Fig. 2: Target strand cleavage catalyzed by nCas9 (H840A) can be reduced by an additional mutation at N863 in the HNH domain.
From: Prime editing with genuine Cas9 nickases minimizes unwanted indels
a WGS results at on-target sites visualized with the IGV viewer. Genomic DNA isolated from HEK293T cells was subjected to in vitro digestion with purified nCas9 (H840A) and nCas9 (H840A + N863A) proteins with appropriate sgRNAs. The addition of an N863A mutation to nCas9 (H840A) removed its target strand-cleaving function, as seen at three different loci. b Circos plots showing genome-wide DSBs detected by Digenome-seq. Genome-wide DSB sites created by purified WT Cas9, nCas9 (H840A), and nCas9 (H840A + N863A) proteins were captured. Arrow heads: on-target sites; height of black bars: Digenome scores. c Number of DSB sites found by Digenome-seq at three loci. d Average numbers of genome-wide DSBs found in samples treated with WT Cas9, nCas9 (H840A), and nCas9 (H840A + N863A). Mean ± SEM (d) was determined three independent experiments. Source data are provided as a Source Data file.
