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Precise genomic deletions using paired prime editing

Abstract

Current methods to delete genomic sequences are based on clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 and pairs of single-guide RNAs (sgRNAs), but can be inefficient and imprecise, with errors including small indels as well as unintended large deletions and more complex rearrangements. In the present study, we describe a prime editing-based method, PRIME-Del, which induces a deletion using a pair of prime editing sgRNAs (pegRNAs) that target opposite DNA strands, programming not only the sites that are nicked but also the outcome of the repair. PRIME-Del achieves markedly higher precision than CRISPR–Cas9 and sgRNA pairs in programming deletions up to 10 kb, with 1–30% editing efficiency. PRIME-Del can also be used to couple genomic deletions with short insertions, enabling deletions with junctions that do not fall at protospacer-adjacent motif sites. Finally, extended expression of prime editing components can substantially enhance efficiency without compromising precision. We anticipate that PRIME-Del will be broadly useful for precise, flexible programming of genomic deletions, epitope tagging and, potentially, programming genomic rearrangements.

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Fig. 1: Precise episomal deletions using PRIME-Del.
Fig. 2: Concurrent programming of deletion and insertion using PRIME-Del.
Fig. 3: Precise genomic deletions using PRIME-Del.
Fig. 4: Characterizing PRIME-Del across the genome.
Fig. 5: Potential advantages of using PRIME-Del in various genome editing applications.

Data availability

Raw sequencing data have been uploaded on the Sequencing Read Archive and made available to the public with associated BioProject accession no. PRJNA692623. Selected plasmids used for programming genomic deletions are available from Addgene (catalog nos. 172655, 172656, 172657 and 172658).

Code availability

Source code for PRIME-Del is available at https://github.com/shendurelab/Prime-del. An interactive webpage for designing pegRNAs for PRIME-Del is available at https://primedel.uc.r.appspot.com.

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Acknowledgements

We thank former and present members of the Shendure lab, including Y. Yin, J. Tomes, S. Domcke, A. Boulgakov, D. Calderon, J. Gehring, S. Srivatsan and L. Starita, for helpful discussions. We thank the David Liu laboratory at Harvard University/Howard Hughes Medical Institute for sharing the prime editing plasmids. We thank J. Gehring, J. Cuperus and the Stanley Fields laboratory at the Department of Genome Sciences, University of Washington, for their help with using a ddPCR instrument. This work was supported by the National Human Genome Research Institute (grant no. 5UM1HG009408-04). J.C. is a Howard Hughes Medical Institute Fellow of the Damon Runyon Cancer Research Foundation (DRG-2403-20). J.S. is an Investigator of the Howard Hughes Medical Institute.

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Authors

Contributions

J.C., C.C.S. and J.S. conceived the project. J.C. designed and performed experiments with guidance from W.C. and J.S., and assistance from W.C., C.C.S., C.L., F.M.C., A.L., R.M.D. and B.M. F.M.C. and W.Y. contributed to validation data. J.C., W.C. and J.S. analyzed the data. W.C. developed the software included in the manuscript. J.C. and J.S. wrote the manuscript with input from the other authors.

Corresponding authors

Correspondence to Junhong Choi or Jay Shendure.

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Competing interests

The University of Washington has filed a patent application based on this work, in which J.C., W.C. and J.S. are listed as inventors. The remaining authors declare no competing interests.

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Peer review information Nature Biotechnology thanks Daesik Kim, Bruce Conklin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–7.

Reporting Summary

Supplementary Tables 1–3

Nucleic acid sequences used in the present study.

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Choi, J., Chen, W., Suiter, C.C. et al. Precise genomic deletions using paired prime editing. Nat Biotechnol 40, 218–226 (2022). https://doi.org/10.1038/s41587-021-01025-z

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