Fig. 1: nCas9 (H840A) can create DSBs.
From: Prime editing with genuine Cas9 nickases minimizes unwanted indels
a A schematic diagram of the anticipated cleavage patterns of WT Cas9, nCas9 (D10A), and nCas9 (H840A). b In vitro plasmid digestion assay. Supercoiled plasmids were digested with nicking endonuclease Nt.BbvCI, restriction enzyme SpeI, and purified WT Cas9, nCas9 (D10A), and nCas9 (H840A) proteins. Nicks or DSBs in supercoiled plasmids generate open-circular or linear forms, respectively. c Relative band intensities of the supercoiled, open circular, and linear forms were calculated using ImageJ software. d Expected cleavage patterns at the HEK4 target site as visualized by the IGV viewer. Red: forward strand; blue: reverse strand; arrow head: cleavage point; blue characters: protospacer adjacent motif (PAM). e The actual cleavage patterns after in vitro digestion of genomic DNA with purified WT Cas9, nCas9 (D10A), and nCas9 (H840A) proteins together with HEK4-targeting sgRNA. Gel image (b) represents one of three independent experiments with similar results. Source data are provided as a Source Data file.
