Fig. 4: Editing efficiencies and specificity of Cas9 variants.

a Schematic of Cas9 domains and corresponding point mutations for Cas9 variants. Red letters indicated mutated amino acids of indicated Cas9 variants. b Editing efficiencies detected by primer-extension-mediated sequencing for Cas9 variants targeting RAG1A in HEK293T cells. Target site sequence was listed above and the red letters indicated the protospacer adjacent motif sequence. Error bars, mean ± SD. Two-tailed t-test, *p < 0.05. c Frequencies of total translocation junctions in 1-kb regions around off-target hotspots for indicated variants targeting RAG1A in HEK293T cells. Total numbers of identified off-target hotspots for each Cas9 variant were showed above the bar. Error bars, mean ± SD. Two-tailed t-test, *p < 0.05; **p < 0.01. d Scatter plot of RAG1A off-target hotspots for indicated variants. y Axis showed frequency of each hotspot per 100,000 editing events (indels plus translocation). Note that the wild-type (WT) libraries presented in b–d were independently prepared from the ones in Figs. 1 and 2. Sequencing reads for the libraries in this figure was less (~60%) and the identified hotspots were only 38, but still more than linear amplification-mediated high-throughput genome-wide translocation sequencing. e–g Editing efficiencies and off-target hotspots for Cas9 variants targeting EMX1 site in HEK293T cells, depicted as described in the legend to b–d