Fig. 1

Analysis of the on-target activities of wild-type (WT) SpCas9 and three high-fidelity SpCas9 variants for seven genomic sites using different sgRNAs. a sgRNA constructs used and tRNA-mediated sgRNA processing. For the U3/U6 promoter, the transcription initiation site starts with A/G, so the transcribed sgRNA carries an A/G at the 5′ end. The sgRNAs are precisely processed from tRNA–sgRNA precursors. Endogenous RNase P and RNase Z cleave the transcripts and release mature sgRNAs. b Comparison of the on-target activities of WT SpCas9 and three variants at five genomic sites (sites 1–5) without A at their 5′ ends using U3:sgRNA-AN₂₀ or U3:tRNA-sgRNA-N₂₀. c Comparison of the corresponding on-target activities at two genomic sites (sites 6 and 7) with A at their 5′ ends using U3:sgRNA-AN₁₉, U3:sgRNA-AN₂₀, or U3:tRNA-sgRNA-N₂₀. Two independent replicates were performed. Solid filled columns indicate replicate 1 and pattern filled columns indicate replicate 2. d Summary of the on-target activities of three SpCas9 variants using U3:sgRNA-AN20 or U3:tRNA-sgRNA-N20 compared to WT SpCas9 in b, c