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Prime editing with genuine Cas9 nickases minimizes unwanted indels

Author

Listed:
  • Jaesuk Lee

    (Institute for Basic Science
    Seoul National University)

  • Kayeong Lim

    (Institute for Basic Science
    Korea Institute of Science and Technology)

  • Annie Kim

    (Institute for Basic Science)

  • Young Geun Mok

    (Institute for Basic Science
    GreenGene Inc)

  • Eugene Chung

    (Institute for Basic Science
    Seoul National University)

  • Sung-Ik Cho

    (Institute for Basic Science
    Seoul National University
    Yonsei University College of Medicine)

  • Ji Min Lee

    (Institute for Basic Science
    Seoul National University)

  • Jin-Soo Kim

    (Institute for Basic Science
    National University of Singapore)

Abstract

Unlike CRISPR-Cas9 nucleases, which yield DNA double-strand breaks (DSBs), Cas9 nickases (nCas9s), which are created by replacing key catalytic amino-acid residues in one of the two nuclease domains of S. pyogenesis Cas9 (SpCas9), produce nicks or single-strand breaks. Two SpCas9 variants, namely, nCas9 (D10A) and nCas9 (H840A), which cleave target (guide RNA-pairing) and non-target DNA strands, respectively, are widely used for various purposes, including paired nicking, homology-directed repair, base editing, and prime editing. In an effort to define the off-target nicks caused by these nickases, we perform Digenome-seq, a method based on whole genome sequencing of genomic DNA treated with a nuclease or nickase of interest, and find that nCas9 (H840A) but not nCas9 (D10A) can cleave both strands, producing unwanted DSBs, albeit less efficiently than wild-type Cas9. To inactivate the HNH nuclease domain further, we incorporate additional mutations into nCas9 (H840A). Double-mutant nCas9 (H840A + N863A) does not exhibit the DSB-inducing behavior in vitro and, either alone or in fusion with the M-MLV reverse transcriptase (prime editor, PE2 or PE3), induces a lower frequency of unwanted indels, compared to nCas9 (H840A), caused by error-prone repair of DSBs. When incorporated into prime editor and used with engineered pegRNAs (ePE3), we find that the nCas9 variant (H840A + N854A) dramatically increases the frequency of correct edits, but not unwanted indels, yielding the highest purity of editing outcomes compared to nCas9 (H840A).

Suggested Citation

  • Jaesuk Lee & Kayeong Lim & Annie Kim & Young Geun Mok & Eugene Chung & Sung-Ik Cho & Ji Min Lee & Jin-Soo Kim, 2023. "Prime editing with genuine Cas9 nickases minimizes unwanted indels," Nature Communications, Nature, vol. 14(1), pages 1-11, December.
  • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-37507-8
    DOI: 10.1038/s41467-023-37507-8
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    References listed on IDEAS

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