Difference between revisions of "Part:BBa K5247135"

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<h3>Experiment 1: Testing the reporter plasmid using designed pegRNAs</h3>
 
<h3>Experiment 1: Testing the reporter plasmid using designed pegRNAs</h3>
 
<h4>Experimental Workflow</h4>
 
<h4>Experimental Workflow</h4>
 +
https://static.igem.wiki/teams/5247/engineering-cycle/rep-it3.svg
 
<p>
 
<p>
 
To this end, we created pegRNAs that target our pPEAR_CFTR plasmid. Apart from a part of the reverse transcriptase template, these pegRNAs were identical to those targeting genomic CFTR.
 
To this end, we created pegRNAs that target our pPEAR_CFTR plasmid. Apart from a part of the reverse transcriptase template, these pegRNAs were identical to those targeting genomic CFTR.
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https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/se-nose.png
 
https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/se-nose.png
<p>Normalized editing efficiency of the pegRNAs according to flow cytometry</p>
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<p>flow cytometry data from</p>
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<br>
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<br>
 
https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild8.png
 
https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild8.png
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<p>Normalized editing efficiency of the pegRNAs according to flow cytometry</p>
  
 
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<h3>Experiment 2: pegRNA Screening</h3>
 
<h4>Experimental Workflow</h4>
 
<h4>Experimental Workflow</h4>
<p>For this second experiment, different 12 additional pegRNAs were designed, all including a 3' motif called trevopreQ1 described as useful for efficient prime editing in literature. Also we tested two different lenth of primer binding sequences (PBS) and three different lenght of reverse transcriptase templates (RTT). All of the pegRNAs were also tested with and without silent edits. Again, we co-transfected the pPEAR_CFTR, PE2 and pegRNA plasmids into HEK293 cells.
+
<p>
 +
For this second experiment, different 12 additional pegRNAs were designed, all including a 3' motif called trevopreQ1 described as useful for efficient prime editing in literature. Also we tested two different lenth of primer binding sequences (PBS) and three different lenght of reverse transcriptase templates (RTT). All of the pegRNAs were also tested with and without silent edits. Again, we co-transfected the pPEAR_CFTR, PE2 and pegRNA plasmids into HEK293 cells.
 
</p>
 
</p>
 
<p>
 
<p>
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</p>
 
</p>
 
https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild9.png
 
https://static.igem.wiki/teams/5247/photos/facs-results-mechanism/bild9.png
 +
 
<p>
 
<p>
We evaluated the functionality of our reporter system by co-transfecting our reporter construct with a pCMV-PE2 prime editor plasmid as well as a plasmid expressing pegRNA that targeted our reporter (see <a onClick={() => goToPagesAndOpenTab('pegrna', '/engineering')}> pegRNA engineering cycle </a>) into HEK293 cells. After 72 h we saw a significant number of cells showing fluorescence.  
+
These results show, that our reporter system is easily applicable for screening pegRNAs with grave similarities to the CFTR target. Since all pegRNAs were found to facilitate some kind of editing, this shows that even low editing efficiencies can be detected. This, alongside the noise-free negative control, aligns with our aim to create a sensitive reporter.
 
</p>
 
</p>
 
                
 
                
                        <p>
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                    " alt="Illustration of pegRNA testing using the pPEAR_CFTR system"/>   
                         
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                        </p>
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                        <figure>
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                          <img src="https://static.igem.wiki/teams/5247/engineering-cycle/rep-it3.svg" alt="Illustration of pegRNA testing using the pPEAR_CFTR system"/>   
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                           <figcaption><b>Figure 4:</b> Illustration of the pegRNA testing using the pPEAR_CFTR system. HEK293 cells transiently transformed with the pPEAR_CFTR plasmid (in the middle) show fluorescence after transformation with a prime editor and a reporter-specific pegRNA expression plasmid. The pDAS12124_PEAR-GFP-preedited is used as an internal positive control and for normalization.</figcaption>
 
                           <figcaption><b>Figure 4:</b> Illustration of the pegRNA testing using the pPEAR_CFTR system. HEK293 cells transiently transformed with the pPEAR_CFTR plasmid (in the middle) show fluorescence after transformation with a prime editor and a reporter-specific pegRNA expression plasmid. The pDAS12124_PEAR-GFP-preedited is used as an internal positive control and for normalization.</figcaption>
 
                         </figure>
 
                         </figure>

Revision as of 13:40, 2 October 2024


DNA fragment for CFTR-specific pegRNA screening

Introduction

In the context of cystic fibrosis, the F508del mutation represents a significant challenge for correction. The efficacy of current gene editing technologies hinges on the availability of precise tools to ensure the success of treatment strategies. In view of the above, we have developed a novel reporter system that is specifically tailored to the F508del mutation in the CFTR gene. The objective is to provide a high degree of comparability to the genomic context of this mutation, while maintaining ease of use. This system allows researchers to test and screen Prime Editors and various pegRNAs (prime editing guideRNAs), particularly in the context of the F508del mutation. By closely mimicking the genomic environment, it is believed that this tool will offer enhanced utility in the selection of optimal Prime Editing strategies.

Design and Functionality

The reporter system has been designed with the specific intention of facilitating a more comparable genomic context for the F508del mutation, particularly for the purpose of testing the efficacy of different pegRNA variants and prime editors. The system provides a highly reliable platform for screening a variety of pegRNAs, thereby facilitating the identification of the most effective variant for correcting the F508del mutation. The system is constructed around a plasmid structure, specifically pDAS12124_PEAR-GFP-GGTdel, from which a modified version of GFP (Green Fluorescent Protein) has been derived. The green fluorescent protein (GFP) is composed of two exons, separated by a Vim gene intron in its natural state. In the absence of the intron, the GFP is expressed and fluoresces. However, the GFP sequence was modified to introduce a three-base-pair deletion, specifically in the junction between Exon 1 and the Vim gene intron. This deletion affects the last base of Exon 1 and the first two bases of the intron, effectively disrupting the splice site. As a result, the intron is no longer correctly spliced out, leading to the expression of a non-functional GFP that does not fluoresce. img src="https://static.igem.wiki/teams/5247/engineering-cycle/rep-it3_strategy.svg

Functionality in Prime Editor and pegRNA testing

The principal feature of the reporter system is its capacity to assess and quantify the efficacy of diverse Prime Editors, with a particular focus on pegRNAs. In its default state, the system expresses a non-functional GFP due to the disruption of the splice site. However, if a Prime Editor successfully restores the mutation to its correct form, the splice site is repaired and functional GFP is expressed, thereby allowing for fluorescent detection. This fluorescence serves as a reliable indicator of successful prime editing.

Conclusion

This reporter system represents a substantial advancement in the study and correction of the CFTR F508del mutation. The design of the system allows for the straightforward screening of an array of Prime Editor and pegRNA constructs, while maintaining a high degree of comparability to the genomic context. By closely emulating the CFTR gene environment, particularly in the context of the F508del mutation, researchers are able to identify the most efficient pegRNAs and Prime Editors, offering a promising approach for developing more effective gene-editing treatments for cystic fibrosis.

Experimental Characterization

Our reporter plasmid was designed based on literature research and theoretical comsiderations. However, for our plasmid to be a useful contribution for other teams to use, its function had to be tested experimentally.

Experiment 1: Testing the reporter plasmid using designed pegRNAs

Experimental Workflow

https://static.igem.wiki/teams/5247/engineering-cycle/rep-it3.svg

To this end, we created pegRNAs that target our pPEAR_CFTR plasmid. Apart from a part of the reverse transcriptase template, these pegRNAs were identical to those targeting genomic CFTR.

I our first experiment, we tested 2 variants of pegRNAs targeting the reporter by co-transfecting the pPEAR_CFTR, a plasmid expressing the PE2 prime editor well as one pegRNA expressing plasmid, respectively into HEK293 cells. The two pegRNAs differed insofar as the one pegRNA introduced one silent edit into the target sequence and the other one three. Additionally, for positive controls we transfected a technical control plasmid as well the unmodified pDAS12124_PEAR-GFP-preedited plasmid, which could be used to determine the transfection efficiency as well as normalize the editing efficiency. As negative controls, our modified plasmid pPEAR_CFTR, pCMV-PE2 and the pegRNA plasmid were transfected individually.

Results



hek-ntc.png

Negative control of the pPEAR_CFTR plasmid transfected individually. No fluorescence could be seen, indicating no noise in the reporting system.


se-nose.png

flow cytometry data from



bild8.png

Normalized editing efficiency of the pegRNAs according to flow cytometry

Experiment 2: pegRNA Screening

Experimental Workflow

For this second experiment, different 12 additional pegRNAs were designed, all including a 3' motif called trevopreQ1 described as useful for efficient prime editing in literature. Also we tested two different lenth of primer binding sequences (PBS) and three different lenght of reverse transcriptase templates (RTT). All of the pegRNAs were also tested with and without silent edits. Again, we co-transfected the pPEAR_CFTR, PE2 and pegRNA plasmids into HEK293 cells.

The pegRNAs lead to differing amounts of cells showing fluorescence, which, assuming comparable transfection efficiencies, indicates varying prime editing efficiency.

bild9.png

These results show, that our reporter system is easily applicable for screening pegRNAs with grave similarities to the CFTR target. Since all pegRNAs were found to facilitate some kind of editing, this shows that even low editing efficiencies can be detected. This, alongside the noise-free negative control, aligns with our aim to create a sensitive reporter.

                    " alt="Illustration of pegRNA testing using the pPEAR_CFTR system"/>  
                          <figcaption>Figure 4: Illustration of the pegRNA testing using the pPEAR_CFTR system. HEK293 cells transiently transformed with the pPEAR_CFTR plasmid (in the middle) show fluorescence after transformation with a prime editor and a reporter-specific pegRNA expression plasmid. The pDAS12124_PEAR-GFP-preedited is used as an internal positive control and for normalization.</figcaption>
                       </figure>
                       <H4 text="Learn" id="learn-head"/>

Our results demonstrate three things: Firstly, the original pDAS12124_PEAR-GFP-preedited plasmid leads to undisrupted expression of eGFP in the transfected cells. Secondly, the modifications that we made to create our own, context specific PEAR plasmid prevented proper expression of eGFP in transfected, unedited cells as planned and notably with no apparent noise. The last and most important insight gained was, that editing of the reporter plasmid using respective pegRNAs successfully restores eGFP expression, proving that our reporter works as intended.

This achievement formed a convenient basis for the following optimization of prime editing in the CFTR F508del locus for us as well as other research groups.

                    </p>
                 </div>

<H3 text="Application in epithelial Cells" id="rep4head"/> <H4 text="Design" id="design-head"/> <p> Although we could show that our PEAR reporter plasmid works in a HEK cell model, according to <a onClick={() => goToPagesAndOpenTab('ignatova', '/human-practices')}> Prof.Dr. Zoya Ignatova </a> insights gained here might still not entirely transfer to cells actively expressing CFTR. As recommended, we applied our reporter to a system closer to a therapeutic target <a onClick={() => goToPageAndScroll ('Cell Culture2H', '/materials-methods')}>CFBE41o-</a>. The cells are derived from bronchial epithelial cells of a cystic fibrosis patient and are homozygous for CFTR F508del.

                       <H4 text="Build" id="build-head"/>

For experimenting in CFBE41o- cells, the same reporter construct was used as for the HEK293 test. However, we used a different prime editor (pCMV-PE6c, see prime editing systems engineering cycle<a onClick={() => goToPagesAndOpenTab('pe-systems', '/engineering')}> prime editing systems circle </a>), and only pegRNAs were used, that proved the most efficient in preceding experiments (see <a onClick={() => goToPagesAndOpenTab('pegrna', '/engineering')}> pegRNA engineering cycle </a>).

                       <H4 text="Test" id="test-head"/>

Similar to the previous cycle, we evaluated the functionality of our reporter system by co-transfecting our reporter construct with a pCMV-PE6c prime editor plasmid as well as a plasmid expressing pegRNA that targeted our reporter this time into CFBE41o- cells. After 72 h we saw a significant number of cells showing fluorescence.

Like with the experiments in HEK cells, we transfected a technical control plasmid as well the unmodified pDAS12124_PEAR-GFP-preedited plasmid as positive controls and our modified plasmid, pCMV-PE6c and the pegRNA plasmid individually as negative controls. Again, the positive controls showed solid fluorescence, while the negative control did not.

                       <figure>
                          <img src="https://static.igem.wiki/teams/5247/engineering-cycle/rep-it5.svg" alt="Illustration of applying the pPEAR_CFTR system to lung epithelial cell lines"/>  
                          <figcaption>Figure 5: Illustration of the pegRNA testing using the pPEAR_CFTR system. CFBE41o- cells transiently transformed with the pPEAR_CFTR plasmid (in the middle) show fluorescence after transformation with a prime editor and a reporter-specific pegRNA expression plasmid. The pDAS12124_PEAR-GFP-preedited is used as an internal positive control and for normalization.</figcaption>
                       </figure>
                       <H4 text="Learn" id="learn-head"/>

This experiment confirms that our reporter can not only be used in cell lines distantly related to patient cells of interest, in our case HEK203 cells, but also works in cells actively expressing CFTR and carrying the mutation. The reporter still showed no noise.

                    </p>

<H3 text="Application in Primary Cells" id="rep5head"/> <H4 text="Design" id="design-head"/> <p> The model closest to application in actual patient cells are human derived primary cells. For our last test of our modified PEAR reporter, we thus chose to use <a onClick={() => goToPageAndScroll ('Cell Culture3H', '/materials-methods')}>human nasal epithelial cells</a> derived from members of our team.

                       <H4 text="Build" id="build-head"/>

For testing our reporter in the human nasal epithelial cells, the same constructs have been used as in the previous iteration with CFBE41o- cells.

                       <H4 text="Test" id="test-head"/>

The experimental setup for this experiment was a scaled down version of the previous cycle with the only altered variable being the cells transfected. In this case, we did not observe any fluorescence, neither in the tested cells, nor the technical or pDAS12124_PEAR-GFP-preedited positive controls.

                       <H4 text="Learn" id="learn-head"/>

In this last experiment, the negative technical positive control implies a failed transfection of the cells. Thus, this attempt did not allow to draw any conclusion regarding the function of our reporter in primary cells. The experiment is to be repeated in the future.

                    </p>

<H3 text="Outlook" id="rep6head"/> <p> Our CFTR contextualized PEAR reporter proved to consistently allow detection of prime editing without notable noise, laying the foundation for optimization of existing and testing of new prime editing systems. Although very versatile in the context of targeting CFTR F508del with the <a onClick={() => goToPagesAndOpenTab('pegRNA-genau-collapsible', '/description')}>spacer of our choice</a>, a wider applicability to other genomic targets and other possible prime editor variants working differently than Cas9-based systems would be favorable. In the original PEAR plasmid however, modification of variable region is quite impractical. Also, as a part the eGFP is RCF[1000] but not RCF[10] BioBrick standard conform and hardly compatible with other parts like our <a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'pe3', path: '/engineering', tabId: 'pe-systems'})}>PreCyse cassette</a>.

                       <H4 text="Design" id="design-head"/>

This is why, as an outlook and contribution for future iGEM teams, we aim to create a more modular and compatible part similar to our PreCyse Casette. For this we made use of the experience gained when cloning pegRNAs. An oligonucleotide-based golden gate cloning site in the region of interest surrounding the 5’ splice donor site allows for cheap and convenient modification of the sequence. The area between the TypeIIS restriction sites is designed as a dropout cassette coding for a fluorescence marker expressed in E. coli, that enables rapid screening for transformants containing correctly digested plasmid backbones. Sequence and Features

Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 933
    Illegal PstI site found at 980
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 933
    Illegal PstI site found at 980
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 933
    Illegal PstI site found at 980
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 933
    Illegal PstI site found at 980
  • 1000
    COMPATIBLE WITH RFC[1000]