Fig. 1

Directed evolution step to enhance Cas nuclease activity. a Schematic and experimental flowchart of the yeast Cas evolution step. Framed sequences correspond to the ON1 and ON2 cassettes carrying the CjCas9 target sites and AAVS1-TS34 and AAVS1-TS32 targets, respectively. Active mutants cleaving both ON1 and ON2 in the ADE2 and TRP1 loci are amplified from colonies growing in ADE − and TRP − conditions and undergo additional rounds of mutagenesis and selection. b Cleavage activity of SpCas9 and CjCas9 are reported as percentages of survival, corresponding to the number of colonies growing in TRP − and ADE − plates with respect to total transformants measured in unrestricted growth conditions. Culture plates shown in Additional file 1: Fig. S1a. c The activity of the CjCas9 variants (MUT) isolated at each round of evolution was compared with CjCas9 WT by measuring the percentages of survival after transformation. Culture plates are shown in Additional file 1: Fig. S1b. d Peaks at each CjCas9 amino acid position represent the frequency of the mutated residues obtained by NGS deep sequencing. Amino acid substitutions with high frequency are reported in the graph. e Editing activity at the endogenous loci in HEK293 cells of CjCas9 variants containing substitutions corresponding to the most frequently mutated amino acids in d. In b and e, data are reported as mean ± standard deviation of n ≥ 3 biologically independent samples. Individual values are represented as empty circles