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Commit 5abd2fa0 authored by Philip Mundt's avatar Philip Mundt
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added links to engineering page

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src/contents/engineering.tsx
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import { ButtonOneEngineering } from "../components/Buttons"; import { ButtonOneEngineering } from "../components/Buttons";
import { LoremShort } from "../components/Loremipsum";
import { openElement } from "../utils/openElement"; import { openElement } from "../utils/openElement";
import { H2, H3, H4, H5, PhilipH3 } from "../components/Headings"; import { H2, H3, H4, H5, PhilipH3 } from "../components/Headings";
import { useTabNavigation } from "../utils/TabNavigation"; import { useTabNavigation } from "../utils/TabNavigation";
...@@ -87,7 +86,7 @@ export function Engineering() { ...@@ -87,7 +86,7 @@ export function Engineering() {
</figure> </figure>
<H4 text="Test" id="test-head"/> <H4 text="Test" id="test-head"/>
<p> <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> </p>
<H4 text="Learn" id="learn-head"/> <H4 text="Learn" id="learn-head"/>
<p> <p>
...@@ -100,7 +99,7 @@ export function Engineering() { ...@@ -100,7 +99,7 @@ export function Engineering() {
<H3 text="Proof of Concept for PEAR" id="rep2head"/> <H3 text="Proof of Concept for PEAR" id="rep2head"/>
<H4 text="Design" id="design-head"/> <H4 text="Design" id="design-head"/>
<p> <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 context”. 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 context”. We reasoned that this system might be flexible, and sensitive enough to build our optimizations strategies upon.
</p> </p>
<H4 text="Build" id="build-head"/> <H4 text="Build" id="build-head"/>
<p> <p>
...@@ -108,7 +107,7 @@ export function Engineering() { ...@@ -108,7 +107,7 @@ export function Engineering() {
</p> </p>
<H4 text="Test" id="test-head"/> <H4 text="Test" id="test-head"/>
<p> <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> </p>
<figure> <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"/> <img src="https://static.igem.wiki/teams/5247/engineering-cycle/rep-it2.svg" alt="Illustration of the proof of concept using the PEAR2in1 system"/>
...@@ -116,7 +115,7 @@ export function Engineering() { ...@@ -116,7 +115,7 @@ export function Engineering() {
</figure> </figure>
<H4 text="Learn" id="learn-head"/> <H4 text="Learn" id="learn-head"/>
<p> <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>
</p> </p>
</div> </div>
...@@ -160,7 +159,7 @@ export function Engineering() { ...@@ -160,7 +159,7 @@ export function Engineering() {
<H3 text="Application in epithelial Cells" id="rep4head"/> <H3 text="Application in epithelial Cells" id="rep4head"/>
<H4 text="Design" id="design-head"/> <H4 text="Design" id="design-head"/>
<p> <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> </p>
<H4 text="Build" id="build-head"/> <H4 text="Build" id="build-head"/>
<p> <p>
...@@ -188,7 +187,7 @@ export function Engineering() { ...@@ -188,7 +187,7 @@ export function Engineering() {
<H3 text="Application in Primary Cells" id="rep5head"/> <H3 text="Application in Primary Cells" id="rep5head"/>
<H4 text="Design" id="design-head"/> <H4 text="Design" id="design-head"/>
<p> <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> </p>
<H4 text="Build" id="build-head"/> <H4 text="Build" id="build-head"/>
<p> <p>
...@@ -208,7 +207,7 @@ export function Engineering() { ...@@ -208,7 +207,7 @@ export function Engineering() {
<p id="rep6"> <p id="rep6">
<H3 text="Outlook" id="rep6head"/> <H3 text="Outlook" id="rep6head"/>
<p> <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> </p>
<H4 text="Design" id="design-head"/> <H4 text="Design" id="design-head"/>
<p> <p>
...@@ -547,7 +546,7 @@ export function Engineering() { ...@@ -547,7 +546,7 @@ export function Engineering() {
<p id="peg1"> <p id="peg1">
<H3 text="Initial pegRNA Design and Silent Edits" id="peg1head"/> <H3 text="Initial pegRNA Design and Silent Edits" id="peg1head"/>
<p> <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> </p>
<H4 text="Design" id="design-head"/> <H4 text="Design" id="design-head"/>
<p> <p>
...@@ -568,7 +567,7 @@ export function Engineering() { ...@@ -568,7 +567,7 @@ export function Engineering() {
</p> </p>
<H4 text="Test" id="test-head"/> <H4 text="Test" id="test-head"/>
<p> <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> </p>
<H4 text="Learn" id="learn-head"/> <H4 text="Learn" id="learn-head"/>
<p> <p>
...@@ -580,7 +579,7 @@ export function Engineering() { ...@@ -580,7 +579,7 @@ export function Engineering() {
<p id="peg2"> <p id="peg2">
<H3 text="Screening of pegRNA variants" id="peg2head"/> <H3 text="Screening of pegRNA variants" id="peg2head"/>
<p> <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> </p>
<H4 text="Design" id="design-head"/> <H4 text="Design" id="design-head"/>
<p> <p>
...@@ -614,11 +613,11 @@ export function Engineering() { ...@@ -614,11 +613,11 @@ export function Engineering() {
</p> </p>
<H4 text="Design" id="design-head"/> <H4 text="Design" id="design-head"/>
<p> <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> </p>
<H4 text="Build" id="build-head"/> <H4 text="Build" id="build-head"/>
<p> <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> </p>
<H4 text="Test" id="test-head"/> <H4 text="Test" id="test-head"/>
<p> <p>
...@@ -646,7 +645,7 @@ export function Engineering() { ...@@ -646,7 +645,7 @@ export function Engineering() {
</p> </p>
<H4 text="Test" id="test-head"/> <H4 text="Test" id="test-head"/>
<p> <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> </p>
<H4 text="Learn" id="learn-head"/> <H4 text="Learn" id="learn-head"/>
<p> <p>
...@@ -676,7 +675,7 @@ export function Engineering() { ...@@ -676,7 +675,7 @@ export function Engineering() {
</p> </p>
<H4 text="Test" id="test-head"/> <H4 text="Test" id="test-head"/>
<p> <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> </p>
<H4 text="Learn" id="learn-head"/> <H4 text="Learn" id="learn-head"/>
<p> <p>
...@@ -703,8 +702,6 @@ export function Engineering() { ...@@ -703,8 +702,6 @@ export function Engineering() {
<div className="eng-box box" > <div className="eng-box box" >
<H2 id="nickase-header" text="Alternative Nickases"></H2> <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> <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>
<div className="box" > <div className="box" >
<p id="nic1"> <p id="nic1">
...@@ -719,6 +716,26 @@ export function Engineering() { ...@@ -719,6 +716,26 @@ export function Engineering() {
<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. 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> </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"/> <H4 text="Build" id="text"/>
<p> <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. 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.
...@@ -744,26 +761,6 @@ export function Engineering() { ...@@ -744,26 +761,6 @@ export function Engineering() {
<p> <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. 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>
<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> </p>
<div className="box" > <div className="box" >
...@@ -809,32 +806,32 @@ export function Engineering() { ...@@ -809,32 +806,32 @@ export function Engineering() {
</p> </p>
<H4 text="Test" id="test-head"/> <H4 text="Test" id="test-head"/>
<p> <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> </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"/> <H4 text="Learn" id="learn-head"/>
<p> <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. 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>
<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> </p>
</div> </div>
<div className="box" > <div className="box" >
...@@ -845,8 +842,29 @@ export function Engineering() { ...@@ -845,8 +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. 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>
<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> </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> <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. 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> </p>
...@@ -862,11 +880,31 @@ export function Engineering() { ...@@ -862,11 +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. 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>
<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>
<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> </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> <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. 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> </p>
...@@ -874,8 +912,49 @@ export function Engineering() { ...@@ -874,8 +912,49 @@ export function Engineering() {
</InfoBox> </InfoBox>
<H4 text="Build" id="build-head"/> <H4 text="Build" id="build-head"/>
<p> <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> </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"/> <H4 text="Test" id="test-head"/>
<p> <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. 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.
...@@ -890,96 +969,7 @@ export function Engineering() { ...@@ -890,96 +969,7 @@ export function Engineering() {
<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. 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>
<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> </p>
</div> </div>
<div className="box" > <div className="box" >
...@@ -995,22 +985,19 @@ export function Engineering() { ...@@ -995,22 +985,19 @@ export function Engineering() {
</p> </p>
<H4 text="Test" id="test-head"/> <H4 text="Test" id="test-head"/>
<p> <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> </p>
<H4 text="Learn" id="learn-head"/> <H4 text="Learn" id="learn-head"/>
<p> <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. 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> </p>
<div className="row align-items-center"> <div className="row align-items-center">
<div className="col"> <figure>
<figure> <img src="https://static.igem.wiki/teams/5247/engineering-cycle/nickase-assay.webp" style={{height:"75%", width:"75%"}}/>
<img src="https://static.igem.wiki/teams/5247/engineering-cycle/nickase-assay.webp" style={{height:"75%", width:"75%"}}/> <figcaption>
<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.
<b>Figure 1:</b> </figcaption>
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. </figure>
</figcaption>
</figure>
</div>
</div> </div>
</p> </p>
</div> </div>
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