Extended Data Fig. 10: Activities of CBE and CBE variants at the indicated Cas9-dependent off-target sites.
a, The Cas9-dependent off-target effects of the CBE and CBE variants. b, The comparison of editing frequencies of CBE and CBE variants at 34 potential off-target sites. P values were calculated by two sided Student’s t-tests, compared with YE1-BE3-FNLS group. Each cell represents the percentage of total sequencing reads with C to T conversion. n = 21 independent experiments for each group. Box-and-whisker plots: center line indicates median, the bottom and top lines of the box represents the first quartile and third quartile of the values, respectively. The bottom and top of the vertical line represent the minimum and maximum value. HEK293T cells were transfected with plasmids expressing BE3, BE3R126E, BE3R132E, YE1-BE3, FE1-BE3, BE3-hA3A, BE3-hA3AY130F, BE3-FNLS and YE1-BE3-FNLS and sgRNAs matching the indicated on-target sequence using Lipofectamine 3000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus the top ten known Cas9 off-target loci for these sgRNAs, as previously determined using the GUIDE-seq method23, 24 and ChIP-seq method25. Sequences of the on-target and off-target protospacers and primers were shown in Extended Data Table 5.
