Fig. 5: Off-target analyses of the LrCas9 nuclease system in rice.

a The Off-target analysis of LrCas9 at an NGAAA PAM site with mismatched protospacers (spacers) through amplicon deep-sequencing. The single base mismatch was underlined and labeled with blue. The correct spacer along with all 20 single-nucleotide mismatched spacers were compared. Each dot represents a biological replicate. Each spacer contains two or three independent experiments (n = 2 or n = 3). Data are presented as mean values ± SD. b The diagram of GUIDE-seq in this study. See details in the “Methods” section. c The dsODN tag (GUIDE) insertion rates at the Os-GG02 site were quantified by deep sequencing. Each dot represents a biological replicate. Each target contains three biological replicates. Data are presented as mean values ± s.d. Data were analyzed by one-way ANOVA with Dunnett’s multiple comparisons tests. ***P < 0.001; ****P < 0.0001. d The insertion direction of GUIDE that integrated at the Os-GG02 site in the rice genome. The insertion rates were quantified by deep sequencing. e The top five off-target sites identified by GUIDE-seq at Os-AG04, Os-CG01, Os-TG02, and Os-GG02. The dot indicated the same nucleotide residues with the on-target site. The ratios were calculated on the right. Total off-target sites were listed in Supplementary Data 3. Source data are provided as a Source Data file.