<p>The modified GFP sequence was cloned into the pDAS12124_PEAR-GFP-preedited plasmid, which was then transfected into HEK cells to initiate the pegRNA screening process. The capacity to observe the restoration of functional GFP provides a definitive indication of the efficacy of both the Prime Editor and the specific pegRNA variant under examination. Furthermore, the considerable degree of similarity between the reporter system and the actual genomic context of the CFTR mutation renders the screening process highly pertinent to the optimisation of specific applications. </p>
</Subesction>
<Subesctiontitle="Conclusion"id="Description3">
<p>This reporter system represents a substantial advancement in the study and correction of the CFTR F508del mutation. The design of the system allows for the straightforward screening of an array of Prime Editor and pegRNA constructs, while maintaining a high degree of comparability to the genomic context. By closely emulating the CFTR gene environment, particularly in the context of the F508del mutation, researchers are able to identify the most efficient pegRNAs and Prime Editors, offering a promising approach for developing more effective gene-editing treatments for cystic fibrosis. </p>
<p>This reporter system represents a substantial advancement in the study and correction of the CFTR F508del mutation. The design of the system allows for the straightforward screening of an array of Prime Editor and pegRNA constructs, while maintaining a high degree of comparability to the genomic context. By closely emulating the CFTR gene environment, particularly in the context of the F508del mutation, researchers are able to identify the most efficient pegRNAs and Prime Editors, offering a promising approach for developing more effective gene-editing treatments for cystic fibrosis.<SupScrollLinklabel="1"/></p>
<p>In connection with the optimisation of prime editing with regard to the F508del mutation, it was necessary to compare different pegRNAs, as their optimal structure always depends on the application context. We therefore designed and cloned 14 variants of pegRNAs for the target of the reporter system and then tested them on the reporter system using the PE2 system. </p>
<p>For pegRNA screening, we co-transfected the HEK293 cells with our modified reporter plasmid, the pegRNA expressing plasmid and pCMV-PE2. We were then able to measure the fluorescence after 72 hours using FACS and evaluate which pegRNA showed the highest efficiency. </p>
{/* Bild */}
{/* Bild 9 */}
<p>We also co-transfected the CFBE41o- with our modified reporter plasmid, the plasmid expressing pegRNA04 as well as pCMV-PE6c. As a result, we observed fluorescence, indicating successful editing of the reporter plasmid. The negative controls transfected with only one of the plasmids each showed no fluorescence, routing out other factors. This gave us validation, that our pegRNAs work not only in HEK, but also in epithelial cells that express CFTR F508del. </p>
{/* Bild */}
<TwoVertical
description="Microscopy results after 24h or 48h. Transfection of pDAS12124-preedited with lipofectamine 3000 was successfully done in CFBE41o- cell line and visible after 48h. CFBE41o- cell line was transfected with pDAS-IDT with Lipofectamine 3000 and afterwards with LNPs including PE6c and pegRNA4 and was after 24h fluorescence visible."
<p>Based on the results, we were able to select 4 possible candidates and one negative example, whose attributes we then used to create pegRNAs for the CFTR target. The next step is to test these pegRNAs using CFBE41o- cells by again co-transfecting these with three plasmids: reporter plasmid, pegRNA expressing plasmid and pCMV-PE6c, and measuring fluorescence after 72 hours. </p>
</Subesction>
...
...
@@ -58,10 +65,10 @@ export function Parts() {
<p>In the next series of experiments, we would like to investigate various mutation candidates, in particular the possible SpuFz1 nickases (BBa_K5247101- BBa_K5247104) and various PlmCasx nickase variants (BBa_K5247105- BBa_K5247107), in more detail using the PE6c system. </p>
{/* Bild Nikase */}
<H4text="nSpuFz1 "/>
<p>The nSpuFz1 variants are expressed in yeast strain Pichia pastoris (SMD1163), which we obtained from Nils Berelsmann{/* [link zu HP-Timeline]. */} In advance, the corresponding genes were cloned into a suitable expression vector, pPIC9K, via Gibson Assembly to ensure efficient expression of the nickases. The cloning as well as the subsequent expression and purification of the nickases were carried out according to a detailed protocol[2] under the expert guidance of Hakan Soytürk[link zu HP timeline]. </p>
<p>The nSpuFz1 variants are expressed in yeast strain Pichia pastoris (SMD1163), which we obtained from Nils Berelsmann{/* [link zu HP-Timeline]. */} In advance, the corresponding genes were cloned into a suitable expression vector, pPIC9K, via Gibson Assembly to ensure efficient expression of the nickases. The cloning as well as the subsequent expression and purification of the nickases were carried out according to a detailed protocol<SupScrollLinklabel="2"/> under the expert guidance of Hakan Soytürk[link zu HP timeline]. </p>
<H4text="nPlmCasX "/>
<p>The nPlmCasX variants are expressed in E. coli strain BL21D3, which we obtained from AG Müller{/* [link zu HP-Timeline] */}. In advance, the corresponding genes were cloned into a suitable expression vector, pZMB1029, via Gibson Assembly to ensure efficient expression of the nickases. The cloning as well as the subsequent expression and purification of the nickases were carried out according to a detailed protocol[2]. </p>
{/* Bild beide */}
<p>The nPlmCasX variants are expressed in E. coli strain BL21D3, which we obtained from AG Müller{/* [link zu HP-Timeline] */}. In advance, the corresponding genes were cloned into a suitable expression vector, pZMB1029, via Gibson Assembly to ensure efficient expression of the nickases. The cloning as well as the subsequent expression and purification of the nickases were carried out according to a detailed protocol<SupScrollLinklabel="2"/>. </p>
{/* Bild beide */}
<p>After successful purification, the isolated nickases are comprehensively analyzed according to verify their activity and efficiency. These analyses will serve to evaluate the functionality and suitability of the nickases for specific applications in prime editing. Subsequently, detailed characterization experiments are planned to determine the properties of the nickases, if functional, including their specificity, editing activity and potential for use in precise gene editing procedures. </p>
<p>Validation of the nickases will be performed in different cell lines to confirm their efficiency and reliability in a cellular context. These validation steps are crucial to further investigate the potential of Prime Guide for therapeutic applications. </p>
@@ -10,5 +10,52 @@ export default function PartSources(){
constbibtexSources=[
`
@article {10.7554/eLife.69504,
article_type = {journal},
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},
editor = {Lapinaite, Audrone and Stainier, Didier YR and Hamilton, Jennifer R},
volume = 11,
year = 2022,
month = {feb},
pub_date = {2022-02-23},
pages = {e69504},
citation = {eLife 2022;11:e69504},
doi = {10.7554/eLife.69504},
url = {https://doi.org/10.7554/eLife.69504},
abstract = {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.},
