Prime editing with genuine Cas9 nickases minimizes unwanted indels

Jaesuk Lee, Kayeong Lim, Annie Kim, Young Geun Mok, Eugene Chung, Sung-Ik Cho, Ji Min Lee and Jin-Soo Kim ()
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Jaesuk Lee: Institute for Basic Science
Kayeong Lim: Institute for Basic Science
Annie Kim: Institute for Basic Science
Young Geun Mok: Institute for Basic Science
Eugene Chung: Institute for Basic Science
Sung-Ik Cho: Institute for Basic Science
Ji Min Lee: Institute for Basic Science
Jin-Soo Kim: Institute for Basic Science

Nature Communications, 2023, vol. 14, issue 1, 1-11

Abstract: 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).

Date: 2023
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DOI: 10.1038/s41467-023-37507-8

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