Figure 4.

Cas9 from closely related CRISPR-Cas systems can substitute the role of S. pyogenes Cas9 in RNA processing by RNase III. (A) Schematic representation of Cas9 from selected bacterial species. The protein sizes and distances between conserved motifs (RuvC and HNH) are drawn in scale. See Supplementary Figure S2. (B) Northern blot analysis of total RNA extracted from S. pyogenes WT, Δcas9 and Δcas9 complemented with pEC342 (backbone vector containing tracrRNA-171 nt and the cas operon promoter from S. pyogenes) or pEC342-based plasmids containing cas9 orthologous genes, probed for tracrRNA and crRNA repeat. Mature forms of S. pyogenes tracrRNA and pre-crRNA are observed only in the presence of S. pyogenes Cas9 WT or closely related Cas9 orthologs from S. mutans and S. thermophilus*.