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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

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

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

flow cytometry data from

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.

Results

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

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.

Experiment 3: Application in epithelial Cells" id="rep4head"

Although we could show that our PEAR reporter plasmid works in a HEK cell model, insights gained here might still not entirely transfer to cells actively expressing CFTR. We applied our reporter to a system closer to a therapeutic target, CFBE41o-. The cells are derived from bronchial epithelial cells of a cystic fibrosis patient and are homozygous for CFTR F508del.

Experimental workflow

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) and only one pegRNA was used, that proved among the most efficient in preceding experiments.

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. 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.

Results

Negative control with only pPEAR_CFTR transformed into the lung epithelial cell line. No fluorescence could be seen, confirming the exceptionally low noise level of our reporter previously shown in HEK cells.

A significant number of cells co-transformed with reporter, pegRNA and prime editor plasmids showed fluorescence. Again, the positive controls showed solid fluorescence, while all the negative control did not. This indicates that our reporter is equally as applicable in this and possibly other cell lines as it is in HEK cells.

Sequence and Features