<p>Prime editing is a is a very precise and safe method. However, depending on the genomic locus targeted, the editing efficiency can be very low. The cystic fibrosis causing CFTR F508del mutation is, as Mattijs Bulcaen[link] stated in our interview, one of, if not the most obvious application of prime editing, considering the large amount of people affected. The lack of publications addressing CFTR target implied, that the mutation might be particularly hard to edit. At low editing efficiency, successful edits are hard, if not impossible to distinguish from the background noise using conventional methods like sanger sequencing or qPCR. As a basis to effectively test our approach and screen for working pegRNAs, we needed a highly sensitive method of detection with as little noise as possible to optimize our prime editing approach for genomic CFTR targeting.</p>
</div>
<divclassName="box">
<pid="rep1">
<H3text="rep1"id="rep1head"/>
<LoremShort></LoremShort>
<H3text="A Fluorescence Reporter"id="rep1head"/>
<H4text="Design"id="design-head"/>
<p>
We reasoned that the easiest way of detecting DNA changes in a cell would be fluorescence. Our initial idea was to create pegRNAs targeting the coding sequence of a fluorescent protein, that would introduce a mutation resulting in a different emission, giving easily detectable feedback of correct editing. The original Aequorea victoria GFP protein differs from avGFP(Y66W), emitting light in a wavelength of around 509 nm (cyan), and avGFP(Y66H), emitting light in a wavelength of around 448 nm (blue) by only one amino acid substitution each.<TabScrollLinktab="tab-reporter"num="1"scrollId="desc-1"/> Prime editing could therefore be visualized by facilitating these substitutions with a prime editor.
</p>
<H4text="Build"id="build-head"/>
<p>
To this end, the wild-type and edited versions of the avGFP were put in contrast and we started searching for potential pegRNAs for editing one into the other.
</p>
<H4text="Test"id="test-head"/>
<p>
When trying to find protospacers for Cas9 and other possible nickases[link], 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 Mattijs Bulcaen[link] 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>
<H4text="Learn"id="learn-head"/>
<p>
From our observations we learned that a reporter system is only of use, if it can really mimic the genomic target of choice. The adjustments to be made to create a pegRNA targeting the genomic target from a pegRNA targeting the reporter should be as minor as possible. This includes a similar spacer and a similar edit to be made.
</p>
</p>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
</div>
<divclassName="box">
<pid="rep2">
<H3text="rep2"id="rep2head"/>
<LoremShort></LoremShort>
<H3text="Proof of Concept for PEAR"id="rep2head"/>
<H4text="Design"id="design-head"/>
<p>
After extensive research we came across the prime editor activity reporter (PEAR) created by Simon et al. (2022)<TabScrollLinktab="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.
</p>
<H4text="Build"id="build-head"/>
<p>
Since none of us had any experience in prime editing before our project, we wanted to test whether we can facilitate prime editing in the first place. To do this and also assess the functionality of the PEAR system, we set up a proof of concept using the PEAR 2in1 system. This plasmid includes not only the eGFP with and intron and disrupted 5’ splice site, but also a pegRNA expression cassette. The pegRNA is designed in a way that, in combination with a prime editing protein complex, corrects the disrupted splicing signal.
</p>
<H4text="Test"id="test-head"/>
<p>
In the experiment, we transfected HEK293 cells (as recommended by Mattijs Bulcaen[link]) 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.
</p>
<H4text="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.
</p>
</p>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
</div>
<divclassName="box">
<pid="rep3">
<H3text="rep3"id="rep3head"/>
<LoremShort></LoremShort>
<H3text="Contextualization of PEAR"id="rep3head"/>
<H4text="Design"id="design-head"/>
<p>
The original PEAR plasmid pDAS12124_PEAR-GFP-preedited that we bought from AddGene represents, as the name suggests, how the reporter should look like after successful editing and can thus be used as a positive control and for normalization. To alter the PEAR plasmid so that it mimics the mutated genomic CFTR target, we first analyzed the region surrounding CFTR F508del mutation. As the mutation is a three base pair deletion, we introduced the very same at the 5’ splicing signal. For this modification to reliably disrupt intron splicing and thus eGFP expression, we effectively removed the GT bases of the intronic 5’ splice donor site as well as the preceding, exonic G base of the 5’ flanking sequence. Secondly, we replaced the intronic region downstream of the four base pair 3’ flanking region with the respective sequence from the CFTR locus. This 27 bp substitute included a PAM sequence, an entire spacer as well as four additional base pairs in between present in the original gene sequence. Lastly, we introduced silent mutations upstream of the 5’ flanking sequence that lowered the GC content. This was to mimic the AT-rich region preceding the F508del mutation in the CFTR gene. This reveals one of the necessary shortcomings of this reporter: Edits upstream of the 5’ donor site are heavily restricted by the eGFP coding sequence.
</p>
<H4text="Build"id="build-head"/>
<p>
We constructed the reporter system by first analyzing the original plasmid to identify appropriate restriction sites. We then digested the plasmid backbone and cloned in a gene synthesis fragment ordered at IDT containing the edits via Gibson Assembly cloning. The correct cloning was validated first by colony PCR and then by sequencing the regions of the plasmid containing the cloning sites and our modifications.
</p>
<H4text="Test"id="test-head"/>
<p>
We evaluated the functionality of our reporter system by co-transfecting our reporter construct with a pCMV-PE2 prime editor plasmid as well as a plasmid expressing pegRNA that targeted our reporter (see pegRNA engineering cycle[link]) into HEK293 cells. After 72 h we saw a significant number of cells showing fluorescence.
</p>
<p>
Additionally, for positive controls we transfected a technical control plasmid as well the unmodified pDAS12124_PEAR-GFP-preedited plasmid, which could be used to determine the transfection efficiency as well as normalize the editing efficiency. As negative controls, our modified plasmid, pCMV-PE2 and the pegRNA plasmid were transfected. The positive controls showed fluorescence, while the negative control did not.
</p>
<H4text="Learn"id="learn-head"/>
<p>
Our results demonstrate three things: Firstly, the original pDAS12124_PEAR-GFP-preedited plasmid leads to undisrupted expression of eGFP in the transfected cells. Secondly, the modifications that we made to create our own, context specific PEAR plasmid prevented proper expression of eGFP in transfected, unedited cells as planned and notably with no apparent noise. The last and most important insight gained was, that editing of the reporter plasmid using respective pegRNAs successfully restores eGFP expression, proving that our reporter works as intended.
</p>
<p>
<b>This achievement formed a convenient basis for the following optimization of prime editing in the CFTR F508del locus for us as well as other research groups.</b>
</p>
</p>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
</div>
<divclassName="box">
<pid="rep4">
<H3text="rep4"id="rep4head"/>
<LoremShort></LoremShort>
<H3text="Application in epithelial Cells"id="rep4head"/>
<H4text="Design"id="design-head"/>
<p>
Although we could show that our PEAR reporter plasmid works in a HEK cell model, according to Prof. Dr. Zoya Ignatova[link] 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.
</p>
<H4text="Build"id="build-head"/>
<p>
For experimenting in CFBE41o- cells, the same reporter construct was used as for the HEK293 test. However, we used a different prime editor (pCMV-PE6c, see prime editing systems engineering cycle[link]), and only pegRNAs were used, that proved the most efficient in preceding experiments (see pegRNA engineering cycle[link]).
</p>
<H4text="Test"id="test-head"/>
<p>
Similar to the previous cycle, we evaluated the functionality of our reporter system by co-transfecting our reporter construct with a pCMV-PE6c prime editor plasmid as well as a plasmid expressing pegRNA that targeted our reporter this time into CFBE41o- cells. After 72 h we saw a significant number of cells showing fluorescence.
</p>
<p>
Like with the experiments in HEK cells, we transfected a technical control plasmid as well the unmodified pDAS12124_PEAR-GFP-preedited plasmid as positive controls and our modified plasmid, pCMV-PE6c and the pegRNA plasmid individually as negative controls. Again, the positive controls showed solid fluorescence, while the negative control did not.
</p>
<H4text="Learn"id="learn-head"/>
<p>
This experiment confirms that our reporter can not only be used in cell lines distantly related to patient cells of interest, in our case HEK203 cells, but also works in cells actively expressing CFTR and carrying the mutation. The reporter still showed no noise.
</p>
</p>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
</div>
<divclassName="box">
<pid="rep5">
<H3text="rep5"id="rep5head"/>
<LoremShort></LoremShort>
<H3text="Application in Primary Cells"id="rep5head"/>
<H4text="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.
</p>
<H4text="Build"id="build-head"/>
<p>
For testing our reporter in the human nasal epithelial cells, the same constructs have been used as in the previous iteration with CFBE41o- cells.
</p>
<H4text="Test"id="test-head"/>
<p>
The experimental setup for this experiment was a scaled down version of the previous cycle with the only altered variable being the cells transfected. In this case, we did not observe any fluorescence, neither in the tested cells, nor the technical or pDAS12124_PEAR-GFP-preedited positive controls.
</p>
<H4text="Learn"id="learn-head"/>
<p>
In this last experiment, the negative technical positive control implies a failed transfection of the cells. Thus, this attempt did not allow to draw any conclusion regarding the function of our reporter in primary cells. The experiment is to be repeated in the future.
</p>
</p>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
</div>
<divclassName="box">
<pid="rep6">
<H3text="rep6"id="rep6head"/>
<LoremShort></LoremShort>
<H3text="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].
</p>
<H4text="Design"id="design-head"/>
<p>
This is why, as an outlook and contribution for future iGEM teams, we created a more modular and compatible part. For this we made use of the experience gained when cloning pegRNAs. An oligonucleotide-based golden gate cloning site in the region of interest surrounding the 5’ splice donor site allows for cheap and convenient modification of the sequence. The area between the TypeIIS restriction sites is designed as a dropout cassette coding for a fluorescence marker expressed in E. coli, that enables rapid screening for transformants containing correctly digested plasmid backbones.
</p>
{/* <H4 text="Build" id="build-head"/>
<p>
</p>
<H4 text="Test" id="test-head"/>
<p>
</p>
<H4 text="Learn" id="learn-head"/>
<p>
</p> */}
</p>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
</div>
<Sectiontitle="References"id="references">
<EngRepsources/>
...
...
@@ -419,42 +509,107 @@ export function Engineering() {
<divclassName="box">
<pid="peg1">
<H3text="peg1"id="peg1head"/>
<LoremShort></LoremShort>
<H4text="Design"id="design-head"/>
<p>
</p>
<H4text="Build"id="build-head"/>
<p>
</p>
<H4text="Test"id="test-head"/>
<p>
</p>
<H4text="Learn"id="learn-head"/>
<p>
</p>
</p>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
</div>
<divclassName="box">
<pid="peg2">
<H3text="peg2"id="peg2head"/>
<LoremShort></LoremShort>
<H4text="Design"id="design-head"/>
<p>
</p>
<H4text="Build"id="build-head"/>
<p>
</p>
<H4text="Test"id="test-head"/>
<p>
</p>
<H4text="Learn"id="learn-head"/>
<p>
</p>
</p>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
</div>
<divclassName="box">
<pid="peg3">
<H3text="peg3"id="peg3head"/>
<LoremShort></LoremShort>
<H4text="Design"id="design-head"/>
<p>
</p>
<H4text="Build"id="build-head"/>
<p>
</p>
<H4text="Test"id="test-head"/>
<p>
</p>
<H4text="Learn"id="learn-head"/>
<p>
</p>
</p>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
</div>
<divclassName="box">
<pid="peg4">
<H3text="peg4"id="peg4head"/>
<LoremShort></LoremShort>
<H4text="Design"id="design-head"/>
<p>
</p>
<H4text="Build"id="build-head"/>
<p>
</p>
<H4text="Test"id="test-head"/>
<p>
</p>
<H4text="Learn"id="learn-head"/>
<p>
</p>
</p>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
</div>
<divclassName="box">
<pid="peg5">
<H3text="peg5"id="peg5head"/>
<LoremShort></LoremShort>
<H4text="Design"id="design-head"/>
<p>
</p>
<H4text="Build"id="build-head"/>
<p>
</p>
<H4text="Test"id="test-head"/>
<p>
</p>
<H4text="Learn"id="learn-head"/>
<p>
</p>
</p>
<p><LoremShort></LoremShort></p>
<p><LoremShort></LoremShort></p>
</div>
<Sectiontitle="References"id="references">
<EngPegsources/>
...
...
@@ -495,67 +650,106 @@ export function Engineering() {
<divclassName="box">
<pid="nic2">
<H3text="Fusion Protein from GtFz1 & SpuFz1"id="nic2head"/>
<H4text="Design"id="text"/>
<p>In our ongoing exploration of Fanzor proteins, we identified another potential candidate, GtFz1, which had a suitable TAM sequence for our target application of correcting the F508del mutation in cystic fibrosis. However, GtFz1 showed low cutting efficiency in the tests reported in the literature. To address this, we devised a strategy to combine the favorable TAM-binding region of GtFz1 with the higher cutting efficiency of SpuFz1. Specifically, we planned to engineer a fusion protein by replacing the TAM-binding domain of SpuFz1 with that of GtFz1. The idea was to create a new endonuclease with the optimal TAM sequence for our application and a robust DNA cutting ability.</p>
<p>Given that we were swapping entire domains rather than just single amino acids, we realized that the fusion protein might not retain the ideal TAM-binding efficiency or cutting efficiency of the original proteins. Our strategy was to create a fusion protein that could bind to the TAM site and perform DNA cutting to a certain extent, albeit weakly. We planned to use directed evolution techniques, such as Phage Assisted Continuous Evolution (PACE), to enhance these functionalities over time. This approach relies on having a starting point with some degree of the desired activity, which can then be incrementally improved through evolution.</p>
@@ -10,5 +10,34 @@ export default function EngRepsources(){
constbibtexSources=[
`
@article{Heim_Prasher_Tsien_1994,
title = {Wavelength mutations and posttranslational autoxidation of green fluorescent protein.},
author = {Heim, R and Prasher, D C and Tsien, R Y},
year = 1994,
month = dec,
journal = {Proceedings of the National Academy of Sciences},
publisher = {Proceedings of the National Academy of Sciences},
volume = 91,
number = 26,
pages = {12501–12504},
doi = {10.1073/pnas.91.26.12501},
abstractnote = {The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is an unusual protein with strong visible absorbance and fluorescence from a p-hydroxybenzylidene-imidazolidinone chromophore, which is generated by cyclization and oxidation of the protein’s own Ser-Tyr-Gly sequence at positions 65-67. Cloning of the cDNA and heterologous expression of fluorescent protein in a wide variety of organisms indicate that this unique posttranslational modification must be either spontaneous or dependent only on ubiquitous enzymes and reactants. We report that formation of the final fluorophore requires molecular oxygen and proceeds with a time constant (approximately 4 hr at 22 degrees C and atmospheric pO2) independent of dilution, implying that the oxidation does not require enzymes or cofactors. GFP was mutagenized and screened for variants with altered spectra. The most striking mutant fluoresced blue and contained histidine in place of Tyr-66. The availability of two visibly distinct colors should significantly extend the usefulness of GFP in molecular and cell biology by enabling in vivo visualization of differential gene expression and protein localization and measurement of protein association by fluorescence resonance energy transfer.}
}
`,`
@article{Simon,
title = {PEAR, a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells},
author = {Simon, Dorottya Anna and Tálas, András and Kulcsár, Péter István and Biczók, Zsuzsanna and Krausz, Sarah Laura and Várady, György and Welker, Ervin},
year = 2022,
month = feb,
journal = {eLife},
publisher = {eLife Sciences Publications, Ltd},
volume = 11,
pages = {e69504},
doi = {10.7554/eLife.69504},
issn = {2050-084X},
abstractnote = {Prime editing is a recently developed CRISPR/Cas9 based gene engineering tool that allows the introduction of short insertions, deletions, and substitutions into the genome. However, the efficiency of prime editing, which typically achieves editing rates of around 10%–30%, has not matched its versatility. Here, we introduce the prime editor activity reporter (PEAR), a sensitive fluorescent tool for identifying single cells with prime editing activity. PEAR has no background fluorescence and specifically indicates prime editing events. Its design provides apparently unlimited flexibility for sequence variation along the entire length of the spacer sequence, making it uniquely suited for systematic investigation of sequence features that influence prime editing activity. The use of PEAR as an enrichment marker for prime editing can increase the edited population by up to 84%, thus significantly improving the applicability of prime editing for basic research and biotechnological applications.},
editor = {Lapinaite, Audrone and Stainier, Didier YR and Hamilton, Jennifer R}
