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Prime editing with genuine Cas9 nickases minimizes unwanted indels

Authors
 Jaesuk Lee  ;  Kayeong Lim  ;  Annie Kim  ;  Young Geun Mok  ;  Eugene Chung  ;  Sung-Ik Cho  ;  Ji Min Lee  ;  Jin-Soo Kim 
Citation
 NATURE COMMUNICATIONS, Vol.14(1) : 1786, 2023-03 
Journal Title
NATURE COMMUNICATIONS
Issue Date
2023-03
MeSH
CRISPR-Cas Systems* / genetics ; DNA ; Deoxyribonuclease I* / metabolism ; INDEL Mutation ; Mutation
Abstract
Unlike CRISPR-Cas9 nucleases, which yield DNA double-strand breaks (DSBs), Cas9 nickases (nCas9s) produce nicks or single-strand breaks in the DNA. Here the authors analyse the on- and off-target nicks generated by these nickases, and show that nCas9 (H840A) but not nCas9 (D10A) can cleave both strands and produce unwanted DNA double-strand breaks. 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).
Files in This Item:
T992023372.pdf Download
DOI
10.1038/s41467-023-37507-8
Appears in Collections:
1. College of Medicine (의과대학) > Dept. of Pharmacology (약리학교실) > 1. Journal Papers
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/199637
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