Gene disruption efficiencies of paired Cas9 nickases vs. double the amount of Cas9 nucleases using sgRNA (5′GX20 or 5′GX19) without control sgRNA in human cells. (A–P) Cas9 paired nickase- or nuclease-driven mutations in the human genes detected by the T7E1 assay. HEK293T were analyzed 3 days after transfection with plasmids encoding paired Cas9 nickases or Cas9 nuclease, 5′GX20 sgRNAs targeting AAVS1-S1 (T1 and B1; A), MET (T1 and B1; B), EMX1-S1 (T1 and B1; C), EMX1-S2 (T1 and B1; D), USP1 (T1 and B1; E), USP3 (T1 and B1; F), USP16 (T1 and B1; G), USP31 (T1 and B1; H), USP33 (T1 and B1; I), USP47 (T1 and B1; J), VEGFA-S1 (T1 and B1; K), and VEGFA-S2 (T1 and B1; L). HEK293T were analyzed 3 days after transfection with plasmids encoding paired Cas9 nickases or Cas9 nuclease, 5′GX19 sgRNAs targeting EMX1-S3 (T1 and B1; M), EMX1-S4 (T1 and B1; N), EMX1-S5 (T2 and B1; O) and MET-S2 (T1 and B1; P). The frequency of Cas9 nuclease- or paired nickases-driven mutations as determined by the T7E1 assay are shown using bar graph. Error bars were derived from three independent experiments (n = 3). For brevity, statistical significance was shown only for comparison between a group-of-interest and the paired nickase group, except for the negative control group; *P< 0.05, **P< 0.01, ***P< 0.001 by paired t-test. ‘+’ and ‘++’ denote 1 and 2 μg concentrations of Cas9 nucleases or paired Cas9 nickases using top or bottom sgRNAs, respectively.