Prime editing enables diverse genomic alterations to be written into target sites without requiring double-strand breaks or donor templates. The design of prime-editing guide RNAs (pegRNAs), which must be customized for each edit, can however be complex and time consuming. Compared with single guide RNAs (sgRNAs), pegRNAs have an additional 3′ extension composed of a primer binding site and a reverse-transcription template. Here we report a web tool, which we named pegFinder (http://pegfinder.sidichenlab.org), for the rapid design of pegRNAs from reference and edited DNA sequences. pegFinder can incorporate sgRNA on-target and off-target scoring predictions into its ranking system, and nominates secondary nicking sgRNAs for increasing editing efficiency. CRISPR-associated protein 9 variants with expanded targeting ranges are also supported. To facilitate downstream experimentation, pegFinder produces a comprehensive table of candidate pegRNAs, along with oligonucleotide sequences for cloning.
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The main data supporting the results in this study are available within the paper and its Supplementary Information. For the pegRNAs that were experimentally tested in this study, all relevant information is provided as Supplementary Information. This information can be used to recreate the pegRNA designs described here, via the pegFinder web portal (http://pegfinder.sidichenlab.org).
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We thank S. Eisenbarth for support. R.D.C. is supported by the Yale NIH Medical Scientist Training Program (MSTP) training grant (no. T32GM136651) and an NIH National Research Service Award (NRSA) fellowship from NCI (no. F30CA250249). J.S.C. is supported by the Yale MSTP training grant from NIH (no. T32GM136651) and an NIH NSRA fellowship from NHLBI (no. F30HL149151). S.C. is supported by Yale SBI/Genetics Startup Fund, NIH/NCI/NIDA (nos. DP2CA238295, 1R01CA231112, U54CA209992-8697, R33CA225498, RF1DA048811), DoD (no. W81XWH-20-1-0072/BC190094), AACR (nos. 499395, 17-20-01-CHEN), Cancer Research Institute (CLIP), V Foundation, Ludwig Family Foundation, Sontag Foundation (DSA), Blavatnik Family Foundation and Chenevert Family Foundation.
The authors declare no competing interests. For full disclosure, S.C. is a co-founder, funding recipient and scientific advisor of EvolveImmune Therapeutics; the company has no relation to this study.
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Supplementary figures and notes.
DNA sequences used as inputs to pegFinder.
Example results produced by pegFinder, detailing candidate pegRNAs and cloning oligos for generating mutant KRAS G12D in human cells.
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Chow, R.D., Chen, J.S., Shen, J. et al. A web tool for the design of prime-editing guide RNAs. Nat Biomed Eng 5, 190–194 (2021). https://doi.org/10.1038/s41551-020-00622-8
Nature Biotechnology (2021)