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A web tool for the design of prime-editing guide RNAs

Nature Biomedical Engineering volume 5, pages 190–194 (2021)Cite this article

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Abstract

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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Fig. 1: pegFinder, a pegRNA designer for CRISPR prime editing.
Fig. 2: Experimental validation of pegRNAs designed by pegFinder.

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Data availability

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).

Code availability

The custom code is available at GitHub (https://github.com/rdchow/pegfinder). The web portal is accessible at http://pegfinder.sidichenlab.org.

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Acknowledgements

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.

Author information

Author notes

  1. These authors contributed equally: Ryan D. Chow, Jennifer S. Chen.

Authors and Affiliations

  1. Department of Genetics, Yale University School of Medicine, New Haven, CT, USA

    Ryan D. Chow, Johanna Shen & Sidi Chen

  2. Systems Biology Institute, Yale University, West Haven, CT, USA

    Ryan D. Chow, Johanna Shen & Sidi Chen

  3. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA

    Ryan D. Chow, Johanna Shen & Sidi Chen

  4. M.D.-Ph.D. Program, Yale University, New Haven, CT, USA

    Ryan D. Chow, Jennifer S. Chen & Sidi Chen

  5. Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA

    Jennifer S. Chen

  6. Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA

    Jennifer S. Chen

  7. Immunobiology Program, Yale University, New Haven, CT, USA

    Jennifer S. Chen & Sidi Chen

  8. Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT, USA

    Sidi Chen

  9. Yale Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT, USA

    Sidi Chen

  10. Department of Neurosurgery, Yale University School of Medicine, New Haven, CT, USA

    Sidi Chen

  11. Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT, USA

    Sidi Chen

  12. Yale Liver Center, Yale University School of Medicine, New Haven, CT, USA

    Sidi Chen

  13. Yale Center for Biomedical Data Science, Yale University School of Medicine, New Haven, CT, USA

    Sidi Chen

  14. Center for RNA Science and Medicine, Yale University School of Medicine, New Haven, CT, USA

    Sidi Chen

Authors
  1. Ryan D. Chow
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  2. Jennifer S. Chen
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  4. Sidi Chen
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Contributions

R.D.C. conceived the pegRNA design tool and developed the pegFinder algorithm. J.S.C. developed the web interface. R.D.C. and J.S. performed experiments. R.D.C. and J.S.C. wrote the manuscript. S.C. provided conceptual advice and supervised the work.

Corresponding author

Correspondence to Sidi Chen.

Ethics declarations

Competing interests

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 information

Supplementary Information

Supplementary figures and notes.

Reporting Summary

Peer review information

Supplementary Dataset 1

DNA sequences used as inputs to pegFinder.

Supplementary Dataset 2

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

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