Cas9 nickases (nCas9s), unlike CRISPR-Cas9 nucleases which create DNA double-strand breaks (DSBs), produce single-strand breaks. This study investigated off-target nicks using Digenome-seq. nCas9 (H840A), unlike nCas9 (D10A), created unwanted DSBs. Further mutagenesis in nCas9 (H840A), particularly the double-mutant nCas9 (H840A + N863A), eliminated DSB induction and reduced unwanted indels. In prime editor systems (PE2, PE3, ePE3), nCas9 (H840A + N854A) significantly increased correct edits while minimizing unwanted indels, achieving high editing purity.

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