Journal Article

Prime editing with genuine Cas9 nickases minimizes unwanted indels

Kim, Annie, Mok, Young Geun, Lee, Ji Min, Kim, Jin-Soo, Cho, Sung-Ik, Lim, Kayeong, Lee, Jaesuk, Chung, Eugene

2023

Overview

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). 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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 13/109 / 38/23 / 38/77 / 42/41 / 45 / 631/337/149 / 631/337/4041/3196 / 82/83 / Amino acids / CRISPR / CRISPR-Cas Systems - genetics / Deoxyribonuclease I - metabolism / Deoxyribonucleic acid / DNA / DNA damage / DNA sequencing / Editing / Homology / Humanities and Social Sciences / INDEL Mutation / multidisciplinary / Mutation / Nicking endonuclease / Nuclease / RNA-directed DNA polymerase / Science / Science (multidisciplinary) / Strands / Whole genome sequencing