Part:BBa_K5247135
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.
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.
Results
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Flow Cytometry analysis of pegRNAs with and without silent edits.
Experiment 2: Screening of pegRNA variants
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.
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Experiment 3: Application in lung epithelial cell lines
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We tried to transfect CFBE41o- cells with pDAS12124-preedited, our internal positive control, to check if a transfection of this cell line is possible. Furthermore we tried to co transfect the CFBE41o- with pPEAR_CFTR, PE6c and pegRNA4.
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Transfection of CFBE41o- with pDAS12124-preedited was successful (see Figure 10). After 24 hours a successful co transfection of pPEAR_CFTR with PE6c and pegRNA4 was visible, although the transfection efficiency was really bad (see Figure 10).
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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."
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Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 933
Illegal PstI site found at 980 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 933
Illegal PstI site found at 980 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 933
Illegal PstI site found at 980 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 933
Illegal PstI site found at 980 - 1000COMPATIBLE WITH RFC[1000]
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