CRISPR-Cas9: Difference between revisions

A comparison of the quaternary complex structures of SaCas9 and SpCas9 revealed that the structurally diverse REC and WED domains recognize distinct structural features of the repeat:anti-repeat duplex, allowing the cognate sgRNAs to be distinguished in a highly specific manner. The SaCas9 WED domain has a new fold comprising a twisted five-stranded β-sheet flanked by four α-helices, and is responsible for the recognition of the distorted repeat:anti-repeat duplex, as described above. In contrast, the SpCas9 WED domain adopts a compact loop conformation and interacts with the repeat:anti-repeat duplex, which is structurally different from that of the SaCas9 sgRNA. The AnCas9 WED domain has yet another different fold containing three antiparallel b-hairpins. These structural differences in the WED domains are consistent with the variations in the sgRNA scaffolds among the CRISPR-Cas9 systems. The REC lobes also contribute to the orthogonal recognition of the sgRNA scaffolds. While the REC lobes of SaCas9 and SpCas9 share structural similarity, the SpCas9 REC lobe has four characteristic insertions (Ins 1–4), which are absent in the SaCas9 REC lobe. Ins 2 (also known as the REC2 domain) does not contact the nucleic acids in the SpCas9 structures and is dispensable for the DNA cleavage activity, consistent with the absence of Ins 2 in SaCas9 (Figures 4E and 4F). Ins 1 and 3 recognize the SpCas9-specific internal loop in the repeat:anti-repeat duplex (Figure 4F and Figure S6C). In particular, Ins 3 interacts with the flipped-out G43 and U44 in the repeat:anti-repeat duplex in base-specific manners (Figure S6C). In addition, Ins 4 interacts with stem loop 1 of the SpCas9 sgRNA, which is shorter than that of the SaCas9 sgRNA. Together, these structural observations demonstrate how the Cas9 orthologs recognize their cognate sgRNAs in orthogonal manners, using specific combinations of the structurally diverse REC and WED domains.