Prime editing with genuine Cas9 nickases minimizes unwanted indels

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).

Automated summary

In this study, the authors analyzed the on- and off-target nicks produced by Cas9 nickases and found that nCas9 (H840A) can cleave both strands and cause unwanted DNA double-strand breaks, while nCas9 (D10A) can only cleave one strand. They also found that incorporating additional mutations into nCas9 (H840A) can further inactivate the HNH nuclease domain and reduce the frequency of unwanted indels caused by error-prone repair of double-strand breaks.