Fig. 1: Comparative analysis of various Cas9 sequences and investigating REC expansion of SpCas9.

a Schematic illustration of REC expansion in the Cas9-evolution-hypothesis. b Domain size of IscB and Cas9s. Crystal structures of all the proteins have been identified. REC, recognition (REC) lobe; BH, bridge helix; PAM, protospacer-adjacent motifs; PI, PAM-Interacting Domain. FnCas9 (PDB:5B2O), SpCas9 (PDB:4OO8), St1Cas9 (PDB:6M0V), SaCas9 (PDB:5AXW), NmeCas9 (PDB:6JDQ), CjCas9 (PDB: 6JOO), OgeuIscB (PDB:7UTN). c Violin plot illustrating distribution of Cas9-sizes in various species. One pot means one sequence. n, number of sequences in each group. d Highlighting evolution of Cas9 proteins in different species. F, Francisella; Sp, Streptococcus pyogenes; St1, Streptococcus thermophilus; Sa, Staphylococcus aureus; Nme, Neisseria meningitidis and Cj, Campylobacter jejuni. e A crystal structure of St1Cas9 (PDB:6M0V). f A crystal structure of SaCas9 (PDB:5AXW). g Schematic of REC expansion from SpCas9. Insert positions are shown in cells. h Workflow for testing Cas9 variants activity in HEK293T cells. Episomal EGFP plasmid was co-transfected with Cas9 and gRNA plasmids to monitor Cas9 activity. i Activities induced by SpCas9 and variants. Data are presented as mean ±s.d. (n = 3). P values were determined by two-way ANOVA Sidak’s multiple comparisons test. j Testing influences of BHs with different lengths on activities. Data are presented as mean ±s.d.(n = 3). P values were determined by two-way ANOVA Sidak’s multiple comparisons test. k Variant Left-REC12 mediated EGFP disruption at different plasmid dosages. Low dosage 1, 2 ng of reporter plasmid; Low dosage 2, 4 ng of reporter plasmid. Data are presented as mean ±s.d. (n = 3). P values were determined by two-way ANOVA Sidak’s multiple comparisons test. l Disruption activities of SpCas9 and GS-Cas9 on endogenous GFP site in HEK293-deGFP cells. Data are presented as mean ±s.d.(n = 3). Source data are provided as a Source Data file.