{"id":"doi:10.1038/s41467-023-37507-8","kind":"article","title":"Prime editing with genuine Cas9 nickases minimizes unwanted indels","authors":["Lee J","Lim K","Kim A","Mok YG","Chung E","Cho SI","Lee JM","Kim JS"],"author_count":8,"year":2023,"published":"2023-03-30","venue":"Nature communications","cited_by":93,"doi":"10.1038/s41467-023-37507-8","arxiv_id":null,"nct_id":null,"source_url":"https://doi.org/10.1038/s41467-023-37507-8","archive_url":"https://space-kitty.com/papers/doi:10.1038/s41467-023-37507-8","updated":null,"pmid":"36997524","pmcid":"PMC10063541","openalex_id":null,"pdf_url":"https://europepmc.org/articles/PMC10063541/pdf","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).","abstract_license":"CC-BY (article license as recorded by Europe PMC; abstract reproduced unmodified apart from markup removal)","license":"CC-BY","is_open_access":true,"retracted":false,"peer_reviewed":null,"categories":[],"details":null,"provenance":"europepmc","attribution":"Abstract of \"Prime editing with genuine Cas9 nickases minimizes unwanted indels\" by Lee J, Lim K, Kim A et al., https://doi.org/10.1038/s41467-023-37507-8, used under its Creative Commons Attribution license (CC-BY); markup removed, otherwise unmodified.","topics":[{"slug":"gene-editing-therapies","name":"CRISPR & Gene-Editing Therapies","domain_slug":"medicine"}],"cited_in_dossiers":[]}