Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

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

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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

Similar content being viewed by others

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.

References

  1. Pickar-Oliver, A. & Gersbach, C. A. The next generation of CRISPR–Cas technologies and applications. Nat. Rev. Mol. Cell Biol. 20, 490–507 (2019).

    Article  CAS  Google Scholar 

  2. Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).

    Article  CAS  Google Scholar 

  3. Liu, Y. et al. Efficient generation of mouse models with the prime editing system. Cell Discov. 6, 27 (2020).

    Article  CAS  Google Scholar 

  4. Lin, Q. et al. Prime genome editing in rice and wheat. Nat. Biotechnol. 38, 582–585 (2020).

    Article  CAS  Google Scholar 

  5. Meier, J. A., Zhang, F. & Sanjana, N. E. GUIDES: sgRNA design for loss-of-function screens. Nat. Methods 14, 831–832 (2017).

    Article  CAS  Google Scholar 

  6. Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR–Cas9. Nat. Biotechnol. 34, 184–191 (2016).

    Article  CAS  Google Scholar 

  7. Listgarten, J. et al. Prediction of off-target activities for the end-to-end design of CRISPR guide RNAs. Nat. Biomed. Eng. 2, 38–47 (2018).

    Article  CAS  Google Scholar 

  8. Moreno-Mateos, M. A. et al. CRISPRscan: designing highly efficient sgRNAs for CRISPR–Cas9 targeting in vivo. Nat. Methods 12, 982–988 (2015).

    Article  CAS  Google Scholar 

  9. Haeussler, M. et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 17, 148 (2016).

    Article  Google Scholar 

  10. Labun, K. et al. CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res. 47, W171–W174 (2019).

    Article  CAS  Google Scholar 

  11. Park, J., Bae, S. & Kim, J.-S. Cas-Designer: a web-based tool for choice of CRISPR–Cas9 target sites. Bioinformatics 31, 4014–4016 (2015).

    Article  CAS  Google Scholar 

  12. Needleman, S. B. & Wunsch, C. D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol. 48, 443–453 (1970).

    Article  CAS  Google Scholar 

  13. Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome targeting with near-PAMless engineered CRISPR–Cas9 variants. Science 368, 290–296 (2020).

    Article  CAS  Google Scholar 

  14. Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).

    Article  CAS  Google Scholar 

Download references

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
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jennifer S. Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Johanna Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sidi Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Additional information

Peer review information Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41551-020-00622-8

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research