Fig. 2

CjCas9 enhancement through screening in mammalian cells. a Schematic representation of the TetR-EGFP reporter cell line. TetR (blue) is constitutively expressed inactivating EGFP (green) expression. TetR inactivation by Cas9 cleavage enables EGFP expression and isolation of fluorescent cells by sorting. b CjCas9 variants isolated from rounds 1 and 2 of the mammalian screening are compared with CjCas9 WT transducing the TetR-EGFP reporter cell line. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test: ns, not significant, ****P < 0.0001. c Editing activity of selected variants obtained from yeast and mammalian selection. Dots represent the fold changes of activity of each variant (from A to Q) with respect to CjCas9 WT targeting EMX, RHO, or CCR5. Extended data are shown in Additional file 1: Fig. S3. The grand mean values of each variant are shown in the graph. Sanger sequences of the variants are reported in Additional file 2: Table S2. In b, data are reported as mean ± standard deviation of n ≥ 3 biologically independent samples. Individual values are represented as empty circles