Extended Data Fig. 4: Cas9-associated PcrIIC1 is a non-toxic protein with 3′–5′ ssDNA nuclease activity.
From: Pro-CRISPR PcrIIC1-associated Cas9 system for enhanced bacterial immunity
a, RNA-seq analysis of gene locus of PIN-associated CRISPR–Cas9 system in Chryseobacterium sp. by PE150 (blue) and SE50 (red) strategies showing the expression of Cas9, PIN, Cas1, Cas2 and RNA components (tracrRNA and CRISPR array). b, Phylogenic analysis of Cas9-associated PIN (green) with other PIN-domain toxin proteins (light blue) and Doc toxin proteins (yellow; outgroups). c, Left, model of in vivo toxin assay in E. coli BW25141 revealed by monitoring optical density (OD) over time. Right, growth curve of E. coli BW25141 containing different plasmids expressing MBP, SUMO, PcrIIC1 or VapC toxin with 1% glucose repressor (top) or 25 mM arabinose inducer (bottom). OD600 was monitored over time. Data are presented as mean ± s.d. (n = 3 biologically independent wells). d, In vitro cleavage of 3′- or 5′-Fam-labelled ssDNA targets by PcrIIC1 revealed by denaturing PAGE, S means substrates and P means cleavage products. Gel shown is representative of three independent experiments with similar results (n = 3 independent replicates). e, In vitro cleavage of two different Fam-labelled dsDNA targets by PcrIIC1 revealed by denaturing PAGE. Gel shown is representative of three independent experiments with similar results (n = 3 independent replicates). f, In vitro cleavage of three different 5′- or 3′-Fam-labelled ssRNA targets by PcrIIC1 revealed by denaturing PAGE. The gel shown is representative of three independent experiments with similar results (n = 3 independent replicates). All aliquots were collected at the following time points: 0, 2, 5, 15, 30, 60, 90 and 120 min. For gel source data, see Supplementary Fig. 1.
