Fig. 2: Identification and characterization of PsCas9, an RNA-guided DNA nuclease.

A Schematic representation of PsCas9 CRISPR locus. Red line: tracrRNA; black rectangles: repeats; diamonds: spacers; black arrow: transcription direction of repeat–spacer array. Note that for simplicity repeat-spacer arrays do not represent the actual number of spacers. B Phylogenetic tree of Cas9 orthologs with depicted Type II-A, II-B and II-C systems (data from⁵³). C Secondary structure of the optimized sgRNA scaffold for PsCas9 visualized by CLC Genomics Workbench. CRISPR repeat:tracrRNA modules are colored in green and blue respectively. D Experimental approach to identify the preferred PAM sequence of PsCas9. A plasmid library consisting of randomized four base pair PAM sequences (red) downstream of a defined target sequence (blue/brown) was used, followed by NGS analysis of the cleaved products. The preferred PAM sequence was calculated by position frequency matrix (PFM) and identified as NGG PAM. The sequence logo was visualized with R using the ggseqlogo package. E In vitro DNA cleavage assay performed by incubating RNP complexes with fluorescently labeled DNA substrate. F, G In vitro DNA cleavage of the EMX1a substrate using sgRNAs with spacer length ranging from 18 to 30 nucleotides (see Supplementary Fig. 4D). Presented are k-fast s⁻¹ (F) and % DNA target cleavage after 10 s and 10 min reaction, respectively (G). Data are presented as mean ± SD, n = 3 (all experimental data points available in Source Data). H In vitro DNA cleavage rates of EMX1a with a mismatch at every single base position along the protospacer (see Supplementary Fig. 4G). Mismatch positions are labeled in 3’ to 5’ direction, with position 1 being directly upstream of the PAM. “no MM” = no mismatch. Data are presented as mean ± SD, n = 2 (all experimental data points available in Source data).