheses, to add restriction sites. We then facilitated restriction cloning of the amplificates into an E. coli protein expression vector provided by the laboratory of or PI Kristian Müller. We subsequently transformed E. coli with the gene fragments of our nickase candidates for CasX and SpuFz. However, the transformant cells did not grow, leading us to suspect that the plasmid backbone we received maybe impaired in some way. Given the timeline, we were not able to complete the testing of our nickase candidates. Our current steps involve troubleshooting regarding the restriction cloning and continuing with protein expression and purification once the issue is resolved.
</p>
<H4 text="Test" id="test-head"/>
<p>
The next phase of our plan, once we overcome the current issues with cloning and successfully overexpress our nickase candidates, would involve conducting an in vitro plasmid cleavage assay (Fig. 13). In this assay, the purified nickases would be combined with their respective guide RNAs and a supercoiled test plasmid. The guide RNAs would direct the nickases to the target sequence on the plasmid. Depending on the results, the plasmid would remain supercoiled if untouched, become relaxed if nicked, or be linearized if cut by an endonuclease. To analyze these outcomes, we would perform gel electrophoresis, where the different conformations of the plasmid (supercoiled, relaxed, or linearized) would migrate differently through the gel. Supercoiled plasmids would migrate the furthest, relaxed plasmids would move the slowest, and linearized plasmids would fall between these two. As controls, we would have used the plasmid in its uncut form, nicked by nCas9 and digested using a restriction enzyme.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
If we could have proceeded with the nickase assays, the readout would allow us to determine whether the tested proteins function as nickases, endonucleases, or remain inactive.
</p>
<div className="row align-items-center">
<OneFigure
pic1="https://static.igem.wiki/teams/5247/engineering-cycle/nickase-assay.webp"
num={13}
description="Theoretical gel electrophoresis results for our nickase assay. Lanes 1 and 8 represent molecular weight ladders, which provide size markers for the plasmid fragments. Lane 2 contains the untreated reporter plasmid, which remains supercoiled and travels the farthest through the gel. Lane 3 serves as a positive control, containing nCas9, gRNA, and the reporter plasmid. The nCas9 nickase nicks the plasmid, relaxing its structure, and as a result, the relaxed circular plasmid moves slower than the supercoiled form. Lane 4 acts as a negative control, containing a restriction enzyme and the reporter plasmid. The enzyme fully cuts the plasmid, linearizing it, and this linear form moves slower than the supercoiled plasmid but faster than the relaxed circular form. Lane 5 includes CasX and the reporter plasmid without gRNA, meaning no cleavage occurs, leaving the plasmid in its supercoiled state, which migrates similarly to the untreated plasmid in lane 2. Lane 6 contains CasX, gRNA, and the reporter plasmid, resulting in full cleavage and plasmid linearization, causing it to migrate similarly to the linear plasmid in lane 4. Finally, lane 7 includes our nickase candidate (either CasX or SpuFz1), gRNA, and the reporter plasmid. Ideally, the candidate would nick the plasmid, resulting in a relaxed circular form that moves similarly to the nicked plasmid in lane 3."
alt1="Theoretical gel electrophoresis results for our nickase assay. Lanes 1 and 8 represent molecular weight ladders, which provide size markers for the plasmid fragments. Lane 2 contains the untreated reporter plasmid, which remains supercoiled and travels the farthest through the gel. Lane 3 serves as a positive control, containing nCas9, gRNA, and the reporter plasmid. The nCas9 nickase nicks the plasmid, relaxing its structure, and as a result, the relaxed circular plasmid moves slower than the supercoiled form. Lane 4 acts as a negative control, containing a restriction enzyme and the reporter plasmid. The enzyme fully cuts the plasmid, linearizing it, and this linear form moves slower than the supercoiled plasmid but faster than the relaxed circular form. Lane 5 includes CasX and the reporter plasmid without gRNA, meaning no cleavage occurs, leaving the plasmid in its supercoiled state, which migrates similarly to the untreated plasmid in lane 2. Lane 6 contains CasX, gRNA, and the reporter plasmid, resulting in full cleavage and plasmid linearization, causing it to migrate similarly to the linear plasmid in lane 4. Finally, lane 7 includes our nickase candidate (either CasX or SpuFz1), gRNA, and the reporter plasmid. Ideally, the candidate would nick the plasmid, resulting in a relaxed circular form that moves similarly to the nicked plasmid in lane 3."
/>
</div>
</p>
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</div>
<div className="box" >
<p id="peg6">
<h3>peg6</h3>
<LoremShort></LoremShort>
<p id="nic6">
<H3 text="Ongoing: SpuFz1 expression in Yeast" id="nic6head"/>
<H4 text="Design" id="design-head"/>
<p>
When talking to <a onClick={() => goToPagesAndOpenTab('saito', '/human-practices')}> Makoto Saito </a>, he told us that expressing SpuFz1 in E. coli did not work for him. He advised us to instead establish a yeast expression system. Since none of us had any experience with using yeast for protein production, we reached out to <a onClick={() => goToPagesAndOpenTab('nberelsmann', '/human-practices')}> Nils Berelsmann </a> at the faculty of chemistry at our university. He was able to provide us with a yeast expression strain as well as the corresponding expression vector. Because we were strongly restricted by time at that point, we then asked <a onClick={() => goToPagesAndOpenTab('hakan', '/human-practices')}> Hakan Soytürk </a> from the biological faculty for help and he offered to facilitate transformation and selection of positive Yeast transformants for us.
</p>
<H4 text="Build" id="build-head"/>
<p>
The workflow for cloning SpuFz1 amplificates into the pPIC9K yeast vector was similar to the cloning into the E. coli expression vector. After multiple attempts of transformations and colony PCRs we found positive clones for two of the nickase variants. Hakan transformed them into the yeast strain for us, but no colonies formed, indicating unsuccessful transformation. Due to time constraints, we were not able to repeat the cloning. However, testing our SpuFz1 and also CasX nickase candidates and eventually using them for prime editing.
</p>
</p>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
</div>
<div className='row align-items-center'>
<div className='col'>
<TwoLinePDF link='https://static.igem.wiki/teams/5247/pdfs/cloning-strategy-spufz1.pdf' name="cloning-strategy-spufz1.pdf"/>
</div>
<div className='seperator-2 col-2'>
</div>
<div className='col'>
<TwoLinePDF link='https://static.igem.wiki/teams/5247/pdfs/cloning-strategy-plmcasx.pdf' name="cloning-strategy-plmcasx.pdf"/>
</div>
</div>
<div className='row align-items-center'>
<div className='col'>
<PDF link='https://static.igem.wiki/teams/5247/pdfs/cloning-strategy-cas9.pdf' name="cloning-strategy-cas9.pdf'"/>
</div>
</div>
</div>
<Section title="References" id="references">
<EngNicksources/>
</Section>
<br/>
<div className="row ">
<div className="col">
<div className="left"><ButtonOneEngineering label="Previous" open="nikase" scrollToId="Nikase"/></div>
<div className="left"><ButtonOneEngineering label="Previous" open="pegrna" scrollToId="pegrna-header"/></div>
</div>
<div className="col button-left">
<div className="right"><ButtonOneEngineering label="Next" open="delivery" scrollToId="Delivery"/></div>
<div className="right"><ButtonOneEngineering label="Next" open="delivery" scrollToId="delivery-header"/></div>
</div>
</div>
</section>
</div>
<div className="enginneeringtab" id="tab-delivery" style={{display: "none"}}>
<section id="Delivery sec" >
<div className="eng-box box" >
<H3 id="Delivery" text="Delivery"></H3>
<p><LoremShort></LoremShort></p>
<H2 id="delivery-header" text="Delivery"></H2>
<p>The design path of our lipid nanoparticle (LNP) for mRNA delivery underwent multiple cycles of research and discussion, marked by important decision points and learnings along the way. By ongoing further improvement, we designed our lungs-specific LNP called AirBuddy with improved stability aspects, becoming more precise in the delivery of our therapeutic cargo LNP by LNP.</p>
<div className="row align-items-center">
<div className="col">
<img src="https://static.igem.wiki/teams/5247/delivery/airbuddy.webp" className="center" style={{maxHeight: "80pt", margin: "auto"}}/>
</div>
</div>
</div>
<div className="box" >
<p id="del1">
<h3>del1</h3>
<LoremShort></LoremShort>
<H3 text="Iteration 1 - AVVs vs LNPs" id="del1head" />
<p>Initially, this project part started with a discussion with <a onClick={() => goToPagesAndOpenTab('kristian', '/human-practices')}> Prof. Dr. Krisitan Müller</a>, PI of our team with expertise in Adeno-associated viruses (AAVs), focusing on whether to pursue LNPs or AAVs for mRNA delivery. The deciding factor leaned towards LNPs, as they offered a significant advantages including less immunogenic potential<TabScrollLink tab="tab-delivery" num="31" scrollId="desc-31"/> and bigger loading capacity<TabScrollLink tab="tab-delivery" num="32" scrollId="desc-32"/>. LNPs loading capacity depends on various factors, but in general they offer a bigger cargo size compared to 4.7 kb for AVVs<TabScrollLink tab="tab-delivery" num="33" scrollId="desc-33"/>. This allows the delivery of bigger mRNA constructs compared to AAVs, which is needed for our Prime Editing construct.</p>
<p><a onClick={() => goToPagesAndOpenTab('weber', '/human-practices')}>Prof. Wolf-Michael Weber and Dr. Jörg Große-Onnebrink</a> from the UKM in Münster were our first point of contact for the development of our LNP for CFTR treatment. Moreover, <a onClick={() => goToPagesAndOpenTab('radukic', '/human-practices')}>Dr. Marco Radukic </a>form Bielefeld University provided us with a very useful cargo, namely minicircle DNA carrying the EYFP gene from <a href="https://www.plasmidfactory.com/custom-dna/minicircle-dna/" title="PlasmidFactory" >PlasmidFactory</a> as a positive control for our experiments. He also helped us establish protocols for LNP synthesis and LNP transfection in our lab.</p>
</p>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
</div>
<div className="box" >
<p id="del2">
<h3>del2</h3>
<LoremShort></LoremShort>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
<H3 text="Interation 2 - Cayman LNP" id="del2head" />
In the first experimental phase, LNPs from <strong>Cayman Chemical LNP Exploration Kit (LNP-102)</strong> consisting of SM-102, 1,2-DSPC, Cholesterol, and DMG-PEG(2000)<TabScrollLink tab="tab-delivery" num="34" scrollId="desc-34"/> were tested with mRNA encoding fluorescent protein to evaluate their transfection efficiency. However, the results indicated good non-lung-specific transfection efficiency, which was a critical factor for the project. This led the team to reconsider their choice of this LNP.
</p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/cayman-lnp-freigestellt.webp"
alt1="Cayman LNP"
num={1}
description="Schematic view of LNP-102 from Cayman Chemical."
/>
</div>
<div className="box" >
<p id="del3">
<h3>del3</h3>
<LoremShort></LoremShort>
<H3 text="Interation 3 - Corden LNP" id="del3head" />
In the next phase, we chose to use a new LNP formulation, namely the <strong>LNP Starter Kit #2</strong><TabScrollLink tab="tab-delivery" num="35" scrollId="desc-35"/> of <a onClick={() => goToPagesAndOpenTab('corden', '/human-practices')}>Corden Pharma</a>, because it offered several advantages over the initial option. The key benefit of this new LNP lies in the use of DOTAP, a cationic lipid that enhances interaction with negatively charged cell membranes in the lungs, improving cellular uptake efficiency. While SM-102 in the Cayman LNP-102 is effective for systemic delivery, it lacks the same specificity for lung tissue. Additionally, Corden Pharma’s plant-based BotaniChol® prevents animal-sourced contamination and helps address the global lipid shortage for vaccine production. mPEG-2000-DSPE provides superior stability and reduces immune system activation over time, making it particularly suitable for pulmonary delivery. This made the new formulation a better choice for safely and effectively targeting lung tissue, especially in delivering therapies for CFTR-related diseases. During this time, the team encountered a paper on capsaicin-chitosan nanoparticles, which explored its use in targeted delivery and higher transfection efficiency. However, after further investigation and consultation of <a onClick={() => goToPagesAndOpenTab('kolonkofirst', '/human-practices')}>Dr. Katharina Kolonko</a>, it was determined that capsaicin was not suitable in our way of pulmonary application.
</p>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
</div>
<br/>
<div className="row ">
<div className="col">
<div className="left"><ButtonOneEngineering label="Previous" open="pegrna" scrollToId="pegRNA"/></div>
</div>
<div className="col button-left">
<div className="right"><ButtonOneEngineering label="Next" open="references" scrollToId="References"/></div>
</div>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/corden-lnp-freigestellt.webp"
alt1="Corden LNP"
num={2}
description="Schematic view of LNP #2 from Corden Pharma with DOTAP as cationic lipid, DSPC as phospholipid and mPEG-2000-DSPE as PEG lipid."
/>
</div>
</section>
</div>
<div className="box" >
<p id="del4">
<H3 text="Interation 4 - Spray-dried SORT LNP called AirBuddy" id="del4head" />
The next design iteration incorporated the insights from Wang's LNP work for building upon SORT principles to make the nanoparticles lung-specific<TabScrollLink tab="tab-delivery" num="36" scrollId="desc-36"/>. The main components include DMG-PEG 2000, cholesterol, DOPE and DOTAP as phospholipids and cationic lipids such as 4A3-SC8. In our LNP development, we carefully considered the use of PEG. While PEG can improve stability, it can also reduce cellular uptake and induce immune responses, necessitating a balanced approach to its inclusion<TabScrollLink tab="tab-delivery" num="37" scrollId="desc-37"/>.
<Collapsible id="Col1" open={false} title="Ambivalence of PEG and our implementation">
<p>
<H4 text="What is PEG and why is it important for LNPs?" id="text" />
Polyethylene glycol (PEG) is an essential component in the formulation of LNPs, which are widely used in drug delivery systems, particularly for mRNA-based therapies like vaccines. PEG-lipids are hybrid molecules consisting of a hydrophilic PEG chain attached to a hydrophobic lipid anchor. This unique structure enables PEG-lipids to interact effectively with both aqueous environments and lipid structures, such as cell membranes and lipid nanoparticles themselves.
<p>PEGylation—attaching PEG to lipids—provides numerous benefits. It increases the stability of LNPs by forming a protective outer layer, preventing aggregation, extending circulation time in the bloodstream, and reducing immune system detection. These advantages are critical in ensuring that the LNPs reach their target cells and deliver the therapeutic payload effectively. </p>
<H4 text="Why is PEG relevant for LNPs in mRNA delivery?" id="text" />
PEG improves the pharmacokinetics of LNPs by extending their systemic circulation time, which is crucial for therapies like mRNA vaccines, where the nanoparticles must remain in the bloodstream long enough to reach their target cells. Additionally, PEG-lipids can reduce the size of LNPs, enhancing their ability to penetrate cell membranes and deliver the therapeutic material efficiently. However, a balance must be struck. Increasing PEG content can lead to smaller, more stable particles, but it may also reduce intracellular delivery and protein expression. Therefore, while PEG boosts circulation and stability, too much can hinder therapeutic effectiveness.
<H4 text="Cytotoxicity and mPEG-2000-DSPE" id="text" />
One challenge with PEGylation is the potential for immune responses, such as the <i>accelerated blood clearance</i> (ABC) phenomenon, where repeated exposure to PEGylated particles leads to faster clearance by the immune system. There are also risks of hypersensitivity reactions like <i>complement activation-related pseudoallergy</i> (CARPA). Thus, selecting the right PEG-lipid type is essential to mitigate these risks.
<p>We collaborated with <a onClick={() => goToPagesAndOpenTab('corden', '/human-practices')}>Corden Pharma</a>, a specialist in LNP technologies, to address these concerns. Based on their recommendations, we opted for <strong>mPEG-2000-DSPE</strong> as our PEG-lipid of choice. This variant minimizes cytotoxicity while providing excellent stability and circulation time. It has also proven effective in reducing immune-related side effects while preserving the integrity and performance of our nanoparticles. </p>
<H4 text="DMG-PEG2000 vs mPEG-2000-DSPE" id="text" />
While mPEG-2000-DSPE has traditionally been used for stabilizing LNPs and enhancing delivery efficiency, we decided to incorporate DMG-PEG2000 into our SORT LNP-based AirBuddy due to its superior properties. DMG-PEG2000 offers better biodegradability and enhanced stability in pulmonary applications. Unlike mPEG-2000-DSPE, which tends to accumulate in the body and may lead to immune activation over time, DMG-PEG2000 is known for its faster clearance and reduced potential for long-term toxicity. For lung-specific delivery, where stability and safety are critical, DMG-PEG2000 ensures that the nanoparticles remain stable long enough to deliver the therapeutic material effectively, but also degrade at a rate that minimizes unwanted immune responses. This makes DMG-PEG2000 a more suitable choice for therapies targeting CFTR-related diseases, where precise and safe delivery to the lungs is essential for treatment success.
<p>Details about the biosafety aspects of our LNP can be read <a onClick={() => goToPageAndScroll ('sort-lnp-and-cytotoxicity', '/safety')}> here </a>. </p>
<H4 text="Conclusion" id="text" />
We use DMG-PEG2000 in our SORT LNP-based AirBuddy because of its superior biodegradability, enhanced stability, and reduced risk of immune system activation. By building on insights from experts and incorporating principles from Wang’s LNP work, we’ve tailored our nanoparticles for lung-specific delivery. This choice ensures that our formulations remain stable long enough to deliver the therapeutic payload effectively while minimizing potential long-term toxicity. This balance is crucial for pulmonary applications, where DMG-PEG2000 outperforms alternatives like mPEG-2000-DSPE, making it the ideal choice for treating CFTR-related lung diseases.
</p>
</Collapsible>
<p>DMG-PEG2000 of the SORT LNP offers better biodegradability and enhanced stability in pulmonary applications - it is known for its faster clearance and reduced potential for long-term toxicity. To ensure we addressed this issue, cytotoxicity tests were performed in addition to the determination of physicochemical properties in cooperation with the <a onClick={() => goToPagesAndOpenTab('biophysik', '/human-practices')}>Physical and Biophysical Chemistry working group of Bielefeld University</a> to characterize the LNPs. More details about the composition of the SORT LNPs and function of the components can be read below.</p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/airbuddy-svg.svg"
alt1="AirBuddy"
num={3}
description="Schematic view of our lung-specific SORT LNP called AirBuddy carrying chitosan-protected pegRNA and mRNA for the assembly of our prime editing technology named PrimeGuide."
/>
<Collapsible id="Col2" open={false} title="Composition of our SORT LNP called Airbuddy // LNP Handbook Cooperation">
<H4 text="Components of AirBuddy" id="text" />
<H5 text="Ionizable Lipid" id="text" />
<p>The primary ingredient, 4A3-SC8 or MC3, are ionizable cationic lipids that forms the core of the LNP. Ionizable cationic lipids become positively charged in acidic environments, such as when a pH change occurs for example in acidic buffers or in the endosome. This allows them to bind to negatively charged nucleic acids and form protective capsules around it. In the endosome these lipids facilitate endosomal escape through electrostatic interactions between the LNPs and the endosomal or cellular membranes.</p>
<H5 text="Helper Lipids" id="text" />
<p>DOTAP (Dioleoyltrimethyl-ammonium propane) is a cationic lipid that makes up 50 % of the total molar lipid ratio. It plays a crucial role in binding to the negatively charged surface of lung epithelial cells. This enhances transfection efficiency and helps make the LNP formulation more lung-specific, improving targeted delivery. The neutral helper lipid DOPE (Dioleoylphosphatidylethanolamine) enhances endosomal escape by fusing with the endosomal membrane and improves transfection efficiency.</p>
<H5 text="Sterol" id="text" />
<p>Cholesterol, is an important cationic lipid, providing structural stability, fluidity and permeability to the LNPs, thereby improving their overall transfection efficiency. </p>
<H5 text="PEGylated Lipids" id="text" />
<p>DMG-PEG (Dimyristoylglycerin-polyethyleneglycol) is an important component by improving the LNP stability and preventing aggregation of the LNPs. </p>
<H4 text="Production Methods" id="text" />
<H5 text="LNP Assembly" id="text" />
<p>Our LNP can be formulated using various methods depending on the scale of production, including pipette mixing, vortex mixing, or microfluidic mixing. For cargo protection the mRNA can be mixed with chitosan for stability improvement before adding with the LNP components. After mixing the lipids with mRNA in carefully controlled ratios, the mixture is typically dialyzed to remove organic solvents like ethanol and citrate buffer. The choice of lipid composition and preparation method influences the tissue-targeting capabilities of the LNPs, allowing for selective delivery to organs like the liver, lungs, or spleen. For more detailed information on formulation methods and lipid selection, refer to our LNP Handbook deigned in <a onClick={() => goToPageAndScroll ('handbook', '/human-practices')}> cooperation with iGEM Team Linköping </a> and others.</p>
<p>Click this Button to gain the LNP Handbook</p>
<DownloadLink url="https://static.igem.wiki/teams/5387/liposomes-handbook.pdf" fileName="liposomes-handbook.pdf" />
<H5 text="Spray drying Procedure" id="text" />
<p>By combining these components with the spray drying method from <a onClick={() => goToPageAndScroll ('rnhale', '/human-practices')}> RNhale </a> we offer a versatile and efficient method for delivering RNA therapeutics to the lung, paving the way for gene therapy, especially our Prime Guide. The effective delivery of the prime editing complex is a crucial point in our project. </p>
<H5 text="Storage" id="text" />
<p>The final LNP solution can be stored at 4 °C for a few days. It is recommended to use the formulated LNPs as soon as possible to maintain consistent results. Storage at RT is not recommended. Storage at freezing temperatures is also not recommended unless optimized cryoprotectants are used.</p>
<div className="enginneeringtab" id="tab-references" style={{display: "none"}}>
<section id="References sec" >
<div className="eng-box box" >
<H3 id="References" text="References"></H3>
<p><LoremShort></LoremShort></p>
</Collapsible>
<p>The final innovation for our LNP to become <strong>AirBuddy</strong> came through consultation with Benjamin Winkeljann from <a onClick={() => goToPagesAndOpenTab('rnhale', '/human-practices')}> RNhale</a>, when the use of spray-drying techniques was discussed. Spray-drying the LNPs, instead of using traditional methods, helps improve stability and eco-friendliness of the product<TabScrollLink tab="tab-delivery" num="38" scrollId="desc-38"/>. Our samples are set for transfer to RNhale for spray-drying, with scheduling aligned to resume promptly after the wiki freeze. Meanwhile, the discussion with <a onClick={() => goToPagesAndOpenTab('moorlach', '/human-practices')}>Benjamin Moorlach</a>, chitosan expert working at FH Bielefeld, provided new ideas for improvement by <strong>complexing the cargo with chitosan</strong> to improve the stability of the cargo during spray drying and nebulization. The positive effect of chitosan-complexing for opimized LNP delivery could be confirmed in our lab. In conclusion, we created a stable LNP for efficient delivery of RNA therapeutics to the lungs since the successful delivery of the prime editing complex via inhalation is key to our project. </p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/big-plan-inhalation-teil-del.webp"
alt1="Flow DEL"
description="Application strategy - AirBuddy is inhaled by the patient, enabling uptake of PrimeGuide RNA in lung epithelial cells via endocytosis. "
num={4}
/>
</p>
</div>
<div className="box" >
<p id="del5">
<H3 text="Outlook" id="del5head" />
Ultimately, through continuous cycles of experimentation, feedback, and optimization, our LNP formulation called AirBuddy was designed using SORT LNPs incorporating chitosan-complexation of the cargo and processing via spray-drying, achieving lung-specificity and improved stability suited for inhalation strategies. We also want to state that for our LNP is further room for improvement. Intensive research led us to the realization that, among other modifications, <strong>antibody conjugation</strong> as a surface modification of our LNP for cell type-specific administration, more specifically club cells<TabScrollLink tab="tab-delivery" num="39" scrollId="desc-39"/> and ionocytes<TabScrollLink tab="tab-delivery" num="40" scrollId="desc-40"/> as most CFTR-expressing lung epithelial cells, would round off our most important aspect of precision.
</p>
</div>
<Section title="References" id="references">
<EngDelsources/>
</Section>
<br/>
<div className="row ">
<div className="row">
<div className="col">
<div className="left"><ButtonOneEngineering label="Previous" open="delivery" scrollToId="Delivery"/></div>
<div className="left"><ButtonOneEngineering label="Previous" open="nickase" scrollToId="nickase-header"/></div>
</div>
<div className="col button-left">
</div>
</div>
</section>
......@@ -411,7 +1197,7 @@ export function EngineeringCycleTab(){
style={{strokeWidth:"15",strokeDasharray:"none",stroke:"#850f78",strokeOpacity:"1"}} />
<text
style={{fontSize:"17.3333px",lineHeight:"0",fontFamily:"Arial",fill:"#000000",fillOpacity:"1",stroke:"none",strokeWidth:"15",strokeLinecap:"round",strokeLinejoin:"round",strokeDasharray:"none",strokeOpacity:"1",paintOrder:"fill markers stroke"}}
style={{fontSize:"17.3333px",lineHeight:"0",fontFamily:"Arial",fill:"var(--offblack)",fillOpacity:"1",stroke:"none",strokeWidth:"15",strokeLinecap:"round",strokeLinejoin:"round",strokeDasharray:"none",strokeOpacity:"1",paintOrder:"fill markers stroke"}}
id="text31"
transform="translate(5.6902194,-0.11551883)"><textPath
xlinkHref="#path25"
......@@ -438,7 +1224,7 @@ export function EngineeringCycleTab(){
style={{stroke:"#a0a7f3",strokeWidth:"15",strokeDasharray:"none",strokeOpacity:"1"}} />
<text
style={{fontSize:"17.3333px",lineHeight:"0",fontFamily:"Arial",fill:"#000000",fillOpacity:"1",stroke:"none",strokeWidth:"6",strokeLinecap:"round",strokeLinejoin:"round",strokeDasharray:"none",strokeOpacity:"1",paintOrder:"fill markers stroke"}}
style={{fontSize:"17.3333px",lineHeight:"0",fontFamily:"Arial",fill:"var(--offblack)",fillOpacity:"1",stroke:"none",strokeWidth:"6",strokeLinecap:"round",strokeLinejoin:"round",strokeDasharray:"none",strokeOpacity:"1",paintOrder:"fill markers stroke"}}
id="text28"
transform="translate(-0.03023506,-5.9602336)"><textPath
......@@ -463,7 +1249,7 @@ export function EngineeringCycleTab(){
style={{strokeWidth:"15",strokeDasharray:"none",stroke:"#f57d22",strokeOpacity:"1"}} />
<text
style={{fontSize:"17.3333px",lineHeight:"0",fontFamily:"Arial",fill:"#000000",fillOpacity:"1",stroke:"none",strokeWidth:"15",strokeLinecap:"round",strokeLinejoin:"round",strokeDasharray:"none",strokeOpacity:"1",paintOrder:"fill markers stroke"}}
style={{fontSize:"17.3333px",lineHeight:"0",fontFamily:"Arial",fill:"var(--offblack)",fillOpacity:"1",stroke:"none",strokeWidth:"15",strokeLinecap:"round",strokeLinejoin:"round",strokeDasharray:"none",strokeOpacity:"1",paintOrder:"fill markers stroke"}}
id="text32"
transform="translate(-5.7110315,1.7453243)"><textPath
xlinkHref="#path24"
......@@ -479,7 +1265,7 @@ export function EngineeringCycleTab(){
<a typeof="button" className="svg-button" onClick={openElement({elementToOpen: "designing", classToHide: "cycletab"})}>
<text
style={{fontSize:"17.3333px",lineHeight:"0",fontFamily:"Arial",fill:"#000000",fillOpacity:"1",stroke:"none",strokeWidth:"15",strokeLinecap:"round",strokeLinejoin:"round",strokeDasharray:"none",strokeOpacity:"1",paintOrder:"fill markers stroke"}}
style={{fontSize:"17.3333px",lineHeight:"0",fontFamily:"Arial",fill:"var(--offblack)",fillOpacity:"1",stroke:"none",strokeWidth:"15",strokeLinecap:"round",strokeLinejoin:"round",strokeDasharray:"none",strokeOpacity:"1",paintOrder:"fill markers stroke"}}
id="text29"
transform="translate(8.4052921,8.8553334)"><textPath
xlinkHref="#path22"
......@@ -495,7 +1281,7 @@ export function EngineeringCycleTab(){
cy="63.214005"
r="20" />
<text
style={{fontSize:"8px",lineHeight:"0",fontFamily:"Arial",opacity:"0.85",fill:"#000000",fillOpacity:"1",strokeWidth:"15",strokeLinecap:"round",strokeLinejoin:"round",strokeDasharray:"none",paintOrder:"fill markers stroke"}}
style={{fontSize:"8px",lineHeight:"0",fontFamily:"Arial",opacity:"0.85",fill:"var(--offblack)",fillOpacity:"1",strokeWidth:"15",strokeLinecap:"round",strokeLinejoin:"round",strokeDasharray:"none",paintOrder:"fill markers stroke"}}
x="50.929825"
y="66.676674"
id="text1">
......
src/contents/example.css
View file @ 3fd292fd
/* :root {
our colours
--text-primary: #850F78;
--mediumpurple: #bc15aa;
--purple: #B85BD1;
--accen-secondary: #F57D22;
--accent-primary: #F4CC1E;
--lightyellow: #fae99e;
--lightblue: #A0A7F3 ;
--verylightblue: #ebecfd;
--offblack: #32232C ;
--cebitecgray: #8295A4;
--offwhite: #e9dff1;
--ourbeige: #FFF6F2;
--darkerbeige: #e2dad7;
--background: #FFF6F2;
igem colours
--igemdarkgreen: #006530;
--igemmediumgreen: #019968;
--igemlightgreen: #99cb9a;
--info-border-color: var(--mediumpurple);
--vp-ct: var(--text-primary);
--info-border-color: var(--accent-primary);
--info-bg-color: var(--lightyellow);
--info-title-color: var(--text-primary);
--info-code-bg-color: var(--lightyellow);
--note-border-color: var(--text-primary);
--note-bg-color: var(--darkoffwhite);
--note-title-color: var(--text-primary);
--note-code-bg-color: var(--darkoffwhite);
--tip-border-color: var(--text-primary);
--tip-bg-color: var(--darkoffwhite);
--tip-title-color: var(--text-primary);
--tip-code-bg-color: var(--darkoffwhite);
--warning-border-color: var(--accen-secondary);
--warning-bg-color: var(--lightorange);
--warning-title-color: var(--text-primary);
--warning-code-bg-color: var(--lightorange);
} */
.example-docu{
background-color: var(--igemlightgreen);
width: fit-content;
......@@ -60,7 +19,7 @@
}
.st196{fill:none;stroke:black;stroke-miterlimit:10;}
.st196{fill:none;stroke:var(--offblack);stroke-miterlimit:10;}
.st455{font-size:auto;}
/* .{
......
src/contents/example.tsx
View file @ 3fd292fd
......@@ -10,7 +10,7 @@ import React from 'react';
import { Bar, Doughnut, PolarArea } from 'react-chartjs-2';
import { Chart as ChartJS, Tooltip, Legend, BarElement, CategoryScale, LinearScale, Title, RadialLinearScale } from 'chart.js';
/* import ProteinViewer from '../components/Fanzorviewer.tsx'; */
import { useTabNavigation } from "../utils/TabNavigation.tsx";
import { useTabNavigation } from "../utils/TabNavigation";
ChartJS.register(
......@@ -168,16 +168,16 @@ export function Example() {
<p> Add a dummy sponsor to this slider.</p>
<SimpleSlider>
<a className="sponsor-container" href="https://bts-ev.de/">
<img className="img-sponsor" src="https://static.igem.wiki/teams/5247/sponsors/bts.png"/>
<img className="img-sponsor side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/bts.png"/>
</a>
<a className="sponsor-container" href="https://www.uni-bielefeld.de/fakultaeten/technische-fakultaet/arbeitsgruppen/multiscale-bioengineering/campusbrauerei/">
<img className="img-sponsor" src="https://static.igem.wiki/teams/5247/sponsors/campus-brauerei-hinterlegt.jpeg"/>
<img className="img-sponsor side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/campus-brauerei-hinterlegt.jpeg"/>
</a>
<a className="sponsor-container" href="www.idtdna.com">
<img className="img-sponsor" src="https://static.igem.wiki/teams/5247/sponsors/idt-logo.png"></img>
<img className="img-sponsor side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/idt-logo.png"></img>
</a>
<a className="sponsor-container" href="https://www.cebitec.uni-bielefeld.de/">
<img className="img-sponsor" src="https://static.igem.wiki/teams/5247/sponsors/cebitec-farbe.png"/>
<img className="img-sponsor side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/cebitec-farbe.png"/>
</a>
</SimpleSlider>
</div>
......
src/contents/experiments.tsx deleted 100644 → 0
View file @ 09dfd67d
import { H2 } from "../components/Headings";
import { useTabNavigation } from "../utils/TabNavigation";
export function Experiments() {
useTabNavigation();
return (
<>
<H2 id="heading" text="Heading" ></H2>
<div className="row mt-4">
<div className="col-lg-8">
</div>
</div>
</>
);
}
src/contents/igem-bielefeld.tsx
View file @ 3fd292fd
import { BackUp } from "../components/Buttons";
import { LoremMedium } from "../components/Loremipsum";
import { PDF } from "../components/Pdfs";
import { BlockQuoteB } from "../components/Quotes";
import { Section, Subesction } from "../components/sections";
import { useTabNavigation } from "../utils/TabNavigation";
......@@ -8,17 +8,23 @@ export function igemBielefeld() {
useTabNavigation();
return (
<>
<Section title="Bielefeld University" id="Bielefeld University">
...
<img src="https://static.igem.wiki/teams/5247/photos/university/bielefeld-3381870.jpg"/>
<Section title="Bielefeld University" id="Bielefeld University">
<img src="https://static.igem.wiki/teams/5247/photos/university/bielefeld-3381870.jpg"/>
<p>
Bielefeld University, established in 1969, is distinguished by its interdisciplinary approach to research and teaching at the highest academic level. The university is constituted of 14 faculties, which encompass a comprehensive range of subject areas, including the humanities, natural and technical sciences, social sciences, and education. In the forthcoming years, the recently established faculty of medicine will serve to further expand the university's interdisciplinary research opportunities. With an enrollment of approximately 25,000 students, including 2,000 international students, Bielefeld University offers 123 different degree programs. The campus is currently undergoing a significant expansion, with the objective of becoming one of the most modern in Europe. The new facilities will offer students an exceptional environment. Bielefeld University actively collaborates with over 300 partner institutions worldwide, fostering global academic exchange. Through its Erasmus+ program, the university enables students and faculties to engage in research and study abroad programs across Europe, Asia, America and beyond. While Bielefeld has traditionally been known for its strong sociology department, recent advances in robotics and biotechnology have brought these fields to the forefront of the university's cutting-edge research. As a result, the Centre for Biotechnology (CeBiTec) has become one of the university's most important institutes.
</p>
<img src="https://static.igem.wiki/teams/5247/photos/university/img-1989.jpeg"/>
<p>
The Centre for Biotechnology (CeBiTec) constitutes a central institution at Bielefeld University, dedicated to interdisciplinary research in the field of life sciences. It is one of the largest research facilities on campus, facilitating innovative projects that span several disciplines. The research areas of focus include large-scale genomics, big data bioinformatics and metabolic engineering of single-cell systems for bioproduction. CeBiTec unites research from a multitude of disciplines, including biotechnology, molecular biology, genome research, systems biology, biochemistry, bioinformatics, and computer science. Additionally, it plays a significant role in academic training and doctoral programmes, as well as serving as a hub for biotechnological initiatives. CeBiTec is situated within the state-of-the-art Laboratory Building G on the campus of Bielefeld University, which opened in 2007. Through ist focus on Cross-disciplinary collaboration, CeBiTec serves as a viral link between academic innovation and real-world applications, shaping the future of biotechnology at Bielefeld University and beyond.
</p>
</Section>
<Section title="History" id="History">
<div className="row">
<div className="col">
<img src="https://static.igem.wiki/teams/5247/sponsors/uni-bielefeld-dunkel.png" style={{width:"40%", height:"60%"}}/>
<img src="https://static.igem.wiki/teams/5247/sponsors/uni-bielefeld-dunkel.png" style={{ height:"100px"}}/>
</div>
<div className="col">
<img src="https://static.igem.wiki/teams/5247/sponsors/cebitec-logo-hinterlegt.png" style={{width:"20%", height:"50%", transform: "scale(1.5)"}}/>
<img src="https://static.igem.wiki/teams/5247/sponsors/cebitec-logo-hinterlegt.png" style={{height:"100px"}}/>
</div>
</div>
<br/>
......@@ -33,20 +39,44 @@ export function igemBielefeld() {
</Section>
<Section title="Steering Committee" id="Steering Committee">
<Subesction title="What is a Steering Committee" id="Steering Committee1">
<LoremMedium/>
</Subesction>
<Subesction title="Jörn" id="Steering Committee2">
<LoremMedium/>
</Subesction>
<BlockQuoteB text="iGEM is the biggest opportunity for young researchers to cross their own boundaries." cite="Prof. Dr. Jörn Kalinowski, Principle Investigator of iGEM Bielefeld since 2010"/>
<Subesction title="What is a Steering Committee?" id="Steering Committee1">
<p>The Steering Committee plays a central role in the resumption and further development of iGEM activities at Bielefeld University. After a pause in 2022 due to financial constraints and changes in participation conditions, the Steering Committee was established to ensure Bielefeld's sustainable participation in future iGEM competitions.</p>
<p>
<div className="row">
<div className="col">
<img src="https://static.igem.wiki/teams/5247/pdfs/steering-commitee.webp" />
</div>
<div className="col">
<p>The Steering Committee consists of five renowned scientists from the Faculties of Biology and Technology: Dr. Petra Lutter, Prof. Dr. Jörn Kalinowski, Prof. Dr. Kristian Müller, Prof. Dr. Karsten Niehaus, and Prof. Dr. Jens Stoye. Each of these experts brings specific expertise crucial for the successful execution of iGEM projects. Petra Lutter has contributed to modeling in previous iGEM projects, while Jörn Kalinowski has been significantly involved in all past iGEM projects. Kristian Müller has been an experienced supporter of the iGEM competitions since their inception, and Karsten Niehaus, former head of the "Genome-based Systems Biology" master’s program, brings extensive knowledge of the scientific foundations of the projects. Jens Stoye, representing bioinformatics at Bielefeld University, contributes his expertise in this area. </p>
</div>
</div>
</p>
<p>The main goal of the Steering Committee is to ensure the successful implementation of future iGEM projects. This includes not only academic support but also organizational leadership, securing funding, and providing the necessary infrastructure. The experts in the Steering Committee are significantly involved in the strategic direction of the projects and offer a platform where ideas, resources, and knowledge are pooled to continue Bielefeld's tradition of successful iGEM participation. </p>
<p>
<div style={{marginBottom: "1rem"}}><PDF link="https://static.igem.wiki/teams/5247/pdfs/igem-broschure.pdf" name="igem-broschure.pdf" /></div>
</p>
<p>
<div className="row">
<div className="col">
<img src="https://static.igem.wiki/teams/5247/pdfs/steering-committee-1.webp"/>
</div>
<div className="col">
<p>In helping the new iGEM Bielefeld team advance their project, the Steering Committee played an indispensable role, particularly by promoting the iGEM principle of "Contribution" and fostering the interdisciplinary nature of the project. The committee emphasized the importance of creating tools, data, and methods that can benefit the global iGEM community. This mindset was reflected in the team’s project design, ensuring that their work not only met local goals but also provided meaningful contributions to future projects. </p>
<p>Moreover, the interdisciplinary nature of the iGEM project was strongly encouraged by the Steering Committee. With members from various scientific fields, the committee facilitated collaboration between disciplines such as biology, bioinformatics, and biotechnology. This interdisciplinary approach allowed the team to tackle complex challenges from multiple perspectives, integrating computational models with experimental biology to drive innovation. This guidance helped the iGEM Bielefeld team develop a more robust and impactful project, aligning with both the scientific goals of the competition and the collaborative spirit of the iGEM community. </p>
</div>
</div>
</p>
</Subesction>
</Section>
<Section title="Our Future" id="Future">
<LoremMedium/>
</Section>
<BackUp/>
</>
);
}
src/contents/impressum.tsx
View file @ 3fd292fd
......@@ -22,7 +22,7 @@ export function Impressum() {
33615 Bielefeld<br />
<br />
<b>Contact</b><br />
E-mail: team2024@igem-bielefeld.de<br />
E-mail: team2024[at]igem-bielefeld[dot]de<br />
<br />
<b>Supervisory Authority</b><br />
Bielefeld University - Center for Biotechnology (CeBiTec)
......
src/contents/index.tsx
View file @ 3fd292fd
......@@ -9,7 +9,6 @@ export * from "./Contribution/contribution.tsx";
export * from "./description.tsx";
export * from "../sidebars/descS.tsx"
export * from "./engineering.tsx";
export * from "./experiments.tsx";
export * from "./notebook.tsx";
export * from "./results.tsx";
// Safety
......@@ -26,15 +25,12 @@ export * from "../headers/attribution-h.tsx"
export * from "../headers/cont-h.tsx"
export * from "../headers/desc-h.tsx"
export * from "../headers/home-h.tsx"
export * from "../headers/exp-h.tsx"
export * from "../headers/hp-h.tsx"
export * from "../headers/imp-h.tsx"
export * from "../headers/note-h.tsx"
export * from "../headers/res-h.tsx"
export * from "../headers/safe-h.tsx"
export * from "../headers/team-h.tsx"
export * from "../headers/wiki-h.tsx"
export * from "../headers/ints-h.tsx"
export * from "../headers/spons-h.tsx"
export * from "../headers/eng-h.tsx"
export * from "../headers/sup-h.tsx"
......
src/contents/interviews.tsx
View file @ 3fd292fd
......@@ -111,11 +111,11 @@ export function Ints() {
<img className="interview-img" src="https://static.igem.wiki/teams/5247/photos/hp/mattijs.jpg"/>
</div>
<div className="col">
<div className="col">
<div className="col ">
<ButtonOne text="Erstes Interview" open="mattijsinv1"></ButtonOne>
</div>
<br/>
<div className="col">
<div className="col ">
<ButtonOne text="Zweites Interview" open="mattijsinv2"></ButtonOne>
</div>
</div>
......@@ -174,7 +174,7 @@ export function Ints() {
/>
<QaBox
q="How many patients do you treat?"
a="We currently have 8 children with cystic fibrosis in our medical practice, which is quite a lot. However, if you compare this number with other diseases, it is rather a small number. We have slightly more children with cystic fibrosis in our practice because we specialize in it, among other diseases."
a="We currently have 8 children with Cystic Fibrosis in our medical practice, which is quite a lot. However, if you compare this number with other diseases, it is rather a small number. We have slightly more children with Cystic Fibrosis in our practice because we specialize in it, among other diseases."
/>
<SpecialQaBox
q="What kind of exercises do you do?">
......@@ -226,8 +226,8 @@ export function Ints() {
a="Pancreatic complaints are rarely treated with physiotherapy, unless it is an inflammation. In such cases, the patient is admitted to a hospital. Massage or taping the intestines with kinesiology tape helps with constipation and works very well. "
/>
<QaBox
q="Are there any special hygiene guidelines for you when working with cystic fibrosis patients? "
a="Hygiene guidelines are very important when working with cystic fibrosis patients. A distinction is made between children with and without infections (Pseudomonas). Regular nasal swabs are taken and only children with or without infections are treated in the practice on any given day. Ventilation, patients wearing masks while infected and disinfection of the facilities are essential. Children infected with multi-resistant germs are not allowed to enter the practice; in such cases, physiotherapists visit the patients' homes. "
q="Are there any special hygiene guidelines for you when working with Cystic Fibrosis patients? "
a="Hygiene guidelines are very important when working with Cystic Fibrosis patients. A distinction is made between children with and without infections (Pseudomonas). Regular nasal swabs are taken and only children with or without infections are treated in the practice on any given day. Ventilation, patients wearing masks while infected and disinfection of the facilities are essential. Children infected with multi-resistant germs are not allowed to enter the practice; in such cases, physiotherapists visit the patients' homes. "
/>
<QaBox
q="Are the specific exercises customized? And if so, how do you know which therapy is the right one for which patient (based on laboratory values, tests, different mutation patterns...)? "
......@@ -239,7 +239,7 @@ export function Ints() {
/>
<QaBox
q="How many physiotherapists offer muco-therapy? "
a="The exact number of physiotherapists offering cystic fibrosis therapy is unknown. However, there are several child therapists in the region providing this therapy. "
a="The exact number of physiotherapists offering Cystic Fibrosis therapy is unknown. However, there are several child therapists in the region providing this therapy. "
/>
<QaBox
q="How are the relatives educated? "
......
src/contents/judging.tsx
View file @ 3fd292fd
import { LoremMedium } from "../components/Loremipsum";
import { H3, H4 } from "../components/Headings";
import { BlockQuoteB } from "../components/Quotes";
import { Section } from "../components/sections";
import { useNavigation } from "../utils";
import { useTabNavigation } from "../utils/TabNavigation";
export function Judging() {
useTabNavigation();
const {goToPagesAndOpenTab} = useNavigation();
return (
<>
<Section title="Overview" id="Overview">
<LoremMedium/>
<BlockQuoteB text="Judging for iGEM is one of the highlights on my professional calendar…" cite="Dr. Nancy Burgess, Director of Judging at iGEM HQ"/>
<p>The iGEM competition celebrates innovation in synthetic biology, offering teams the chance to compete for a range of awards based on their achievements in
various categories. Judging is based on how well teams integrate scientific rigor with societal impact, safety, and creativity. We aim to compete for
several prestigious awards, including <b>Best Integrated Human Practices</b>, <b>Safety and Security Award</b>, and <b>Best New Basic Part</b>. Additionally, we seek recognition for our <b>therapeutic project</b> and will strive for excellence in <b>Best Wiki</b>, <b>Best Presentation</b>, and the <b>iGEMers prize</b>. </p>
<div className="row align-items-center">
<div className="col">
<img src="https://static.igem.wiki/teams/5247/photos/other/therapeutic-award.svg" />
</div>
<div className="col">
<img src="https://static.igem.wiki/teams/5247/photos/other/ihp-award.svg" />
</div>
<div className="col">
<img src="https://static.igem.wiki/teams/5247/photos/other/safety-award.svg" />
</div>
<div className="col">
<img src="https://static.igem.wiki/teams/5247/photos/other/basic-part-award.svg" />
</div>
<div className="col">
<img src="https://static.igem.wiki/teams/5247/photos/other/wiki-award.svg" />
</div>
</div>
</Section>
<Section title="Best New Part" id="Best New Part">
<LoremMedium/>
</Section>
<Section title="Safety and Security" id="Safety and Security">
<LoremMedium/>
<div className="row align-items-center">
<div className="col">
<iframe title="Bielefeld-CeBiTec: Next-Generation Prime Editing as Cystic Fibrosis Gene Therapy (2024) - Team Presentation [English]" width="560" height="315" src="https://video.igem.org/videos/embed/479e7f99-6931-47bc-9193-d4367beba4f2" frameBorder="0" allowFullScreen sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
</div>
</div>
<Section title="Best Therapeutic Project" id="Best Therapeutic Project">
<p>Our project is a dual therapeutic approach targeting Cystic Fibrosis (CF), specifically the most common mutation, F508del. We aim to develop a curative solution by correcting the genetic defect using Prime Editing Technology while enhancing the folding of the CFTR protein. Additionally, we developed a Lipid Nanoparticle for the cell-specific targeting of lung epithelial cells. This project directly addresses the unmet therapeutic needs of CF patients, providing a long-term and potentially curative solution. </p>
<h4>Has the team clearly defined the therapeutic problem they are addressing? </h4>
<p>We are addressing Cystic Fibrosis (CF), a severe genetic disorder caused by mutations in the CFTR gene, particularly the F508del mutation. This mutation leads to thick mucus accumulation in vital organs, causing chronic infections and damage, particularly in the lungs. Our focus on the F508del mutation, which affects around 90 % of CF patients in Europe and beyond, ensures our project targets a well-defined and widespread therapeutic need. </p>
<h4>How well is the therapeutic mechanism of action understood and demonstrated? </h4>
<p>Our project employs a dual approach: </p>
<ol>
<li>orrecting the F508del mutation using our Prime Editing Technology PrimeGuide, a well understood gene editing tool designed to repair defective genes</li>
<li>delivering of our mRNA therapeutic via specialized Lipid Nanoparticles AirBuddy, ensuring direct application in lung epithelial cells</li>
</ol>
<h4>Has the team validated the effectiveness of their therapeutic approach? </h4>
<p>We tested the therapeutic efficacy <i>in vitro</i> using patient-derived primary epithelial cells in Air Liquid Culture (ALI) and Apical-Out Organoids carrying the F508del mutation. These cells were treated with PreCyse to assess the genetic correction, while downstream applications will validate the improved folding and function of the CFTR protein. Our experimental design allows us to gather proof-of-concept data, demonstrating the viability of both gene correction and efficient delivery. </p>
<h4>Does the project consider off-target effects or unintended consequences?</h4>
<p>By incorporating FANZOR, a new eukaryotic RNA-binding DNA-endonucleases, we enhance the precision of Prime Editing. Site-directed mutagenesis enable nickasification of our CasX and FANZOR candidates. This enables the usage as Prime Editor, while enhancing efficiency and decreasing PE complex size. We have designed our system to minimize unintended consequences, ensuring that our therapeutic intervention is safe for clinical application. </p>
<h4>What potential does the project have for real-world therapeutic applications? </h4>
<p>Our dual approach has significant potential for real-world therapeutic applications. PreCyse provides a curative genetic solution, improve immediate physiological functions. The project addresses a crucial need in CF therapy by targeting both the root genetic cause and the resulting protein dysfunction, offering a more comprehensive solution than current treatments. </p>
<h4>How well did the team integrate safety into their therapeutic design? </h4>
<p>Safety is a priority in our project. The use of FANZOR enhances the specificity of our gene-editing approach, reducing the likelihood of off-target gene modifications. Additionally, we are committed to rigorous <i>in vitro</i> testing before advancing to <i>in vivo</i> models, ensuring that our therapeutic interventions do not pose risks to patients. For this, we developed an CF-specific fluorescence-based Reporter System mimicking the CF-specific genomic alteration. </p>
<h4>To what extent has the team considered patient impact? </h4>
<p>We have closely aligned our project with patient needs by targeting the F508del mutation, which affects a significant portion of CF patients worldwide. Our therapeutic solution aims to improve quality of life by addressing the root cause of CF and reducing the burden of lifelong treatments. We are also in contact with CF patients and have gathered insights from healthcare providers to ensure our therapy is patient-centric. PreCyse offers a long-time solution, while being cost-efficient and easy-to-use. </p>
<p>Our innovative therapeutic project leverages the power of gene editing and protein modulation to offer a potentially curative solution for Cystic Fibrosis, particularly for the prevalent F508del mutation. With a well-validated scientific foundation, comprehensive safety measures, and a strong focus on patient needs, we believe our project exemplifies the qualities deserving of the <b>Best Therapeutic Project Award</b>. Our dual approach provides a template for future genetic disease treatments, paving the way for more effective and personalized medical solutions. </p>
</Section>
<Section title="Best Integrated Human Practice" id="Best Integrated Human Practice">
<LoremMedium/>
<p>Our team’s project at Bielefeld-CeBiTec integrates Human Practices into every aspect of our work, ensuring that our synthetic biology innovation aligns with societal needs, ethical considerations, and environmental sustainability. By proactively engaging with a broad range of stakeholders, including scientists, regulatory bodies, and community groups, we shaped our project to have meaningful and responsible impacts. Our commitment to understanding the broader implications of our work positions us as a strong candidate for the <b>Best Integrated Human Practices Award</b>. </p>
<h4>Integration of Human Practices </h4>
<p>We actively engaged with a diverse array of stakeholders throughout our project, from CF patients like <a onClick={() => goToPagesAndOpenTab('maxfirst', '/human-practices')}>Max Beckmann</a> to clinicians such as <a onClick={() => goToPagesAndOpenTab('olariu', '/human-practices')}>Dr. Olariu</a> , physiotherapists like <a onClick={() => goToPagesAndOpenTab('westhoffinv', '/human-practices')}>Katrin Westhoff</a>, and industry experts including <a onClick={() => goToPagesAndOpenTab('rnhale', '/human-practices')}>Dr. Benjamin Winkeljann</a> and <a onClick={() => goToPagesAndOpenTab('kolonkofirst', '/human-practices')}>Dr. Katharina Kolonko</a>. Each of these collaborations directly informed and shaped our therapeutic solution. Max Beckmann’s firsthand experience provided us with invaluable insights into the daily challenges of living with CF, helping us align our gene therapy with patient needs. Additionally, clinicians and physiotherapists guided us toward a treatment strategy that is practical, accessible, and effective for different patient demographics. </p>
<h4>Inspiration to Others </h4>
<p>Our project serves as a model for how synthetic biology can be leveraged to meet real-world needs while maintaining a patient-centered focus. By integrating feedback from patients and medical professionals, we demonstrate that cutting-edge science can coexist with empathy and responsibility. Our focus on inclusivity, scalability, and addressing global disparities in CF treatment sets a precedent for future iGEM teams looking to make a meaningful impact on health challenges. </p>
<h4>Documentation for Future Teams </h4>
<p>We have meticulously documented every aspect of our Human Practices integration, providing future teams with a clear roadmap for how to incorporate stakeholder feedback into a therapeutic project. This includes detailed case studies on our interactions with CF patients, medical professionals, and industry experts, along with the adjustments made to our project based on their input. Our thorough documentation ensures that others can learn from our approach and build upon our findings. </p>
<h4>Thoughtful Implementation </h4>
<p>Our project has been deeply informed by ethical, environmental, and societal considerations. For example, our consultation with Prof. Dr. Olariu emphasized the importance of mental health in CF care, leading us to incorporate psychosocial elements into our therapy design. Additionally, we aligned our solution with global healthcare disparities, ensuring our gene therapy can benefit underrepresented populations. Through engagement with regulatory bodies, we ensured that our project complies with all relevant biosafety and legal standards. </p>
<h4>Incorporation of Diverse Stakeholder Views </h4>
<p>We engaged with a broad range of stakeholders, including patients, healthcare providers, researchers, and regulatory experts, ensuring that each perspective played a role in shaping the final therapeutic solution. For instance, feedback from physiotherapist Katrin Westhoff confirmed the necessity of an inhalation-based therapy that is easy for younger patients to use. Collaborations with industry experts like Dr. Benjamin Winkeljann enabled us to optimize the technical aspects of our treatment for scalability and environmental sustainability. </p>
<h4>Creating a Responsible and Beneficial Project </h4>
<p>Our gene therapy for CF addresses not only the scientific challenges but also the societal need for accessible, patient-centered healthcare solutions. By incorporating human practices at every stage, we ensured that our project is ethically responsible and beneficial to society. The feedback from diverse stakeholders helped us refine our approach, ensuring that the solution is sustainable, inclusive, and addresses the global disparities in CF care. The long-term positive impact on the CF community—both in terms of health outcomes and accessibility—demonstrates our project’s commitment to responsible innovation. </p>
<p>Our project exemplifies the seamless integration of Human Practices into every aspect of synthetic biology research. Through collaborations with patients, healthcare professionals, and experts across different fields, we have developed a gene therapy for Cystic Fibrosis that is scientifically innovative, ethically sound, and deeply aligned with the needs of the global CF community. Our commitment to inclusivity, ethical reflection, and environmental sustainability makes us a strong candidate for the <b>Best Integrated Human Practices Award</b>, showcasing our dedication to responsible and impactful synthetic biology. </p>
</Section>
<Section title="Safety & Security" id="Safety & Security">
<p>Our project focuses on advancing biosafety and biosecurity in synthetic biology through the development and implementation of robust safety mechanisms. As part of our PreCyse project, aimed at developing a prime-editing complex to correct the F508del mutation in Cystic Fibrosis (CF), we place great emphasis on safety at every stage of research. With a commitment to responsible innovation, we have ensured that all phases of our work adhere to the highest safety standards, aiming to minimize both environmental and human health risks. Our approach incorporates novel containment systems, rigorous validation processes, and carefully planned checkpoints during experiments to push the boundaries of biosafety in synthetic biology. </p>
<h4>Contribution to Biosafety and Biosecurity </h4>
<p>We developed a comprehensive biosafety plan to minimize risks associated with our synthetic biology project. This includes biocontainment measures and protocols to prevent the accidental release of genetically modified organisms (GMOs) into the environment. By integrating both physical and genetic safeguards, we have ensured that our project contributes to safer synthetic biology applications. For example, our final construct will be tested in primary cultures of nasal epithelial cells from CF patients and healthy individuals, with carefully planned checkpoints for continuous monitoring and timely adjustments. </p>
<p>As part of our commitment to advancing safe and ethical practices in synthetic biology and patient care, we have made several key contributions to support our project and the wider community. Firstly, we developed a <b>questionnaire to evaluate the medical history of Cystic Fibrosis (CF) patients</b>, which has been specifically adapted for the collection of primary human nasal epithelial cells (hNECs). This tool ensures that essential health information is gathered in a systematic and ethical manner, aiding the accurate collection of samples while safeguarding patient well-being. </p>
<p>Secondly, we created <b>Best Practices for safe primary culture handling</b>. This guide outlines the necessary safety protocols for working with primary cell cultures, ensuring that all procedures are conducted with minimal risk of contamination and exposure to harmful pathogens. These practices promote safety for both laboratory personnel and the integrity of the biological materials.</p>
<p>Additionally, we developed a <b>Hygiene Concept for Immunocompromised Individuals</b> in consultation with a CF patient. This concept addresses the specific needs of individuals with weakened immune systems, such as those with CF, HIV, or certain cancers. It focuses on reducing health risks in high-foot-traffic public spaces, especially public restrooms, by implementing tailored hygiene measures. These guidelines aim to protect immunocompromised individuals by creating safer environments within university settings and beyond, contributing to broader public health initiatives. </p>
<h4>Characterization and Validation </h4>
<p>We rigorously tested the safety mechanisms built into our project like the disruption of the PAM sequence and pegRNA design. These mechanisms were validated through controlled experiments, ensuring reliable performance under various conditions. Furthermore, to ensure the safety and precision of our results, we introduced a series of carefully planned experimental milestones. These allow for the continuous validation of the Prime Editing complex, addressing potential issues immediately, which minimizes risk and improves the overall quality of our work. </p>
<h4>Building on Existing Knowledge </h4>
<p>Our work builds upon established biosafety frameworks, specifically improving known genetic containment systems. We incorporated lessons from previous iGEM teams and academic research to refine these tools, making them more effective in controlling gene flow and reducing unintended consequences. By drawing from the existing knowledge base, we ensured our biosafety mechanisms, such as riboswitch and PAM disruption, are both scalable and reliable, significantly advancing biosafety technologies. </p>
<h4>Risk Management </h4>
<p>From the outset, we conducted a thorough risk assessment to identify potential hazards, including those associated with handling GMOs and gene transfer. In response, we implemented stringent lab protocols and biocontainment systems to ensure the safety of our team and the surrounding environment. This risk management approach extends throughout the lifecycle of the project, from the design to the final validation stages. We also adhered to good laboratory practices, such as sterilization and controlled access, which ensures compliance with all relevant biosafety regulations. </p>
<h4>Real-World Applications </h4>
<p>We designed our project with real-world biosafety concerns in mind, particularly the potential environmental impact of GMOs. Our approach ensures that our technology can be safely applied outside the lab, with biocontainment strategies that prevent unintended release. For instance, by designing lipid nanoparticles (LNPs) that selectively fuse with lung epithelial cells, we reduce the risk of unwanted off-target interactions. Additionally, we considered dual-use concerns, ensuring our technology cannot be easily misappropriated for harmful purposes, making our project a model for responsible synthetic biology. </p>
<h4>Check-Ins and iGEM Compliance </h4>
<p>In alignment with iGEM’s emphasis on biosafety, we submitted Check-Ins for the components and organisms not covered by the iGEM White list. These formal evaluations ensured that all aspects of our project were thoroughly assessed and approved by the iGEM Safety Committee. We maintained active communication with the committee to ensure compliance with all iGEM standards, reflecting our dedication to biosafety and biosecurity. </p>
<h4>Laboratory and Safety Practices </h4>
<p>All experiments were conducted at Bielefeld University in Prof. Dr. Kristian Müller's laboratory, following BSL-1 (and BSL-2 if needed) standard operating procedures. The team participated in mandatory safety briefings and adhered to rigorous safety measures, including regulations concerning hazardous substances, genetic engineering, and the handling of biological materials. Our lab activities were meticulously planned to minimize risk and ensure data integrity. </p>
<p>By building on existing knowledge, rigorously testing our biosafety measures, and proactively managing risks, we have developed a project that significantly contributes to the field of biosafety and biosecurity. Our comprehensive approach ensures that our technology can be safely applied in real-world scenarios, and we believe this sets a new standard for responsible synthetic biology. Our efforts, particularly in the development of the Prime Editing complex for CF and the associated biosafety protocols, merit recognition for the <b>Safety and Security Award</b>. </p>
</Section>
<Section title="Best New Basic Part" id="Best New Basic Part">
<p>Our project focuses on optimizing prime editing for the Cystic Fibrosis-causing CFTR F508del mutation, which represents one of the most significant applications for prime editing. Prime editing is a precise and safe gene editing technique, but its efficiency varies greatly depending on the genomic locus. Through our development of a context-specific Prime Editor Activity Reporter (PEAR) system, we have successfully optimized this advanced technology, paving the way for more effective genomic targeting, not just for Cystic Fibrosis but for future synthetic biology applications as well. </p>
<h4> Engineering and Testing for Contextual Precision </h4>
<p>The challenges posed by low editing efficiency and the difficulty in distinguishing successful edits from background noise led us to design a highly sensitive reporter system. After an initial unsuccessful attempt using a fluorescent reporter, we shifted to the PEAR system proved to be much more adaptable and relevant to our needs. </p>
<h4>Iterative Development </h4>
<p>In our first attempt, we explored a fluorescence-based reporter that targeted GFP. While this helped us visualize prime editing in action, we quickly realized that the distance of the mutation site from the PAM sequence in the CFTR gene meant that this system wasn’t suitable for our specific genomic target. We then moved to the PEAR system, which was more flexible and sensitive, as its editing factors aligned with those required for genomic contexts. </p>
<p>Through multiple iterations, including tests in HEK293 cells, epithelial cells, and human-derived primary cells, we refined our system. Our experiments proved the successful use of the PEAR reporter in detecting prime editing activity for CFTR F508del with minimal noise, making it ideal for detecting edits in specific genomic loci with high sensitivity. </p>
<h4>Modularity and Broader Applications </h4>
<p>Recognizing the limitations of the original PEAR plasmid, especially in terms of modification flexibility and compatibility with BioBrick standards, we created a more modular and accessible system. Our new version includes an oligonucleotide-based golden gate cloning site, enabling quick modification and broader applications to other genomic targets and prime editor variants. </p>
<p>Our updated PEAR system is now compatible with RFC[1000] standards, offering future iGEM teams a versatile tool that can be applied to a wide range of editing scenarios. This is not only a significant advancement for CFTR-related research but also for any gene editing project requiring high precision and sensitivity. </p>
<h4>Contribution to the Synthetic Biology Community </h4>
<p>We believe our part contributes significantly to the synthetic biology community by providing an optimized system that can be easily adapted to various gene editing contexts. By improving the modularity and ease of use, we are confident that this part will be a valuable tool for future teams looking to optimize their own prime editing approaches. </p>
<p>Our work has demonstrated that prime editing can be made more efficient and reliable, especially in difficult-to-edit regions like CFTR. Through rigorous testing and validation, we ensured that our part meets high standards for both experimental reliability and community utility. As a result, we position ourselves as strong candidates for the <b>Best New Basic Part Award</b>.</p>
</Section>
<Section title="Conclusion" id="Conclusion">
<p>In conclusion, our project exemplifies the best of synthetic biology, combining cutting-edge science with ethical responsibility and a deep
commitment to societal impact. By integrating human practices into every stage of our work, ensuring the highest standards of biosafety, and contributing
valuable tools to the community, we have set a new standard for innovation in the field. Our therapeutic approach for Cystic Fibrosis has the potential
to revolutionize treatment for this life-threatening condition, while our contributions to biosafety and part development will benefit the broader
synthetic biology ecosystem. We are confident that our project is deserving of recognition in multiple award categories, including <b>Best Therapeutic Project</b>, <b>Best Integrated Human Practices</b>, <b>Safety and Security Award</b>, and <b>Best New Basic Part</b>. Through our work, we hope to inspire future iGEM teams to pursue solutions that are both scientifically excellent and socially responsible. </p>
</Section>
<Section title="Judging Session" id="Judging Session">
<div className="row align-items-center">
<div className="col">
<iframe title="Bielefeld-CeBiTec: Judging Session (2024)" width="560" height="315" src="https://video.igem.org/videos/embed/d90ef3d2-1bb5-426e-8ab7-8e644d1a22a5" frameBorder="0" allowFullScreen sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
</div>
</div>
<H3 text="Judging Feedback" id="Judging Session1"></H3>
<H4 text="Judge 1"></H4>
<div className="row feedbackbfh">
<div className="col b-lg">
<ul>
<li>
I could see engineering principles and mindsets even in the presentation video. It is a very well-engineered and designed project. Every progress started with some hypotheses. The team tested many hypotheses; many didn’t work but they were able to learn and improve.
</li>
<li>
Documentation is very detailed in general. Specifically, the project description comes with reviews of cystic fibrosis and some basic terms so that anyone with basic molecular biology knowledge would be able to understand the project. The graphics, where there are, also facilitate understanding of technology.
</li>
<li>
The human practices are amazing, significantly influencing key project decisions. They went beyond obvious issues (e.g., how health insurance might cover gene therapies, ethics associated with handling patient-derived cells, biobank), showing compelling evidence that it is responsible and good. The same can be said on the team’s safety work.
</li>
</ul>
</div>
<div className="col b-lo">
<ul>
<li>
I don’t see how the proposed new basic part BBa_K5247135 can be useful for the wider community. It is designed with a very specific purpose in mind, of which the characterization is also not done in calibrated units. Basically, one can test different pegRNAs for the F508del mutation in the CFTR gene with fluorescence of the internal positive control arbitrarily set as 100%.
</li>
<li>
I appreciate the extensive documentation of human practices. The way different feedback or research was incorporated is clear but the rationale for the stakeholders chosen is not always clear. It becomes sort of a laundry list of everything that affected the team with too many sections and subtitles.
</li>
<li>
I’m impressed by the fact that the team has access to some sophisticated technology like cryogenic electron microscopy but it’s not clear why these characterizations are necessary, i.e., what the research question is. In fact, the team would then observe various problems associated with these methods. For example, “for SEM analysis, the samples were dried and observed under vacuum, which probably have affected the structure and shape of the LNPs.” If this is the case, then why would the team do it from the beginning?
</li>
<li>
The contributions of the safety work (i.e. primary cultures, biosafety measures, innovative safety mechanisms and regulatory compliance), particularly to what extent they build on existing resources and standards, particularly those from the iGEM community, are not very clear.
</li>
</ul>
</div>
</div>
<H4 text="Judge 2"></H4>
<div className="row feedbackbfh">
<div className="col b-lg">
<p>I enjoyed browsing through the Wiki, very clear and easy to understand and consume. Commendable demonstration of human practices and good integration of feedback. The Project Presentation is professionally done, enjoyable to watch. I highly encourage the tactic of team members walking around and engaging with judges/other teams. I loved the confidence. Wiki has mobile version, which is well appreciated. </p>
</div>
<div className="col b-lo">
<p>I enjoyed the Calendar feature in Project Documentation, but the dates did not always work. There are still some Lorem Ipsum paragraphs remaining (Results, for instance). Visuals in the Registry part are too big and are not well visible.</p>
</div>
</div>
<H4 text="Judge 3"></H4>
<div className="row feedbackbfh">
<div className="col b-lg">
<p>Very well done team Bielefed-CeBiTec. I thought that your project design was very well thought out and was able to clearly see the impact on quality of life that your project would help restore in CF patients. Over the course of this past year, you have done an impressive amount of work, and I think tailoring your project to not only focus on accuracy of your guide RNA but also ensuring the best possible delivery mechanisms to ensure cell uptake was a smart move if you were to track this for market. I also appreciated just how thorough your wiki presents your work, and the amount of effort put into your human practices, engaging with not only members of academia and healthcare, but actual people living with CF. Each page sufficiently attributed and cited original ideas, and the flow from top to bottom of the page made logical sense and made my life as a judge easier. Well done overall, and best of luck as you continue working with this!</p>
</div>
<div className="col b-lo">
<p>There were a couple of notes that stood out to me regarding your wiki and promotional video that I would like to highlight for next year's team. With your individual part designs and part pages, I did not see sufficient data within the part page itself to get excited about the work and how the part can help future iGEMers. Based on your wiki, I know you did a lot of testing and have the capacity to demonstrate part success; however, remember to present your research items in a way that is mindful to someone who is outside of your experimental realm. Ask yourself the following questions: 1) What specifically was successful about my part design? 2) If quantifying data and showing that data in another means (e.g. microscopy images) do these images match? 3) What caveats and troubleshooting would be necessary to bring this part further? Another note, and it is minor, but in your human practices and promotion of the BFH European meetup, you bring attention to connections and participation by two high-profile iGEM members (Nemanja - VP, Tracy - Former Director of Judging). While it is okay to demonstrate their attendance (using videos of them, describing what they do), it feels borderline like favoritism in that they were showing up to your event, in particular with you highlighting their attendance, not necessarily what they presented on and how it impacted you and the other teams in attendance. Be mindful of this going forward.</p>
</div>
</div>
<H4 text="Judge 4"></H4>
<div className="row feedbackbfh">
<div className="col b-lg">
<p>Your project is very good; I like the approach, as we are used to seeing more invasive approaches in medicine. Your project was also very well defended; you answered the questions confidently, which caught my attention. You never hesitated. It was a lot of work, and you managed to shape it well. I hope you can continue with the project and turn it into a startup</p>
</div>
<div className="col b-lo">
<p>It would be useful to delve into the potential challenges related to the biocompatibility and degradability of chitosan nanoparticles in the human body, as ensuring long-term safety and efficacy is essential for therapies of this kind. It would also be interesting to explore methods and experiments to further improve the stability of the nanoparticles, evaluate different pH conditions, and determine which peripheral cells or tissues could be harmed or could benefit. Let's remember that the human body is not an isolated system; many of these interactions may lead to allergies, unwanted responses, or affect the organism's suitability. Cultivating peripheral tissues with the same system would also be a very intriguing approach.</p>
</div>
</div>
<H4 text="Judge 5"></H4>
<div className="row feedbackbfh">
<div className="col b-lg">
<p>You demonstrated a strong commitment to biosafety by outlining detailed safety protocols and addressing both environmental and human health risks. Your proactive approach to identifying potential hazards and implementing safety measures highlights your responsibility in managing synthetic biology risks. Great work!</p>
</div>
<div className="col b-lo">
<p>Your Materials and Methods section provides a solid foundation of your technical work, but could benefit from more detailed explanations of some experimental design choices. Additionally, including a section that discusses challenges encountered and how you have addressed it would offer valuable insights for future teams attempting to replicate or build on yourwork. This would also help illustrate the adaptability and problem-solving aspects of your project</p>
</div>
</div>
<H4 text="Judge 6"></H4>
<div className="row feedbackbfh">
<div className="col b-lg">
<p>The integration of Prime Editing and the AirBuddy delivery system showcases a cutting-edge approach to gene therapy. This not only demonstrates technical innovation but also highlights your commitment to improving treatment efficacy and precision.</p>
</div>
<div className="col b-lo">
<p>While the therapy aims for long-term correction, discussing how you plan to evaluate durability and stability of the genetic correction over time could provide a clearer understanding of the treatment’s potential benefits.</p>
</div>
</div>
</Section>
</>
......
src/contents/methods.tsx
View file @ 3fd292fd
import { Section } from "../components/sections";
import { Section, Subesction } from "../components/sections";
import { useTabNavigation } from "../utils/TabNavigation";
import {H4} from "../components/Headings";
import MethodSources from "../sources/methods-sources";
import { useNavigation } from "../utils";
import { OneFigure } from "../components/Figures";
export function Methods() {
const {goToPagesAndOpenTab} = useNavigation ();
useTabNavigation();
return (
<>
<Section title="Introduction" id="Introduction">
...
<p>This section highlights the key materials and methods pivotal to advancing our project with the primary goal to develop an efficient prime editing technology to correct the F508del mutation in the CFTR gene by the delivery to lung epithelial cells using optimized lipid nanoparticles (LNPs) via pulmonary administration. We utilized patch clamp electrophysiology to precisely measure ion channel activity, providing crucial insights into cellular function and the impact of genetic modifications on CFTR performance. Additionally, our cell culture models of lung epithelial cells allowed us to test both the delivery and efficacy of our gene-editing system under conditions that closely mimic the <i>in vivo</i> environment. To ensure that our LNPs were both effective and safe, we performed extensive LNP cytotoxicity and characterization experiments, evaluating their biocompatibility, stability, and efficiency in delivering the editing technology. Each of these methodologies was carefully selected to optimize the delivery process and maximize the therapeutic potential of our approach.</p>
</Section>
<Section title="Patch Clamp" id="Patch Clamp">
<H4 text="Patch Clamp: A Key Tool in Electrophysiology"></H4>
<p>The patch clamp technique is a highly sensitive method for measuring ionic currents through individual ion channels in cells, making it a cornerstone of electrophysiological research. Initially developed by Erwin Neher and Bert Sakmann in the 1970s [1], this technique has evolved into various configurations, including the Whole-Cell and Single-Channel recordings [2], which provide critical insights into the functional properties of ion channels. </p>
<H4 text="Principles of the patch clamp technique"></H4>
<Subesction title="Patch Clamp: A Key Tool in Electrophysiology" id="Patch Clamp1">
<p>The patch clamp technique is a highly sensitive method for measuring ionic currents through individual ion channels in cells, making it a cornerstone of electrophysiological research. Initially developed by Erwin Neher and Bert Sakmann in the 1970s [1], this technique has evolved into various configurations, including the Whole-Cell and Single-Channel recordings [2], which provide critical insights into the functional properties of ion channels. </p>
</Subesction>
<Subesction title="Principles of the Patch Clamp Technique" id="Patch Clamp2">
<p>Patch clamp recording involves the use of a glass micropipette which is manufactured from a glass capillary through the use of a Micropipette Puller. The micropipette is then filled with an electrolyte solution, which is subsequently brought into contact with the cell membrane. By applying gentle suction, a high-resistance seal called giga seal is formed between the pipette tip and the membrane patch. This enables the measurement of ionic currents with minimal noise interference [3]. <strong>Whole-Cell Configuration</strong> records currents from the entire cell by rupturing the membrane patch, accessing the intracellular environment, and is useful for analysing overall ion channel activity and cellular responses. <strong>Single-Channel Recording</strong> measures currents through individual ion channels without rupturing the membrane, enabling high-resolution study of channel conductance, gating, and selectivity [2].</p>
<div className="figure-wrapper">
<figure>
<iframe title="Bielefeld-CeBiTec: Patch Clamp Measurement (2024)" width="560" height="315" src="https://video.igem.org/videos/embed/0d948e57-5997-430a-a2df-815b71a2fc67?autoplay=1" frameBorder="0" allowFullScreen={true} sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
<figcaption> <b>Figure 1.</b> Microscopic recording of micropipette sealing of a HEK293 cell </figcaption>
</figure>
<div className="row align-items-center">
<div className="col">
<iframe title="Bielefeld-CeBiTec: Patch Clamp Measurement (2024)" width="560" height="315" src="https://video.igem.org/videos/embed/0d948e57-5997-430a-a2df-815b71a2fc67?autoplay=1" frameBorder="0" allowFullScreen={true} sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
</div>
</div>
<figcaption> <b>Figure 1. </b> Microscopic recording of micropipette sealing of a HEK293 cell. </figcaption>
</figure>
</div>
<p>The success of patch clamp experiments heavily depends on the composition of the solutions used. Typically, two main types of solutions are employed: The <strong>Pipette Solution</strong> in the micropipette mimics the intracellular environments, while the <strong>Bath Solution</strong> surrounds the cell and usually contains components that replicate the extracellular environment. Both solutions are meticulously designed to reflect the physiological conditions under which the cells operate, thereby ensuring that the measurements accurately reflect ion channel activity in a natural setting [2].</p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/for-wiki-texts/meth-patch-clamp/bild-meth-patch-clamp.png"
alt1="Patch Clamp Setup."
description="Patch Clamp setup."
num={2}
/>
</Subesction>
<Subesction title="Application in CFTR gene Prime Editing validation" id="Patch Clamp3">
<p>In our ongoing research project focusing on the treatment of Cystic Fibrosis, our patch clamp measurements, performed in collaboration with Dr. Oliver Dräger from the Cellular Neurophysiology working group at Bielefeld University, serve as a powerful validation tool for the assessment of the functional correction of the CFTR gene, particularly the common F508del mutation, via prime editing. The patch clamp technique can be employed in this context to measure the resulting chloride ion channel activity which is altered by the mutation [4]. Whole-Cell recordings were performed to assess whether the corrected CFTR channels function similarly to those in healthy cells. If the chloride ion currents in the edited cells approach levels of healthy cells, this would strongly suggest successful gene editing and validate the functionality of our therapeutic approach.</p>
</Subesction>
</Section>
<Section title="Cell Culture" id="Cell Culture">
<Subesction title="HEK293 and HEK293T cell lines" id="Cell Culture1">
<p>For testing our prime editing approach, we needed an easy-to-handle cell line with a measurable high expression of CFTR and the CFTR F508del mutation. When talking to Mattijs Bulcaen from the Laboratory of Molecular Virology and Gene Therapy at KU Leuven, he recommended to use HEK293T cell lines overexpressing CFTR they had used. HEK293 cells are a very common immortalized human cell line derived from the kidneys of a female embryo. They are particularly suited to research due to their convenient handling and transfection properties. Basic HEK293 cells were provided to us by the Cellular and Molecular Biotechnology working group at Bielefeld University led by Prof. Dr. Kristian Müller, who is also one of the Principal Investigators of our team. HEK293T cells express an additional tsA1609 allele of the SV40 large T-antigen, allowing for replication of vectors containing the SV40 origin of replication[5]. Besides the native CFTR gene, which is not expressed in HEK cells, the HEK293T cell lines used in Leuven carry another copy of the gene embedded in an expression cassette. The cassette includes a CMV promoter, which is a standard promoter used for gene overexpression in human cells derived from the human Cytomegalovirus[6], as well as a puromycin resistance co-expressed with the CFTR allowing for continuous selection of CFTR expressing cells. The whole construct was stably inserted into the genome using lentiviral transduction[7][8]. </p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/for-wiki-texts/meth-used-cells/mikroskopie-hek293t.png"
alt1="Phase contrast image of HEK293T at 20x magnification."
description="Phase contrast image of HEK293T at 20x magnification."
num={3}
bg="white"
/>
</Subesction>
<Subesction title="CFBE41o- cell line" id="Cell Culture2">
<p>The CFBE41o- cell line, derived from bronchial epithelial cells of a one-year-old Cystic Fibrosis patient, serves as a vital model for studying Cystic Fibrosis. These cells closely mimic the physiological environment of the airway epithelium, allowing for more accurate studies on how CFTR mutations affect cell function and response to treatments. They were immortalized through calcium-phosphate-mediated transfection using a replication-defective pSVori plasmid that carries the simian virus 40 large T-antigen (SV40-LT). The plasmid's defective origin of replication prevents viral propagation, thus preserving essential physiological characteristics of the cells while enabling them to develop differentiated morphologies. CFBE41o- cells are homozygous for the F508del CFTR mutation [9]. We are happy we got this cell line with permission from <a onClick={() => goToPagesAndOpenTab('ignatova', '/human-practices')}>Prof. Dr. Ignatova</a>, who is leader of a working group at the Institute for Biochemistry and Molecular Biology of Hamburg University and an iGEM supporter since a long time [10]. </p>
</Subesction>
<Subesction title="Human nasal epithelial cells (hNECs)" id="Cell Culture3">
<p>Human nasal epithelial cells were obtained by nasal brushing, a minimally invasive method. These cells function/act as primary cultures. Cultivated in air-liquid interface (ALI) cultures and apical-out airway organoids (AOAO), they serve as a suitable model to visualise the functional epithelium of the airways in a differentiated form. The <i>in vivo</i> aspects of an airway disease, such as CF, can be modelled using donors with those airway diseases [11]. This model is therefore particularly suitable for testing our prime editing complex. </p>
<div className="figure-wrapper">
<figure>
<img src="https://static.igem.wiki/teams/5247/photos/for-wiki-texts/meth-patch-clamp/bild-meth-patch-clamp.png" alt="Patch clamp setup"/>
<figcaption><b>Figure 2.</b> Patch clamp setup</figcaption>
</figure>
<H4 text="Application in CFTR gene prime editing validation"></H4>
<p>In our ongoing research project focusing on the treatment of cystic fibrosis (CF), our patch clamp measurements, performed in collaboration with Dr. Oliver Dräger from the Cellular Neurophysiology working group at Bielefeld University, serve as a powerful validation tool for the assessment of the functional correction of the CFTR gene, particularly the common F508del mutation, via prime editing. The patch clamp technique can be employed in this context to measure the resulting chloride ion channel activity which is altered by the mutation [4]. Whole-Cell recordings were performed to assess whether the corrected CFTR channels function similarly to those in healthy cells. If the chloride ion currents in the edited cells approach levels of healthy cells, this would strongly suggest successful gene editing and validate the functionality of our therapeutic approach.</p>
<div className="row align-items-center">
<div className="col">
<iframe title="Bielefeld-CeBiTec: ALI cell culture (2024) [English]" width="500" height="315" src="https://video.igem.org/videos/embed/ff557f5a-94be-45e6-90ca-0affa14423e3?autoplay=1&amp;muted=1" frameBorder="0" allowFullScreen={true} sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
</div>
<div className="col">
<iframe title="Bielefeld-CeBiTec: AOAO cell culture (2024) [English]" width="500" height="315" src="https://video.igem.org/videos/embed/058d83cf-ab09-476e-9ab2-30cd114fbc0c?autoplay=1&amp;muted=1" frameBorder="0" allowFullScreen={true} sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
</div>
</div>
<figcaption>
<div className="row align-items-center">
<div className="col">
<b>Figure 4.</b> ALI cultures of hNECs: The active cilia beat frequency of differentiated human nasal epithelial cells (hNECs) in air-liquid interface (ALI) culture is visible. This ciliary movement is crucial for mucociliary transport, which contributes to the clearance of particles and pathogens in the respiratory tract.
</div>
<div className="col">
<b>Figure 5.</b> Apical-Out Airway Organoid (AOAO) culture: Visible apical-out airway organoids in action. These 3D structures, which mimic the airway epithelium, allow detailed study of cellular processes such as mucociliary transport and secretory activities, in which cilia and vesicles play a key role.
</div>
</div>
</figcaption>
</figure>
</div>
</Subesction>
</Section>
<Section title="LNPs" id="LNPs">
<Subesction title="Cytotoxicity Tests" id="Cytotoxicity Tests">
<H4 text="Assessing the Safety of Our LNPs "></H4>
<p>Ensuring the safety and thorough characterization of our LNPs was a central part of our project, as these particles are intended for use in biological systems. We implemented a comprehensive range of assays and techniques to assess their biosafety and physical properties, ensuring their suitability for applications such as drug delivery and gene therapy. Below is an overview of the key steps we took in our assessment.</p>
<H4 text="MTT Assay"></H4>
<div className='row align-items-center'>
<div className='col'>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/integrated-human-practices/mttassay.webp"
alt1="PC1"
description="MTT Assay: Formation of purple formazan crystals by living cells."
num={6}
/>
</div>
<div className='col'>
<p>To evaluate the cytotoxicity of our LNPs, we conducted an MTT assay, which measures the metabolic activity of cells. This assay is based on the ability of living cells to reduce MTT, a yellow tetrazolium salt, into purple formazan crystals through NAD(P)H-dependent enzymes. Cells were treated with various concentrations of LNPs, and after dissolving the formazan crystals with DMSO, we measured absorbance. Higher absorbance values indicate greater cell viability. Our results showed no significant reduction in cell viability across all LNP concentrations, demonstrating that the LNPs did not induce cytotoxic effects. This finding is crucial for ensuring that the LNPs are safe for biological use, supporting their potential in clinical applications such as drug delivery and gene therapy. Overall, the MTT assay provided strong evidence of the biocompatibility of our LNPs. </p>
</div>
</div>
<H4 text="Proliferation Assay to Monitor Long-Term Safety"></H4>
<p>In addition to assessing immediate cytotoxicity, we also evaluated the long-term safety of the LNPs by conducting a proliferation assay. This assay tracked cell division and growth over time to determine whether the LNPs impacted cellular function. Our results showed that LNP-treated cells had similar growth rates to untreated controls, indicating that the LNPs do not interfere with normal cell processes. This further confirms their biocompatibility and suitability for use in biological systems.</p>
</Subesction>
<Subesction title="Flow Cytometry" id="flow cytometry">
<p>To assess the transfection efficiency of our LNPs, we used flow cytometry. This method involved tagging the LNPs with fluorescent markers and measuring their ability to deliver genetic material into target cells. The flow cytometry results provided quantitative insights into how effectively the LNPs transfected cells, helping us optimize their design for gene therapy applications. </p>
</Subesction>
<Subesction title="In-Depth Characterization of LNPs" id="In-Depth Characterization of LNPs">
<H4 text="Dynamic Light Scattering (DLS) and Zeta Potential"/>
<p>The hydrodynamic radius (𝑅𝐻) of the vesicles and LNPs was determined through angle-dependent photon correlation spectroscopy (PCS) at 𝑇=20°C. Samples were measured in NMR tubes using a 3D LS Spectrometer Pro (LS Instruments, Fribourg, Switzerland), which was equipped with a HeNe Laser (632.8 nm, 1145P; JDSU, Milpitas, CA, USA), a decaline index-matching vat, an automated goniometer, and two detectors. Measurements were performed in a 3D cross-mode to eliminate multiple scattering effects, covering a scattering angle range of 30° to 120° in increments of 10°, with a measuring time of three intervals of 120 s per angle. The autocorrelation function of the scattered light intensity was generated using a multiple-τ digital correlator and analyzed via inverse Laplace transformation (CONTIN) to determine the mean relaxation rate (Γ). From these data, the hydrodynamic radius (𝑅𝐻) was calculated using the Stokes–Einstein equation:
𝑅𝐻=𝑘𝐵⋅𝑇/6𝜋𝜂𝐷𝑇 where 𝑘𝐵 is the Boltzmann constant, T is the temperature, η is the solvent viscosity, and DT
is the translational diffusion coefficient. The value of 𝐷𝑇 was obtained from the slope of the linear relationship between the relaxation rate (Γ) and
the squared magnitude of the scattering vector (𝑞2) as defined by:Γ =𝐷𝑇⋅𝑞2Γ.
The viscosity of water was calculated based on the temperature to provide accurate measurements for the given conditions.
To complement the PCS analysis, dynamic light scattering (DLS) was used to determine the size distribution and polydispersity index (PDI) of the LNPs. DLS measurements confirmed that the LNPs had a consistent size distribution with minimal aggregation, which is crucial for their stability and effectiveness. Furthermore, we assessed the zeta potential of the LNPs to evaluate their surface charge. A high zeta potential value indicated that the LNPs were stable in suspension, a necessary condition for maintaining their functionality in biological environments.
Overall, the combination of PCS, DLS, and zeta potential measurements provided a comprehensive characterization of the LNPs, confirming their hydrodynamic properties, stability, and suitability for drug delivery applications. </p>
<div className='row align-items-center'>
<div className='col'>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/lab/dls-methods.webp"
alt1="Dynamic Light Scattering (DLS) measurement set-up."
description="Dynamic Light Scattering (DLS) measurement setup."
num={7}
/>
</div>
</div>
<H4 text="SEM and Cryo-EM for Structural Analysis"></H4>
<div className='row align-items-center'>
<div className='col'>
<p>For the cryogenic electron microscopy (Cryo-EM) analysis, samples were vitrified on holey carbon TEM grids (Lacey Carbon Film coated, 200 Mesh; Science Services, München, Germany) using a Leica blotting and plunging device (Leica EM GP, Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany). The grids were rapidly plunged into liquid ethane cooled by liquid nitrogen to ensure sufficiently fast cooling. After vitrification, the grids were transferred to a cryo transfer and tomography holder (Fischione Model 2550, E.A. Fischione Instruments, Pittsburgh, USA).
TEM images were acquired using a JEOL JEM-2200FS electron microscope (JEOL, Freising, Germany) equipped with a cold field emission electron gun, operated at an acceleration voltage of 200 kV. All images were captured digitally using a bottom-mounted camera (Gatan OneView, Gatan, Pleasanton, USA) and processed with a digital imaging processing system (Digital Micrograph GMS 3, Gatan, Pleasanton, USA).
In addition to Cryo-EM, we employed scanning electron microscopy (SEM) to further characterize the morphology and surface structure of the LNPs. SEM provided high-resolution images that confirmed the spherical shape and uniformity of the LNPs.</p>
</div>
<div className='col'>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/plasmatem.webp"
alt1="PC1"
description="Sample preparation for SEM: Sputtering in Argon plasma."
num={8}
/>
</div>
</div>
</Subesction>
<Subesction title="Conclusion" id="Conclusion">
<H4 text="Importance of Safety in LNP Development"></H4>
<p>Testing the safety of our LNPs was a critical step in their development. LNPs are increasingly being used in cutting-edge therapies, such as mRNA vaccines and targeted drug delivery systems. For these technologies to be viable, the nanoparticles must not harm the cells they are intended to interact with. The MTT and proliferation assays provided robust data, confirming the biocompatibility of our LNPs and reinforcing their potential for safe use in further research and clinical applications. </p>
</Subesction>
</Section>
<Section title="Cell Culture" id="Cell Lines">
<H4 text="HEK293 and HEK293T cell lines"></H4>
<p>For testing our prime editing approach, we needed an easy-to-handle cell line with a measurable high expression of CFTR and the CFTR F508del mutation. When talking to Mattijs Bulcaen from the Laboratory of Molecular Virology and Gene Therapy at KU Leuven, he recommended to use HEK293T cell lines overexpressing CFTR they had used. HEK293 cells are a very common immortalized human cell line derived from the kidneys of a female embryo. They are particularly suited to research due to their convenient handling and transfection properties. Basic HEK293 cells were provided to us by the Cellular and Molecular Biotechnology working group at Bielefeld University led by Prof. Dr. Kristian Müller, who is also one of the Principal Investigators of our team. HEK293T cells express an additional tsA1609 allele of the SV40 large T-antigen, allowing for replication of vectors containing the SV40 origin of replication.[2] Besides the native CFTR gene, which is not expressed in HEK cells, the HEK293T cell lines used in Leuven carry another copy of the gene embedded in an expression cassette. The cassette includes a CMV promoter, which is a standard promoter used for gene overexpression in human cells derived from the human Cytomegalovirus[4], as well as a puromycin resistance co-expressed with the CFTR allowing for continuous selection of CFTR expressing cells. The whole construct was stably inserted into the genome using lentiviral transduction.1,3 </p>
<figure>
<img src="https://static.igem.wiki/teams/5247/photos/for-wiki-texts/meth-used-cells/mikroskopie-hek293t.png" alt="Phase contrast image of HEK293T at 20x magnification"/>
<figcaption> <b>Figure 3.</b>Phase contrast image of HEK293T at 20x magnification</figcaption>
</figure>
<H4 text="CFBE41o- cell line "></H4>
<p>The CFBE41o- cell line, derived from bronchial epithelial cells of a one-year-old cystic fibrosis patient, serves as a vital model for studying cystic fibrosis. These cells closely mimic the physiological environment of the airway epithelium, allowing for more accurate studies on how CFTR mutations affect cell function and response to treatments. They were immortalized through calcium-phosphate-mediated transfection using a replication-defective pSVori plasmid that carries the simian virus 40 large T-antigen (SV40-LT). The plasmid's defective origin of replication prevents viral propagation, thus preserving essential physiological characteristics of the cells while enabling them to develop differentiated morphologies. CFBE41o- cells are homozygous for the F508del-CFTR mutation [1]. We are happy we got this cell line with permission from Prof. Dr. Zoya Ignatova, who is leader of a working group at the Institute for Biochemistry and Molecular Biology of Hamburg University and an iGEM supporter since a long time [6]. </p>
<H4 text="Human nasal epithelial cells (hNECs)"></H4>
<p>Human nasal epithelial cells were obtained by nasal brushing, a minimally invasive method. These cells function/act as primary cultures. Cultivated in air-liquid interface (ALI) cultures and apical-out airway organoids (AOAO), they serve as a suitable model to visualise the functional epithelium of the airways in a differentiated form. The in vivo aspects of an airway disease, such as CF, can be modelled using donors with those airway diseases (5) This model is therefore particularly suitable for testing our prime editing complex. </p>
<Section title="References" id="References">
<MethodSources/>
</Section>
</>
);
}
\ No newline at end of file
}
\ No newline at end of file
src/contents/notebook.tsx
View file @ 3fd292fd
import H1 from "../components/Headings";
import { DownloadImageButton } from "../components/Buttons";
import H2 from "../components/Headings";
import { useTabNavigation } from "../utils/TabNavigation";
export function Notebook() {
useTabNavigation();
return (
<>
<div className="row mt-4">
<div className="col-lg-8">
<strong>
<H1 text="Ich bin ein Header!"/>
</strong>
<i>
<p> Ich bin ein Paragraph. </p>
</i>
<div>
<H2 text="Lab Journals and Protocol collection" id="notebookH"/>
<div className="eng-box box" >
<p>Here you can have a detailed look at our lab work - just klick the front pages of the Lab Journals and Protocol Collection to download them!
</p>
</div>
<p></p>
<div className='row'>
<div className="col">
<DownloadImageButton url="https://static.igem.wiki/teams/5247/pdfs/laboratory-notebook-1-proof-of-concept-for-pe.pdf" fileName="laboratory-notebook-1-proof-of-concept-for-pe.pdf">
<img src="https://static.igem.wiki/teams/5247/lab-journals/titelseite-lab-book-1-proof-of-concept-pe.webp" style={{height: "75%", width: "auto"}}/>
</DownloadImageButton>
</div>
<div className="col">
<DownloadImageButton url="https://static.igem.wiki/teams/5247/pdfs/laboratory-notebook-2-engineering-our-prime-editing-tool-primeguide.pdf" fileName="laboratory-notebook-2-engineering-our-prime-editing-tool-primeguide.pdf">
<img src="https://static.igem.wiki/teams/5247/lab-journals/titelseite-lab-book-2-engineering-pe.webp" style={{height: "75%", width: "auto"}}/>
</DownloadImageButton>
</div>
</div>
<div className='row'>
<div className="col">
<DownloadImageButton url="https://static.igem.wiki/teams/5247/pdfs/lab-book-3-primary-cultures.pdf" fileName="lab-book-3-primary-cultures.pdf">
<img src="https://static.igem.wiki/teams/5247/lab-journals/titelseite-lab-book-3-primary-cell-culture.webp" style={{height: "75%", width: "auto"}}/>
</DownloadImageButton>
</div>
<div className="col">
<DownloadImageButton url="https://static.igem.wiki/teams/5247/pdfs/lab-book-4-lnp-design-airbuddy.pdf" fileName="lab-book-4-lnp-design-airbuddy.pdf">
<img src="https://static.igem.wiki/teams/5247/lab-journals/titelseite-lab-book-4-lnp.webp" style={{height: "75%", width: "auto"}}/>
</DownloadImageButton>
</div>
</div>
</>
<div className='row'>
<div className="col">
<DownloadImageButton url="https://static.igem.wiki/teams/5247/pdfs/lab-book-5-downstream-experiments.pdf" fileName="lab-book-5-downstream-experiments.pdf">
<img src="https://static.igem.wiki/teams/5247/lab-journals/titelseite-lab-book-5-downstream.webp" style={{height: "75%", width: "auto"}}/>
</DownloadImageButton>
</div>
<div className="col">
<DownloadImageButton url="https://static.igem.wiki/teams/5247/pdfs/protocol-collection-igem-2024.pdf" fileName="protocol-collection-igem-2024.pdf">
<img src="https://static.igem.wiki/teams/5247/lab-journals/titelseite-lab-book-sop.webp" style={{height: "75%", width: "auto"}}/>
</DownloadImageButton>
</div>
</div>
</div>
);
}
src/contents/partners.tsx
View file @ 3fd292fd
......@@ -96,34 +96,34 @@ export function Partners() {
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="https://www.zymoresearch.com/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/zymo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/zymo.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://www.stemcell.com/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/stemcell-logo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/stemcell-logo.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://www.plasmidfactory.com/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/plasmidfactory.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/plasmidfactory.png"/>
</a>
</div>
</div>
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="https://www.drwolffgroup.com/en/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/logo-wolff.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/logo-wolff.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://snapgene.com">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/snapgene.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/snapgene.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://bio.nrw.de/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/bionrw-logo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/bionrw-logo.png"/>
</a>
</div>
</div>
......@@ -136,17 +136,17 @@ export function Partners() {
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="https://www.promega.com">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/promega-gelb.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/promega-gelb.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://www.microsynth.com">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/microsynth-logo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/microsynth-logo.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://www.neb.com/en/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/neb-logo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/neb-logo.png"/>
</a>
</div>
</div>
......@@ -165,43 +165,43 @@ export function Partners() {
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="https://www.gip.com/home/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/gip.png" />
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/gip.png" />
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://www.jenabioscience.com/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/jbs-dunkelgruen-text.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/jbs-dunkelgruen-text.png"/>
</a>
</div>
</div>
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="https://v-bio.ventures/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/vbio-logo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/vbio-logo.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://www.mn-net.com/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/mn-logo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/mn-logo.png"/>
</a>
</div>
</div>
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="https://www.fiz-biotech.de/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/fiz-logo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/fiz-logo.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://www.cellsignal.com/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/cell-signaling-technology-logo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/cell-signaling-technology-logo.png"/>
</a>
</div>
</div>
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="https://gasb.de/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/gasb-logo.jpg"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/gasb-logo.jpg"/>
</a>
</div>
</div>
......@@ -214,41 +214,41 @@ export function Partners() {
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="https://www.asimov.com/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/asimov-colorful.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/asimov-colorful.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://www.uni-bielefeld.de/fakultaeten/technische-fakultaet/arbeitsgruppen/multiscale-bioengineering/campusbrauerei/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/campus-brauerei.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/campus-brauerei.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://algenium.de/algenium/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/algenium.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/algenium.png"/>
</a>
</div>
</div>
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="https://2024.igem.wiki/gu-frankfurt/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/logos-team/other-teams/gu-frankfurt-logo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/logos-team/other-teams/gu-frankfurt-logo.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://bts-ev.de/">
<img className="img-sponsor" src="https://static.igem.wiki/teams/5247/sponsors/bts.png"/>
<img className="img-sponsor side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/bts.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://2024.igem.wiki/hamburg/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/logos-team/other-teams/igem-hamburg-logo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/logos-team/other-teams/igem-hamburg-logo.png"/>
</a>
</div>
</div>
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="https://www.stud-scicom.de/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/studscicom-logo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/studscicom-logo.png"/>
</a>
</div>
</div>
......@@ -279,22 +279,22 @@ export function Partners() {
<div className="row align-items-center">
<div className="col">
<a className="sponsor-container" href="https://www.carlroth.de/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/roth.jpg"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/roth.jpg"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://www.uni-bielefeld.de/fakultaeten/technische-fakultaet/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/techfak.jpg"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/techfak.jpg"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://www.sarstedt.com/en/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/sarstedt-logo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/sarstedt-logo.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://cordenpharma.com/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/corden-pharma-logo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/corden-pharma-logo.png"/>
</a>
</div>
</div>
......@@ -304,12 +304,12 @@ export function Partners() {
</div>
<div className="col">
<a className="sponsor-container" href="https://www.capricorn-scientific.com/en">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/capricorn-logo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/capricorn-logo.png"/>
</a>
</div>
<div className="col">
<a className="sponsor-container" href="https://www.twistbioscience.com/">
<img className="img-sponsor-partner-page" src="https://static.igem.wiki/teams/5247/sponsors/twist-bioscience-logo.png"/>
<img className="img-sponsor-partner-page side-margins-auto" src="https://static.igem.wiki/teams/5247/sponsors/twist-bioscience-logo.png"/>
</a>
</div>
<div className="col">
......
src/contents/parts.tsx
View file @ 3fd292fd
import { LoremMedium } from "../components/Loremipsum";
import { Section, Subesction } from "../components/sections";
import { PartTable } from "../components/Table";
import { useTabNavigation } from "../utils/TabNavigation";
import { BasicParts, CompositeParts } from "../data/parts";
import { BasicParts } from "../data/parts";
import { H4 } from "../components/Headings";
import PartSources from "../sources/part-sources";
import { SupScrollLink } from "../components/ScrollLink";
import { OneFigure, TwoVertical } from "../components/Figures";
export function Parts() {
useTabNavigation();
let headcols = ["Part Name", "Registry Code", "Part Description", "length [bp]", "type"]
return (
return (
<div className="col">
<Section title="Introduction" id="Introduction">
<Subesction title="Description" id="Introduction1">
<LoremMedium/>
<Section title="Description" id="Description">
<Subesction title="Introduction" id="Description1">
<p>In the context of Cystic Fibrosis, the F508del mutation represents a significant challenge for correction. The efficacy of current gene editing technologies hinges on the availability of precise tools to ensure the success of treatment strategies. In view of the above, we have developed a novel reporter system that is specifically tailored to the F508del mutation in the CFTR gene. The objective is to provide a high degree of comparability to the genomic context of this mutation, while maintaining ease of use. This system allows researchers to test and screen Prime Editors and various pegRNAs (prime editing guideRNAs), particularly in the context of the F508del mutation. By closely mimicking the genomic environment, it is believed that this tool will offer enhanced utility in the selection of optimal Prime Editing strategies. </p>
</Subesction>
<Subesction title="Characterization" id="Introduction2">
<LoremMedium/>
<Subesction title="Prime Editor and pegRNA Testing" id="Description2">
<p>The principal feature of the reporter system is its capacity to assess and quantify the efficacy of diverse Prime Editors, with a particular focus on pegRNAs. In its default state, the system expresses a non-functional GFP due to the disruption of the splice site. However, if a Prime Editor successfully restores the mutation to its correct form, the splice site is repaired and functional GFP is expressed, thereby allowing for fluorescent detection. This fluorescence serves as a reliable indicator of successful prime editing. </p>
<p>The modified GFP sequence was cloned into the pDAS12124_PEAR-GFP-preedited plasmid, which was then transfected into HEK cells to initiate the pegRNA screening process. The capacity to observe the restoration of functional GFP provides a definitive indication of the efficacy of both the Prime Editor and the specific pegRNA variant under examination. Furthermore, the considerable degree of similarity between the reporter system and the actual genomic context of the CFTR mutation renders the screening process highly pertinent to the optimisation of specific applications. </p>
</Subesction>
<Subesction title="Conclusion" id="Description3">
<p>This reporter system represents a substantial advancement in the study and correction of the CFTR F508del mutation. The design of the system allows for the straightforward screening of an array of Prime Editor and pegRNA constructs, while maintaining a high degree of comparability to the genomic context. By closely emulating the CFTR gene environment, particularly in the context of the F508del mutation, researchers are able to identify the most efficient pegRNAs and Prime Editors, offering a promising approach for developing more effective gene-editing treatments for Cystic Fibrosis.<SupScrollLink label="1"/> </p>
</Subesction>
</Section>
<Section title="Process" id="Process">
<Subesction title="EC" id="Process1">
<LoremMedium/>
<Section title="Characterization" id="Characterization">
<Subesction title="Design and Functionality" id="Characterization1">
<p>The reporter system has been designed with the specific intention of facilitating a more comparable genomic context for the F508del mutation, particularly for the purpose of testing the efficacy of different pegRNA variants and prime editors. The system provides a highly reliable platform for screening a variety of pegRNAs, thereby facilitating the identification of the most effective variant for correcting the F508del mutation</p>
<p>The system is constructed around a plasmid structure, specifically pDAS12124_PEAR-GFP_GGTdel_edited, from which a modified version of GFP (Green Fluorescent Protein) has been derived. The green fluorescent protein (GFP) is composed of two exons, separated by a Vim gene intron in its natural state. In the absence of the intron, the GFP is expressed and fluoresces. However, the GFP sequence was modified to introduce a three-base-pair deletion, specifically in the junction between Exon 1 and the Vim gene intron. This deletion affects the last base of Exon 1 and the first two bases of the intron, effectively disrupting the splice site. As a result, the intron is no longer correctly spliced out, leading to the expression of a non-functional GFP that does not fluoresce. </p>
</Subesction>
<Subesction title="Design and Build" id="Process2">
<LoremMedium/>
<Subesction title="Adaptions for CFTR F508del mutation comparibility" id="Characterization2">
<p>In addition to the introduction of the three-base-pair deletion, the intron sequence was further altered with the objective of enhancing the comparability of the system to the CFTR genomic context. Specifically, 27 base pairs were replaced downstream of the splice site with a sequence derived from the CFTR gene in the region of the F508del mutation. This modification guarantees that the gRNA spacer employed in our system is identical to the one found in the actual genomic context of the CFTR mutation. </p>
<p>The only notable differences between the system and the genomic sequence are observed in the RTT (Reverse Transcript Template) and PBS (Primer Binding Site), which have been calibrated with silent mutations to maintain comparability in GC content with the native CFTR gene. These silent mutations do not affect the encoded protein but optimise the system's mimicry of the CFTR gene. </p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/thaw/reporter-insert.webp"
alt1="Illustration of our constructed reporter system"
description="Illustration of our constructed reporter system"
num={1}
/>
</Subesction>
</Section>
<Section title="Experiments" id="Experiments">
<Subesction title="Cloning" id="Experiments1">
<LoremMedium/>
<p>The synthesised fragment was cloned into pDAS12124_PEAR-GFP-preedited plasmid using Gibson assembly, thus providing a vector with which the desired tests could be performed in HEK293 cells. The correctness of the cloning was determined by two methods: the correct size of the cloned plasmid was confirmed by gel electrophoresis, while the correct orientation and complete cloning were confirmed by Sanger sequencing. </p>
<H4 text="Workflow "/>
<p>The creation and validation of the CF-specific reporter system commenced with the selection and subsequent outgrowth of E. coli DH5α strains that contain the pDAS12124 plasmid. The initial stage of the process entails the isolation and purification of the pDAS12124 plasmid through the utilisation of conventional plasmid preparation methodologies, thereby ensuring its sterility and facilitating seamless downstream applications. Subsequent to the design of the CF-specific reporter system, the sequence was obtained from IDT. Upon its receipt, the fragment was amplified through polymerase chain reaction (PCR) to produce a sufficient quantity of material for the subsequent cloning phases. With the reporter system fragment ready, the pDAS12124_PEAR-GFP-preedited plasmid was digested using NheI and XhoI restriction enzymes. This cuts out the GFP cassette, creating the required entry point for the integration of the DNA fragment of the reporter system. To prevent the backbone from re-ligating, the sample is treated with phosphatase, ensuring the plasmid remains open for the upcoming Gibson assembly. </p>
<p>Subsequently, a purification process is conducted to extract the plasmid backbone and concentrate the samples. This facilitates the integration of the amplified reporter system into the prepared pDAS12124_PEAR-GFP-preedited backbone, which is then subjected to the Gibson assembly process. This assembly process results in the creation of the novel pDAS12124_PEAR-GFP_GGTdel_edited plasmid, which incorporates the CF-specific reporter system. </p>
<p>Subsequently, the pDAS12124_PEAR-GFP_GGTdel_edited plasmid is transformed into E. coli DH5α cells for propagation. To confirm the successful integration of the reporter fragment, colony PCR (cPCR) is performed on the transformed colonies. The positive colonies, identified by cPCR, are selected and grown in LBCm50 medium for further analysis. </p>
<p>The final validation step involves preparing the pDAS12124_PEAR-GFP_GGTdel_edited plasmid from the positive colonies and verifying the correct insertion of the reporter fragment using Sanger sequencing. This ensures the fragment is inserted in the correct orientation and that the CF-specific reporter system has been successfully constructed without any errors. </p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/thaw/cloning-reporter.webp"
alt1=""
description="Cloning strategy of our reporter system for functional expression"
num={2}
/>
</Subesction>
<Subesction title="Nikase-Assay" id="Experiments2">
<LoremMedium/>
<Subesction title="pegRNA Screening" id="Experiments2">
<p>In connection with the optimisation of prime editing with regard to the F508del mutation, it was necessary to compare different pegRNAs, as their optimal structure always depends on the application context. We therefore designed and cloned 14 variants of pegRNAs for the target of the reporter system and then tested them on the reporter system using the PE2 system. </p>
<p>For pegRNA screening, we co-transfected the HEK293 cells with our modified reporter plasmid, the pegRNA expressing plasmid and pCMV-PE2. We were then able to measure the fluorescence after 72 hours using FACS and evaluate which pegRNA showed the highest efficiency. </p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild9.png"
alt1=""
description="Percentage of fluorescent HEK293 cells 72 h after transfection with various pegRNAs (pegRNA1-14) normalized to pDAS12124 pre-edited as internal positive control as result of flow cytometry analysis"
num={3}
/>
<p>We also co-transfected the CFBE41o- with our modified reporter plasmid, the plasmid expressing pegRNA04 as well as pCMV-PE6c. As a result, we observed fluorescence, indicating successful editing of the reporter plasmid. The negative controls transfected with only one of the plasmids each showed no fluorescence, routing out other factors. This gave us validation, that our pegRNAs work not only in HEK, but also in epithelial cells that express CFTR F508del. </p>
<TwoVertical
description="Microscopy results after 24h or 48h. Transfection of pDAS12124-preedited with lipofectamine 3000 was successfully done in CFBE41o- cell line and visible after 48h. CFBE41o- cell line was transfected with pDAS-IDT with Lipofectamine 3000 and afterwards with LNPs including PE6c and pegRNA4 and was after 24h fluorescence visible."
num={4}
bg="white"
alt1=""
pic1="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild10-1.png"
pic2="https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild10-2.png"
/>
<p>Based on the results, we were able to select 4 possible candidates and one negative example, whose attributes we then used to create pegRNAs for the CFTR target. The next step is to test these pegRNAs using CFBE41o- cells by again co-transfecting these with three plasmids: reporter plasmid, pegRNA expressing plasmid and pCMV-PE6c, and measuring fluorescence after 72 hours. </p>
</Subesction>
<Subesction title="Activity Experiments" id="Experiments3">
<LoremMedium/>
<Subesction title="Future Experiment: Nickase Assay" id="Experiments3">
<p>In the next series of experiments, we would like to investigate various mutation candidates, in particular the possible SpuFz1 nickases (BBa_K5247101- BBa_K5247104) and various PlmCasx nickase variants (BBa_K5247105- BBa_K5247107), in more detail using the PE6c system. </p>
{/* Bild Nikase */}
<H4 text="nSpuFz1 "/>
<p>The nSpuFz1 variants are expressed in yeast strain Pichia pastoris (SMD1163), which we obtained from Nils Berelsmann{/* [link zu HP-Timeline]. */} In advance, the corresponding genes were cloned into a suitable expression vector, pPIC9K, via Gibson Assembly to ensure efficient expression of the nickases. The cloning as well as the subsequent expression and purification of the nickases were carried out according to a detailed protocol<SupScrollLink label="2"/> under the expert guidance of Hakan Soytürk[link zu HP timeline]. </p>
<H4 text="nPlmCasX "/>
<p>The nPlmCasX variants are expressed in E. coli strain BL21D3, which we obtained from AG Müller{/* [link zu HP-Timeline] */}. In advance, the corresponding genes were cloned into a suitable expression vector, pZMB1029, via Gibson Assembly to ensure efficient expression of the nickases. The cloning as well as the subsequent expression and purification of the nickases were carried out according to a detailed protocol<SupScrollLink label="2"/> . </p>
{/* Bild beide */}
<p>After successful purification, the isolated nickases are comprehensively analyzed according to verify their activity and efficiency. These analyses will serve to evaluate the functionality and suitability of the nickases for specific applications in prime editing. Subsequently, detailed characterization experiments are planned to determine the properties of the nickases, if functional, including their specificity, editing activity and potential for use in precise gene editing procedures. </p>
<p>Validation of the nickases will be performed in different cell lines to confirm their efficiency and reliability in a cellular context. These validation steps are crucial to further investigate the potential of Prime Guide for therapeutic applications. </p>
</Subesction>
</Section>
<Section title="Parts Collection" id="Parts Collection">
<Subesction title="Plasmids" id="Parts Collection1">
<LoremMedium/>
</Subesction>
<Subesction title="Basic Parts" id="Parts Collection2">
<Subesction title="Basic Parts" id="Parts Collection1">
<PartTable cols={headcols} data={BasicParts}/>
</Subesction>
<Subesction title="Composite Parts" id="Parts Collection3">
<PartTable cols={headcols} data={CompositeParts}/>
</Subesction>
</Section>
<Section title="References" id="References">
<ol>
<PartSources/>
</ol>
</Section>
</div>
);
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