Fig. 3

Characterization of the highly enhanced variant UltraCjCas9. a Domain localization of the five amino acid substitutions (V35A, E189G, F214I, A492V, and T913S) characterizing UltraCjCas9. See Additional file 1: Fig. S4 for amino acid positions in the CjCas9 structure. b Sequence logo representations of the PAM preference of CjCas9 and UltraCjCas9, obtained using an in vitro PAM determination assay. Heatmaps representing each nucleotide combination are reported in Additional file 1: Fig. S5. c Editing efficiency in the endogenous genomic loci of HEK293 cells with CjCas9 WT, enCjCas9, and UltraCjCas9. PAM sequences are reported in Additional file 1: Fig. S6a. d Graphical summary of editing activities of CjCas9 WT (pink), enCjCas9 (blue), and UltraCjCas9 (yellow) in c. Empty circles in the box plots represent the average percentages of indels for each genomic locus. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test: ns, not significant, *P < 0.05, **P < 0.01. Central line, median; box limits, upper and lower quartiles; whiskers, × 1.5 interquartile range; n = 23 independent loci. e Comparison of SpCas9 and UltraCjCas9 editing activity in the indicated endogenous genomic loci. Statistical significance was assessed using a two-sided t-test corrected using the Holm-Šídák method for each locus. ns, not significant. f GUIDE-Seq analysis performed with CjCas9 WT and UltraCjCas9. GUIDE-seq read counts of each on- and off-target in HEK293T cells are shown on the right side (extended data in Additional file 2: Table S7–S10). In c and e, data are reported as mean ± standard deviation of n ≥ 3 biologically independent samples. Individual values are represented as empty circles