diff --git a/src/contents/Human Practices/Further Engagement/Entrepreneurship.tsx b/src/contents/Human Practices/Further Engagement/Entrepreneurship.tsx
index 62d5766e419c024921c810aa8118a854356d6582..10348a2e4504f871ee127a836a532637c7242c91 100644
--- a/src/contents/Human Practices/Further Engagement/Entrepreneurship.tsx
+++ b/src/contents/Human Practices/Further Engagement/Entrepreneurship.tsx
@@ -1,6 +1,5 @@
import { ButtonOneWithScroll } from "../../../components/Buttons";
import { H4, H5 } from "../../../components/Headings";
-import { LoremShort } from "../../../components/Loremipsum";
export function HPEntrepreneur(){
@@ -26,7 +25,14 @@ export function HPEntrepreneur(){
<p>That is why in this section we focus on the aspects of entrepreneurship that are crucial for the potential successful realisation of our project to develop new therapies for cystic fibrosis. A pivotal moment was our interview with Nicole Friedlein, which gave us valuable insights into the challenges and opportunities in the field of biomedical innovation. The discussions in the interview encouraged us to look more closely at the regulatory requirements, which is why one team member completed a GxP course and subsequently trained the team in this area. In addition, we conducted further interviews in the area of entrepreneurship to gain a better understanding of the practical aspects of business development. These experiences not only enriched the scientific depth of our project, but also sharpened our perspective on the practical implementation and market launch of new therapies.
</p>
<H4 id="ent-heading" text="Our Entrepreneurship"/>
- <LoremShort/>
+ <p>In conclusion, the entrepreneurial journey of developing RNA-based gene therapy for cystic fibrosis, as outlined in our experiences and interviews with industry founders, demonstrates that entrepreneurship is not only an interesting possibility but a necessary avenue to transform scientific innovation into real-world solutions. Our approach has been shaped by the challenges and opportunities in the biotech field, from understanding regulatory frameworks like GxP to navigating complex market dynamics and funding challenges. </p>
+ <p>Through key interviews, such as the one with Nicole Friedlein, we have gained insights into the pivotal role of regulatory standards in scaling our project. The completion of GxP training by one team member reflects our commitment to ensuring compliance with Good Laboratory Practice (GLP) and Good Manufacturing Practice (GMP), both of which are essential for advancing from proof-of-concept to clinical trials. This foundation is crucial for building investor confidence and meeting regulatory requirements.</p>
+
+ <p>Additionally, market evaluations reveal a significant opportunity for our therapy, particularly targeting the unmet needs of cystic fibrosis patients who do not respond to current treatments like CFTR modulators. The growing gene therapy market presents a strong case for our innovation, although we are aware of the competitive landscape dominated by companies like Vertex Pharmaceuticals. Our unique value lies in providing a more permanent solution for patients not served by existing treatments. </p>
+
+ <p>Interviews with founders from companies such as PlasmidFactory and RNhale have provided valuable lessons on transitioning from research to commercialization. The importance of building networks, securing diverse funding sources, and maintaining flexibility to adapt to market feedback are key takeaways that will guide our next steps. Establishing strategic partnerships and seeking early engagement with regulatory bodies will be essential as we prepare for clinical trials and eventual market entry.</p>
+
+ <p>To align our long-term vision of revolutionizing cystic fibrosis treatment with immediate milestones, we will continue optimizing our lipid nanoparticle delivery system, pursuing regulatory compliance, and engaging with the cystic fibrosis community to refine our product. Our focus on both the scientific and business aspects ensures that we are building a strong foundation for success in bringing this innovative therapy to market, improving the lives of patients with cystic fibrosis. </p>
</div>
<div id="ent-interview" className="ent-interview" style={{display: "none"}}>
diff --git a/src/contents/engineering.tsx b/src/contents/engineering.tsx
index d67e237989db27afe23b976b18ae6d8c9b8ebfe6..bf75728670f25a2db7ba12b1b3664507bfecd7ac 100644
--- a/src/contents/engineering.tsx
+++ b/src/contents/engineering.tsx
@@ -86,7 +86,7 @@ export function Engineering() {
</figure>
<H4 text="Test" id="test-head"/>
<p>
- When trying to find protospacers for Cas9 and other possible nickases<a onClick={() => goToPagesAndOpenTab('nickase', '/engineering')}> 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[link] 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.
+ When trying to find protospacers for Cas9 and other possible nickases<a onClick={() => goToPagesAndOpenTab('nickase', '/engineering')}> 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>
@@ -99,7 +99,7 @@ export function Engineering() {
<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 fluorescence activated cell sorting (FACS). 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 contextâ€. We reasoned that this system might be flexible, and sensitive enough to build our optimizations strategies upon.
+ 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 contextâ€. 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>
@@ -107,7 +107,7 @@ export function Engineering() {
</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 pCMV-PE2 prime editor[link PE systems] 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.
+ 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: 'pe1', path: '/engineering', tabId: '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"/>
@@ -115,7 +115,7 @@ export function Engineering() {
</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 Transfection Optimization[link]). Secondly, the reporter had to be modified in a way that resembles the genomic CFTR target.
+ 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>
@@ -159,7 +159,7 @@ export function Engineering() {
<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 CFBE41o-[link]. The cells are derived from bronchial epithelial cells of a cystic fibrosis patient and are homozygous for CFTR F508del.
+ 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>
@@ -187,7 +187,7 @@ export function Engineering() {
<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 human nasal epithelial cells[link] derived from members of our team.
+ 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>
@@ -207,7 +207,7 @@ export function Engineering() {
<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 spacer of our choice[link], 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 PreCyse cassette[link].
+ 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>
@@ -546,7 +546,7 @@ export function Engineering() {
<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 modified PEAR reporter[link]. focused on the incorporation of silent edits into the reverse transcriptase template.
+ 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>
@@ -567,7 +567,7 @@ export function Engineering() {
</p>
<H4 text="Test" id="test-head"/>
<p>
- These two variants were then tested against each other using our reporter plasmid system[link] and a PE2 <a onClick={() => goToPagesAndOpenTab('pe-systems', '/engineering')}> 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.
+ 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>
@@ -579,7 +579,7 @@ export function Engineering() {
<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 FACS, we selected the three most effective candidates.
+ 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>
@@ -613,11 +613,11 @@ export function Engineering() {
</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 CFBE41o- epithelial cells lines[link] homozygous for the CFTR F508del mutation.
+ 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 PE6c prime editor[link]. 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.
+ 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>
@@ -645,7 +645,7 @@ export function Engineering() {
</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="4" scrollId="desc-4"/><TabScrollLink tab="tab-pegrna" num="5" scrollId="desc-5"/>. 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 FACS 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.
+ 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="4" scrollId="desc-4"/><TabScrollLink tab="tab-pegrna" num="5" scrollId="desc-5"/>. 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>
@@ -675,7 +675,7 @@ export function Engineering() {
</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 FACS 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.
+ 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>
@@ -702,8 +702,6 @@ export function Engineering() {
<div className="eng-box box" >
<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>
- <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="nic1">
@@ -718,6 +716,26 @@ export function Engineering() {
<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>
+ <figure>
+ <div className="row align-items-center">
+ <div className="col">
+ <img src="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-3d-model.webp"/>
+ </div>
+ <div className="col">
+ <img src="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-3d-model-zinc-finger.webp"/>
+ </div>
+ </div>
+ <figcaption>
+ <div className="row align-items-center">
+ <div className="col">
+ <b>Figure 1:</b> Schematic illustration of SpuFz1 (PDB code: 8GKH) visualized in ChimeraX
+ </div>
+ <div className="col">
+ <b>Figure 2:</b> 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
+ </div>
+ </div>
+ </figcaption>
+ </figure>
<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.
@@ -743,26 +761,6 @@ export function Engineering() {
<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>
- <div className="row align-items-center">
- <div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-3d-model.webp"/>
- <figcaption>
- <b>Figure 1:</b>
- Schematic illustration of SpuFz1 (PDB code: 8GKH) visualized in ChimeraX
- </figcaption>
- </figure>
- </div>
- <div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-3d-model-zinc-finger.webp" style={{height:"70%", width:"80%"}}/>
- <figcaption>
- <b>Figure 2:</b>
- 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
- </figcaption>
- </figure>
- </div>
- </div>
</p>
<div className="box" >
@@ -808,32 +806,32 @@ export function Engineering() {
</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. X). This comparison revealed significant differences, particularly in the TNB domain, indicating that the zinc finger plays a crucial structural role (Fig. 3).
+ 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>
+ <figure>
+ <div className="row align-items-center">
+ <div className="col">
+ <img src="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-vs-zf-nikase.webp"/>
+ </div>
+ <div className="col">
+ <img src="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-vs-zf-nikase-zinc-finger.webp"/>
+ </div>
+ </div>
+ <figcaption>
+ <div className="row align-items-center">
+ <div className="col">
+ <b>Figure 3:</b> 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.
+ </div>
+ <div className="col">
+ <b>Figure 4:</b> 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
+ </div>
+ </div>
+ </figcaption>
+ </figure>
<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>
- <div className="row align-items-center">
- <div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-vs-zf-nikase.webp"/>
- <figcaption>
- <b>Figure 1:</b>
- 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.
- </figcaption>
- </figure>
- </div>
- <div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-wt-vs-zf-nikase-zinc-finger.webp"/>
- <figcaption>
- <b>Figure 2:</b>
- 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
- </figcaption>
- </figure>
- </div>
- </div>
</p>
</div>
<div className="box" >
@@ -844,8 +842,29 @@ export function Engineering() {
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="5" scrollId="desc-5"/>. 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="1" scrollId="desc-1"/>. 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="6" scrollId="desc-6"/> (Fig. X1 and X2). 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.
+ In addition to these insights, we noticed a significant phylogenetic relationship between Fanzor endonucleases, like SpuFz1, and Cas12 endonucleases<TabScrollLink tab="tab-nickase" num="1" scrollId="desc-1"/>. 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="6" scrollId="desc-6"/> (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>
+ <figure>
+ <div className="row align-items-center">
+ <div className="col">
+ <img src="https://static.igem.wiki/teams/5247/engineering-cycle/cas12-nikase.webp"/>
+ </div>
+ <div className="col">
+ <img src="https://static.igem.wiki/teams/5247/engineering-cycle/cas12-nikase-close-up.webp"/>
+ </div>
+ </div>
+ <figcaption>
+ <div className="row align-items-center">
+ <div className="col">
+ <b>Figure 5:</b> 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
+ </div>
+ <div className="col">
+ <b>Figure 6:</b> Close-up of Cas12a (PDB code: 8SFH) - arginine (R) (1226th amino acid in the primary structure) is colored purple
+ </div>
+ </div>
+ </figcaption>
+ </figure>
+
<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>
@@ -861,11 +880,31 @@ export function Engineering() {
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. X7). 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.
+ 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. X8). 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.
+ 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>
+ <figure>
+ <div className="row align-items-center">
+ <div className="col">
+ <img src="https://static.igem.wiki/teams/5247/engineering-cycle/ascas12a-nuc-domain.webp"/>
+ </div>
+ <div className="col">
+ <img src="https://static.igem.wiki/teams/5247/engineering-cycle/ascas12a-nuc-domain-close-up.webp"/>
+ </div>
+ </div>
+ <figcaption>
+ <div className="row align-items-center">
+ <div className="col">
+ <b>Figure 7:</b> 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.
+ </div>
+ <div className="col">
+ <b>Figure 8:</b> 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.
+ </div>
+ </div>
+ </figcaption>
+ </figure>
<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>
@@ -873,8 +912,49 @@ export function Engineering() {
</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. X3 and Fig. X4) and CasX (Fig. X5 and Fig. X6) 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.
+ 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>
+ <figure>
+ <div className="row align-items-center">
+ <div className="col">
+ <img src="https://static.igem.wiki/teams/5247/engineering-cycle/casx-nikase.webp"/>
+ </div>
+ <div className="col">
+ <img src="https://static.igem.wiki/teams/5247/engineering-cycle/casx-nikase-close-up.webp"/>
+ </div>
+ </div>
+ <figcaption>
+ <div className="row align-items-center">
+ <div className="col">
+ <b>Figure 9:</b> 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
+ </div>
+ <div className="col">
+ <b>Figure 10:</b> 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
+ </div>
+ </div>
+ </figcaption>
+ </figure>
+
+ <figure>
+ <div className="row align-items-center">
+ <div className="col">
+ <img src="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-nikase.webp"/>
+ </div>
+ <div className="col">
+ <img src="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-nikase-close-up.webp"/>
+ </div>
+ </div>
+ <figcaption>
+ <div className="row align-items-center">
+ <div className="col">
+ <b>Figure 11:</b> 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
+ </div>
+ <div className="col">
+ <b>Figure 12:</b> Close-up of SpuFz1 (PDB code: 8GKH) - the two arginines (R) (564th and 568th amino acid in the primary structure) are purple in color
+ </div>
+ </div>
+ </figcaption>
+ </figure>
<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.
@@ -889,96 +969,7 @@ export function Engineering() {
<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>
- <div className="row align-items-center">
- <div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/cas12-nikase.webp"/>
- <figcaption>
- <b>Figure 1:</b>
- 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
- </figcaption>
- </figure>
- </div>
- <div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/cas12-nikase-close-up.webp"/>
- <figcaption>
- <b>Figure 2:</b>
- Close-up of PlmCasX (PDB code: 7WAZ) - arginine (R) (1226th amino acid in the primary structure) is colored purple
- </figcaption>
- </figure>
- </div>
- </div>
- <div className="row align-items-center">
- <div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/casx-nikase.webp"/>
- <figcaption>
- <b>Figure 3:</b>
- 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
- </figcaption>
- </figure>
- </div>
- <div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/casx-nikase-close-up.webp"/>
- <figcaption>
- <b>Figure 4:</b>
- 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
- </figcaption>
- </figure>
- </div>
- </div>
- <div className="row align-items-center">
- <div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-nikase.webp"/>
- <figcaption>
- <b>Figure 5:</b>
- 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
- </figcaption>
- </figure>
- </div>
- <div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/spufz-nikase-close-up.webp"/>
- <figcaption>
- <b>Figure 6:</b>
- Close-up of SpuFz1 (PDB code: 8GKH) - the two arginines (R) (564th and 568th amino acid in the primary structure) are purple in color
- </figcaption>
- </figure>
- </div>
- </div>
-
-
- <figure>
- <div className="row align-items-center">
- <div className="col">
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/ascas12a-nuc-domain.webp"/>
-
- </div>
- <div className="col">
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/ascas12a-nuc-domain-close-up.webp"/>
-
- </div>
- </div>
- <figcaption>
- <div className="row align-items-center">
- <div className="col">
- <b>Figure 7:</b>
- 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.
- </div>
- <div className="col">
- <b>Figure 8:</b>
- 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.
- </div>
- </div>
- </figcaption>
-
- </figure>
-
-
</p>
</div>
<div className="box" >
@@ -994,22 +985,19 @@ export function Engineering() {
</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.X). 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.
+ 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">
- <div className="col">
- <figure>
- <img src="https://static.igem.wiki/teams/5247/engineering-cycle/nickase-assay.webp" style={{height:"75%", width:"75%"}}/>
- <figcaption>
- <b>Figure 1:</b>
- 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.
- </figcaption>
- </figure>
- </div>
+ <figure>
+ <img src="https://static.igem.wiki/teams/5247/engineering-cycle/nickase-assay.webp" style={{height:"75%", width:"75%"}}/>
+ <figcaption>
+ <b>Figure 13:</b> 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.
+ </figcaption>
+ </figure>
</div>
</p>
</div>
diff --git a/src/contents/methods.tsx b/src/contents/methods.tsx
index 9619b93d2af5af414fd1dc3007cc96fc80738bf3..13e8169e513cf07d451d9cb271e252be58bdb532 100644
--- a/src/contents/methods.tsx
+++ b/src/contents/methods.tsx
@@ -83,14 +83,14 @@ export function Methods() {
</figcaption>
</figure>
</div>
- <div className='col'>
+ <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="Fluorescence-Activated Cell Sorting (FACS)" id="FACS">
+ <Subesction title="Flow cytometry" id="FACS">
<p>To assess the transfection efficiency of our LNPs, we used fluorescence-activated cell sorting (FACS). This method involved tagging the LNPs with fluorescent markers and measuring their ability to deliver genetic material into target cells. FACS provided quantitative insights into how effectively the LNPs transfected cells, helping us optimize their design for gene therapy applications. </p>
</Subesction>
diff --git a/src/contents/results.tsx b/src/contents/results.tsx
index 29f05c1faff9713e8f2322e10dab6cdecaa59f5b..2b7c0c76518266d3581a0ffe15366b9aa7fb6340 100644
--- a/src/contents/results.tsx
+++ b/src/contents/results.tsx
@@ -271,9 +271,9 @@ export function Results() {
Description here
</figcaption>
</figure>
- <H5 text="FACS"/>
+ <H5 text="Flow cytometry"/>
<div className="row align-items-center">
- <p>We performed FACS analysis 72 h post-transfection to evaluate the transfection efficiency of the SORT LNP in HEK293. The relative percentage of fluorescent cells was determined by measuring the percentage of FITC-A+ cells, followed by normalization to the negative control and fold change calculation.</p>
+ <p>We performed flow cytometries analysis 72 h post-transfection to evaluate the transfection efficiency of the SORT LNP in HEK293. The relative percentage of fluorescent cells was determined by measuring the percentage of FITC-A+ cells, followed by normalization to the negative control and fold change calculation.</p>
<div className="col">
<figure>
<img src="https://static.igem.wiki/teams/5247/delivery/results/sortlnp-facs.png" alt="SORTFACS" style={{maxHeight: "200pt"}}/>
diff --git a/src/data/hptimelinedata.tsx b/src/data/hptimelinedata.tsx
index 4f53f92f9268b5c0db1a21393fc7aa31469882ee..772c72d187dd689cc84209b58a5fa559fc0c9c4d 100644
--- a/src/data/hptimelinedata.tsx
+++ b/src/data/hptimelinedata.tsx
@@ -121,6 +121,7 @@ const pics: { [key: string]: string } = {
labsupply: "https://static.igem.wiki/teams/5247/photos/hp/labsupply-info.webp",
hamburg: "https://static.igem.wiki/teams/5247/photos/hp/gruppenbild-hamburg.webp",
sriram: "https://static.igem.wiki/teams/5247/photos/sriram.svg",
+ logo: "https://static.igem.wiki/teams/5247/logos-team/precyse-no-slogan.png"
};
@@ -342,17 +343,40 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
{
vorname: "Looking for expertise",
nachnname: "",
- pictureurl: pics['placeholder'],
+ pictureurl: pics['logo'],
+ job: "Team iGEM",
+ affiliation: "Bielfeld CeBiTec 2024",
tag: "Milestone",
heading: "Identifying key experts in cystic fibrosis and prime editing",
interviewtabid: "experts",
cardtext: "",
- quote: "",
- aimofcontact: "",
- insights: "",
- implementation: "",
+ quote: "Focusing on gene therapy for cystic fibrosis has been a meaningful journey for our team. Collaborating with experts has enriched our understanding and helped us refine our approach, especially in exploring prime editing. We're eager to turn our plans into reality and make a real impact",
+ quoteNachname:"Kanthak, Teammember",
+ quoteVorname: "Kai",
type: "meta",
- summary: "",
+ summary: [<p>After our team came together and thoroughly explored various project ideas, we decided to focus on gene therapy for cystic fibrosis, largely due to a personal connection with a close friend affected by the condition. Up until that point, we had not yet developed a concrete concept, so we sought to engage with experts in order to broaden our understanding of the latest advancements in gene therapy.
+ In addition to grasping the importance of a functional gene therapy, we delved into different strategies regarding the underlying mechanisms and the best delivery methods for the therapy. While the general topic of our project was clear, we now faced the challenge of working out the details. At this stage, we decided to consult further experts in the field of cystic fibrosis to deepen our knowledge and refine our approach.
+ Through connections with the University Hospital Münster and our local hospital, we aimed to gain a comprehensive overview of the clinical applications of gene therapy and the current research in cystic fibrosis. These consultations with specialists allowed us to acquire valuable insights into different therapeutic options and laid the groundwork for our own exploration of potential strategies, particularly in the area of prime editing as a promising treatment avenue.</p>,
+ <ul>
+ <li>
+ <strong>Team Formation & Research:</strong> Chose gene therapy for cystic fibrosis and explored mechanisms and delivery strategies.
+ </li>
+ <li>
+ <strong>Expert Engagement:</strong> Consulted with specialists to refine approach, focusing on prime editing.
+ </li>
+ <li>
+ <strong>Medical Collaboration:</strong> Gained clinical insights through partnerships with hospitals.
+ </li>
+ <li>
+ <strong>Project Development:</strong> Developed detailed plans for gene therapy, incorporating expert feedback.
+ </li>
+ <li>
+ <strong>Testing & Future Application:</strong> Plan to test strategies and prepare for potential clinical trials.
+ </li>
+ </ul>
+
+ ],
+
months: "April"
},
{
@@ -464,21 +488,33 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
},
{
vorname: "Exploring new ideas",
- nachnname: "x",
- pictureurl: pics['placeholder'],
+ nachnname: "",
+ pictureurl: pics['logo'],
tag: "Milestone",
- affiliation: "",
- heading: "Further brainstorming on approaches",
- interviewtabid: "brainstorming",
+ job: "Team iGEM",
+ affiliation: "Bielfeld CeBiTec 2024",
+ heading: "Further brainstorming on approaches and specifing the project",
+ interviewtabid: "exploring",
cardtext: "",
- quote: "x",
- quoteNachname:"x",
- quoteVorname: "d",
- aimofcontact: "d",
- insights: "d",
- implementation: "d",
+ quote: "One of the most valuable aspects of our project is the feedback we've received. It truly motivates us to make a difference in the community.",
+ quoteNachname:"Sanfilipo, Teammember",
+ quoteVorname: "Liliana",
type: "meta",
- summary: "d",
+ summary:[<p>After receiving valuable feedback from both clinical and academic experts, we decided to focus on optimizing Prime Editing strategies for cystic fibrosis treatment. Both experts not only encouraged us in our approach but also provided insightful feedback, which we will integrate into our future project design. Through these discussions, we learned that current treatment strategies are urgently needed in real life but are limited in precision and efficiency.
+ Additionally, we gained key insights into lung-specific delivery methods, which inspired us to pursue lung-specific correction of the CFTR gene, a critical aspect of cystic fibrosis therapy.
+ At this stage, we are eager to expand our perspectives by seeking input from industry and business professionals, while also striving to increase our local impact. To evaluate this impact, we plan to develop a survey aimed at understanding the interest in gene therapy and the community’s knowledge of cystic fibrosis within our local area. This will help us gauge awareness and ensure our project addresses both scientific and societal needs effectively.</p>>
+ <ul>
+ <li>
+ <strong>Expert Feedback Integration:</strong> Refined the project focus on optimizing Prime Editing strategies and lung-specific gene delivery based on clinical and academic insights.
+ </li>
+ <li>
+ <strong>Focus on Lung-Specific Correction:</strong> Shifted toward lung-specific CFTR gene correction for cystic fibrosis treatment.
+ </li>
+ <li>
+ <strong>Community Engagement Plan:</strong> Initiated plans for a local survey to assess awareness of cystic fibrosis and interest in gene therapy, aiming to increase local impact.
+ </li>
+ </ul>
+ ],
months: "may"
},
{
@@ -544,17 +580,39 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
{
vorname: "Documenting progress",
nachnname: "",
- pictureurl: pics['placeholder'],
+ pictureurl: pics['logo'],
tag: "Milestone",
+ job: "Team iGEM",
+ affiliation: "Bielfeld CeBiTec 2024",
heading: "Tracking progress in expert search and idea development",
interviewtabid: "progress",
cardtext: "",
- quote: "",
- aimofcontact: "",
- insights: "",
- implementation: "",
+ quote: "I believe our focus on a human-centric approach is crucial. It ensures that our research not only advances science but also prioritizes the needs and safety of the patients we aim to help.",
+ quoteNachname:"Lange, Teammember",
+ quoteVorname: "Kaya",
type: "meta",
- summary: "",
+ summary: [<p>
+ Early in our research, we committed to a human-centric approach, recognizing the importance of working according to Good Laboratory Practice (GLP) standards. At this point, we also identified key Special Prizes to aim for, including the Biosafety and Security Award, Best New Basic Part, and Best Integrated Human Practices. To further advance our project, we partnered with Stemcell Technologies, whose technical support provided valuable insights. This collaboration allowed us to explore the cultivation of primary human nasal epithelial cells, which are crucial for testing our synthetic biology components and Prime Editing technologies. In line with this, we expanded our biosafety standards to ensure compliance with higher safety levels and enhanced our understanding of preclinical trial-like experiments. Moreover, we are committed to raising awareness within our local community while also seeking to broaden our impact internationally. This dual focus helps us ensure both the safety and societal relevance of our project as we move forward.
+ </p>,
+
+ <ul>
+ <li>
+ <strong>Commitment to Human-Centric Approach:</strong> Early in the project, the decision to follow a human-centric approach ensured that societal impact, ethical considerations, and the needs of patients would be central to all scientific developments.
+ </li>
+ <li>
+ <strong>Selection of Special Prizes (Biosafety and Security, Best Integrated Human Practices):</strong> Prioritizing biosafety, security, and human practices from the start highlighted the project’s focus on safety, ethical responsibility, and community engagement, aligning with broader human-centric goals.
+ </li>
+ <li>
+ <strong>Collaboration with Stemcell Technologies:</strong> Partnering with industry leaders provided technical expertise that allowed the team to deepen their understanding of human cell cultivation and gene editing, ensuring that the project’s technological developments were informed by real-world applications.
+ </li>
+ <li>
+ <strong>Expansion of Biosafety Standards:</strong> Extending biosafety protocols to mimic preclinical trial conditions reinforced the commitment to safe, ethical research practices and laid the groundwork for clinical relevance, demonstrating responsibility toward future patients.
+ </li>
+ <li>
+ <strong>Local and International Community Engagement:</strong> Efforts to raise awareness at both local and global levels ensured that the project was not only scientifically sound but also socially responsible, with a focus on educating and involving the public in the conversation around cystic fibrosis and gene therapy.
+ </li>
+ </ul>
+ ],
months: "may"
},
{
@@ -683,18 +741,32 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
{
vorname: "Integrate Insights",
nachnname: "",
- pictureurl: pics['placeholder'],
+ pictureurl: pics['logo'],
job: "Team iGEM",
affiliation: "Bielfeld CeBiTec 2024",
tag: "Milestone",
- heading: "Getting Acquainted with Cystic Fibrosis",
+ heading: "From Insights to Impact: Enhancing Awareness and Prime Editing for Cystic Fibrosis",
interviewtabid: "inisghts",
cardtext: "",
quoteNachname: "Köhler, Teammember",
quoteVorname: "Vera",
- quote: "Firstly, we discussed various project ideas, including the use of magnetic microswimmers for targeted medical applications, gene editing approaches for cystic fibrosis, treatments for muscular dystrophy and combating cyanobacteria with algae.",
+ quote: "I’m excited about our partnership with Mukoviszidose e.V. Deutschland to raise awareness in our community. Educating people about cystic fibrosis and gene therapy is essential, and I believe our scientific advancements will have a broader impact beyond just this condition.",
type: "meta",
- summary: "",
+ summary: [<p>
+ During the early stages of our project, we discovered through our survey that while many participants were open to trying gene therapies, they lacked adequate knowledge about them. Additionally, most respondents were unfamiliar with cystic fibrosis, highlighting the need for greater awareness. Driven by a desire to educate the people of Bielefeld, we collaborated with Mukoviszidose e.V. Deutschland to support the Muko Move campaign, a successful initiative aimed at raising awareness about cystic fibrosis. On the scientific front, we elevated our project to a new level. With valuable feedback from Mattijs Bulcaen at the University of Leuven, we incorporated a novel RNA structural element into our Prime Editing complex, significantly improving its efficiency. After successfully optimizing the pegRNA, we moved forward with enhancing the Prime Editing protein complex. Our goal is to make Prime Editing not only safer but also easier to apply, so that our research can benefit more than just cystic fibrosis patients, ultimately broadening the impact of our work.
+ </p>,
+
+ <ul>
+ <li>
+ <strong>Survey Insights:</strong> Many participants were motivated to try gene therapies but lacked knowledge about them and cystic fibrosis, highlighting the need for better public education.
+ </li>
+ <li>
+ <strong>Awareness Campaign:</strong> Partnered with Mukoviszidose e.V. Deutschland to support the Muko Move campaign, raising awareness about cystic fibrosis in the local community.
+ </li>
+ <li>
+ <strong>Scientific Advancements:</strong> Improved the efficiency of Prime Editing by incorporating a novel RNA structural element, with further efforts to optimize the Prime Editing protein complex for broader applicability beyond cystic fibrosis.
+ </li>
+ </ul>],
months: "june"
},
{
@@ -1050,7 +1122,7 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
{
vorname: "Close the Loop",
nachnname: "",
- pictureurl: pics['placeholder'],
+ pictureurl: pics['logo'],
job: "Team iGEM",
affiliation: "Bielfeld CeBiTec 2024",
tag: "Milestone",
@@ -1369,7 +1441,7 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
{
vorname: "Present evidence",
nachnname: "",
- pictureurl: pics['placeholder'],
+ pictureurl: pics['logo'],
job: "Team iGEM",
affiliation: "Bielfeld CeBiTec 2024",
tag: "Milestone",
@@ -1568,7 +1640,7 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
{
vorname: "Connect and Share",
nachnname: "",
- pictureurl: pics['placeholder'],
+ pictureurl: pics['logo'],
job: "Team iGEM",
affiliation: "Bielfeld CeBiTec 2024",
tag: "Milestone",
@@ -1976,37 +2048,35 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
{
vorname: "Grand Jambooree in Paris",
nachnname: "",
- pictureurl: pics['placeholder'],
+ pictureurl: pics['logo'],
job: "Team iGEM",
affiliation: "Bielfeld CeBiTec 2024",
tag: "Milestone",
- heading: "Brainstorming and selection of ideas and concepts",
+ heading: "Preparations for the Grand Jambooree in Paris",
interviewtabid: "jamboree",
cardtext: "",
- quote: "",
- aimofcontact: "",
- insights: "",
- implementation: "",
+ quote: "I’m really nervous about the judging session. It feels like all our hard work is leading up to this moment, and I just hope we make a strong impression!",
+ quoteNachname: "Guckes, Teammember",
+ quoteVorname: "Isabell",
type: "meta",
- summary: "",
+ summary: "As the team gears up for the Grand Jambooree in Paris, our focus has been on finalizing our presentation materials, including a dynamic presentation video that showcases our project and research findings. We are preparing for various formats of engagement, such as presentations and talks, where we’ll share our insights with fellow participants. Additionally, our poster session will provide an opportunity for interactive discussions, and we are bracing for the judging sessions that will critically evaluate our work.",
months: "october"
},
{
vorname: "Carry It Forward",
nachnname: "",
- pictureurl: pics['placeholder'],
+ pictureurl: pics['logo'],
job: "Team iGEM",
affiliation: "Bielfeld CeBiTec 2024",
tag: "Milestone",
- heading: "Brainstorming and selection of ideas and concepts",
+ heading: "The future of our project PreCyse",
interviewtabid: "forward",
cardtext: "",
- quote: "",
- aimofcontact: "",
- insights: "",
- implementation: "",
+ quote: "I hope to start my Master's thesis with this approach to build on the team's hard work and make a difference.",
+ quoteNachname: "Lange, Teammember",
+ quoteVorname: "Kaya",
type: "meta",
- summary: "",
+ summary: [<p>As we move forward with our project, our intention is to continue our research and development at our university. We believe that by building on our current findings, we can make significant contributions to the field. Additionally, we are considering reaching out to Mattjis Bulcaen at KU Leuven for potential collaboration in the future, as their expertise could further enhance our work.</p>],
months: "october"
},