Fig. 5: AcrIIA4 blocks Cas9 off-target activity less effectively.

a Editing efficiencies detected by primer-extension-mediated sequencing for Cas9:RAG1A in HEK293T cells with indicated mass ratios of Cas9 over AcrIIA4. Error bars, mean ± SD. Two-tailed t-test, **p < 0.01. b Frequencies of translocation junctions in 1-kb regions around off-target hotspots for Cas9:RAG1A in HEK293T cells with indicated ratios of Cas9 over AcrIIA4. Total numbers of identified off-target hotspots for indicated samples were showed above the bars. Error bars, mean ± SD. Two-tailed t-test, *p < 0.05; **p < 0.01. c Composition of indels and off-target junctions for Cas9:RAG1A libraries with indicated ratios of Cas9 over AcrIIA4. Total junction numbers from pooled three libraries were indicated above the bars. Note that the total bar length of each library was normalized to the same though inhibitor-treated samples always contained less junctions than untreated ones. d–f Editing efficiencies and off-target hotspots in other Cas9-targeting loci with 1:1 Cas9 over AcrIIA4, depicted as described in the legend to a–c. Note that for 1:1 ratio described in a–c, the amount of plasmid DNA used for transfection for Cas9:RAG1A, AcrIIA4, and blank were 2, 2, and 4 μg, respectively; while for 1:1 in d–f, the amounts were 2 μg, 2 μg and 0, which led to a higher transfection efficiency of Cas9:RAG1A