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Precise genomic deletions using paired prime editing


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.

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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 An interactive webpage for designing pegRNAs for PRIME-Del is available at


  1. Knott, G. J. & Doudna, J. A. CRISPR–Cas guides the future of genetic engineering. Science 361, 866–869 (2018).

    Article  CAS  Google Scholar 

  2. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  Google Scholar 

  3. Canver, M. C. et al. Characterization of genomic deletion efficiency mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells. J. Biol. Chem. 289, 21312–21324 (2014).

    Article  Google Scholar 

  4. Byrne, S. M., Ortiz, L., Mali, P., Aach, J. & Church, G. M. Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells. Nucleic Acids Res. 43, e21 (2015).

    Article  Google Scholar 

  5. Gasperini, M. et al. CRISPR/Cas9-mediated scanning for regulatory elements required for HPRT1 expression via thousands of large, programmed genomic deletions. Am. J. Hum. Genet. 101, 192–205 (2017).

    Article  CAS  Google Scholar 

  6. Gasperini, M. et al. A genome-wide framework for mapping gene regulation via cellular genetic screens. Cell 176, 1516 (2019).

    Article  CAS  Google Scholar 

  7. Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat. Biotechnol. 36, 765–771 (2018).

    Article  CAS  Google Scholar 

  8. Zuccaro, M. V. et al. Allele-specific chromosome removal after Cas9 cleavage in human embryos. Cell (2020).

  9. Mehta, A. & Haber, J. E. Sources of DNA double-strand breaks and models of recombinational DNA repair. Cold Spring Harb. Perspect. Biol. 6, a016428 (2014).

    Article  Google Scholar 

  10. Diao, Y. et al. A tiling-deletion-based genetic screen for cis-regulatory element identification in mammalian cells. Nat. Methods 14, 629–635 (2017).

    Article  CAS  Google Scholar 

  11. Zhu, S. et al. Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR–Cas9 library. Nat. Biotechnol. 34, 1279–1286 (2016).

    Article  CAS  Google Scholar 

  12. Khosravi, M. A. et al. Targeted deletion of BCL11A gene by CRISPR–Cas9 system for fetal hemoglobin reactivation: a promising approach for gene therapy of beta thalassemia disease. Eur. J. Pharmacol. 854, 398–405 (2019).

    Article  CAS  Google Scholar 

  13. Dastidar, S. et al. Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells. Nucleic Acids Res. 46, 8275–8298 (2018).

    Article  CAS  Google Scholar 

  14. 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 

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

    Article  CAS  Google Scholar 

  16. Kivioja, T. et al. Counting absolute numbers of molecules using unique molecular identifiers. Nat. Methods 9, 72–74 (2011).

    Article  Google Scholar 

  17. Dominissini, D. et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485, 201–206 (2012).

    Article  CAS  Google Scholar 

  18. Watry, H. L. et al. Rapid, precise quantification of large DNA excisions and inversions by ddPCR. Sci. Rep. 10, 14896 (2020).

    Article  CAS  Google Scholar 

  19. Verkerk, A. J. et al. Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell 65, 905–914 (1991).

    Article  CAS  Google Scholar 

  20. Tippens, N. D. et al. Transcription imparts architecture, function and logic to enhancer units. Nat. Genet. 52, 1067–1075 (2020).

    Article  Google Scholar 

  21. Mandal, P. K. et al. Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. Cell Stem Cell 15, 643–652 (2014).

    Article  CAS  Google Scholar 

  22. 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 

  23. Kweon, J. et al. Engineered prime editors with PAM flexibility. Mol. Ther. (2021).

  24. Leibowitz, M. L. et al. Chromothripsis as an on-target consequence of CRISPR–Cas9 genome editing. Nat. Genet. (2021).

  25. Schene, I. F. et al. Prime editing for functional repair in patient-derived disease models. Nat. Commun. (2020).

  26. Owens, D. D. G. et al. Microhomologies are prevalent at Cas9-induced larger deletions. Nucleic Acids Res. 47, 7402–7417 (2019).

    Article  CAS  Google Scholar 

  27. Kim, D. Y. et al. Unbiased investigation of specificities of prime editing systems in human cells. Nucleic Acids Res. (2020).

  28. El-Brolosy, M. A. et al. Genetic compensation triggered by mutant mRNA degradation. Nature 568, 193–197 (2019).

    Article  CAS  Google Scholar 

  29. Ma, Z. et al. PTC-bearing mRNA elicits a genetic compensation response via Upf3a and COMPASS components. Nature 568, 259–263 (2019).

    Article  CAS  Google Scholar 

  30. Concordet, J.-P. & Haeussler, M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 46, W242–W245 (2018).

    Article  CAS  Google Scholar 

  31. 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 

  32. Kim, H. K. et al. Predicting the efficiency of prime editing guide RNAs in human cells. Nat. Biotechnol. (2020).

  33. McKenna, A. & Shendure, J. FlashFry: a fast and flexible tool for large-scale CRISPR target design. BMC Biol. 16, 74 (2018).

    Article  Google Scholar 

  34. Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    Article  CAS  Google Scholar 

  35. Chen, W. et al. Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair. Nucleic Acids Res. 47, 7989–8003 (2019).

    Article  CAS  Google Scholar 

  36. Zhang, J., Kobert, K., Flouri, T. & Stamatakis, A. PEAR: a fast and accurate Illumina Paired-End reAd mergeR. Bioinformatics 30, 614–620 (2014).

    Article  CAS  Google Scholar 

  37. Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).

    Article  CAS  Google Scholar 

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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 and Affiliations



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).

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