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Prime editing with genuine Cas9 nickases minimizes unwanted indels

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Title
Prime editing with genuine Cas9 nickases minimizes unwanted indels
Author(s)
Jaesuk Lee; Kayeong Lim; Annie Kim; Young Gun Mok; Eugene Chung; Sung-Ik Cho; Ji Min Lee; Jin Soo Kim
Publication Date
2023-03
Journal
Nature Communications, v.14, no.1
Publisher
Nature Research
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). © 2023, The Author(s).
URI
https://pr.ibs.re.kr/handle/8788114/13302
DOI
10.1038/s41467-023-37507-8
ISSN
2041-1723
Appears in Collections:
Center for Genome Engineering(유전체 교정 연구단) > 1. Journal Papers (저널논문)
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