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Deletion and replacement of long genomic sequences using prime editing


Genomic insertions, duplications and insertion/deletions (indels), which account for ~14% of human pathogenic mutations, cannot be accurately or efficiently corrected by current gene-editing methods, especially those that involve larger alterations (>100 base pairs (bp)). Here, we optimize prime editing (PE) tools for creating precise genomic deletions and direct the replacement of a genomic fragment ranging from ~1 kilobases (kb) to ~10 kb with a desired sequence (up to 60 bp) in the absence of an exogenous DNA template. By conjugating Cas9 nuclease to reverse transcriptase (PE-Cas9) and combining it with two PE guide RNAs (pegRNAs) targeting complementary DNA strands, we achieve precise and specific deletion and repair of target sequences via using this PE-Cas9-based deletion and repair (PEDAR) method. PEDAR outperformed other genome-editing methods in a reporter system and at endogenous loci, efficiently creating large and precise genomic alterations. In a mouse model of tyrosinemia, PEDAR removed a 1.38-kb pathogenic insertion within the Fah gene and precisely repaired the deletion junction to restore FAH expression in liver.

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Fig. 1: PEDAR mediates large target deletion and simultaneous insertion at an endogenous genomic locus.
Fig. 2: Flexibility of PEDAR in programming a larger deletion and insertion in HEK293T cells.
Fig. 3: PEDAR generates an in-frame deletion to restore mCherry expression in TLR cells.
Fig. 4: PEDAR corrects the pathogenic insertion in a tyrosinemia I mouse model.

Data availability

A Reporting Summary for this article is available as a Supplementary Information file. The raw gel images underlying Figs. 1c,d, 2b,c,f,g, 3d and 4f and Supplementary Figs. 2d,e, 3a,b,e,f and 4a,b are provided as a Source Data file and an additional Supplementary Data file, respectively. The NCBI ClinVar database is accessible at The raw DNA sequencing data are available at the NCBI Sequence Read Archive database under accession numbers PRJNA746292 and PRJNA746489Source data are provided with this paper.


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We thank C. Mello, P. Zamore, S. Wolfe, T. Flotte and E. Sontheimer for discussions and E. Haberlin for editing the manuscript. We thank E. Sontheimer (UMass Medical School) for providing the HEK293T-TLR cell line and M. Grompe (Oregon Health & Science University) for providing the FahΔExon5 mice. We thank Y. Liu, Y. Gu and E. Kittler in the UMass Morphology, Flow Cytometry and Deep Sequencing Cores for support. W.X. was supported by grants from the National Institutes of Health (DP2HL137167, P01HL131471 and UG3HL147367), American Cancer Society (129056-RSG-16-093), the Lung Cancer Research Foundation and the Cystic Fibrosis Foundation. T.J. was supported by grants from National Institutes of Health (K99HL153940).

Author information

Authors and Affiliations



T.J. and W.X. designed the study. T.J. performed experiments. T.J., X.-O.Z. and Z.W. analyzed the data. T.J. and W.X. wrote the manuscript with comments from all authors.

Corresponding author

Correspondence to Wen Xue.

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Competing interests

UMass has filed a patent application on this work. W.X. is a consultant for the Cystic Fibrosis Foundation Therapeutics Lab. The other authors declare no competing interests.

Additional information

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

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

Supplementary information

Supplementary Information

Supplementary Figs. 1–8 and Tables 1–3.

Reporting Summary

Supplementary Data

Unprocessed gel and blot images for Supplementary Figs. 2d,e, 3a,b,e,f and 4a,b.

Source data

Source Data Fig. 1

Unprocessed gels and blots for Fig. 1c,d.

Source Data Fig. 2

Unprocessed gels and blots for Fig. 2b,c,f,g.

Source Data Fig. 3

Unprocessed gels and blots for Fig. 3d.

Source Data Fig. 4

Unprocessed gels and blots for Fig. 4f.

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Jiang, T., Zhang, XO., Weng, Z. et al. Deletion and replacement of long genomic sequences using prime editing. Nat Biotechnol 40, 227–234 (2022).

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