Mechanism of type II-B CRISPR-Cas enhanced specificity

The constant threat of foreign genetic elements destroying bacteria has driven the evolution of diverse defense mechanisms including clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) systems. The Cas9 endonuclease from Streptococcus pyogenes (SpCas9) has revolutionized genome editing, as it can be programmed by an RNA sequence to target and cleave DNA. However, SpCas9 is prone to deleterious off-target effects, prompting efforts to develop and discover high-fidelity variants. In the search for a high-fidelity Cas9, many intrinsically specific Cas9 orthologs have been found in nature. One such ortholog is from the human pathogen Francisella novicida (FnCas9), yet the molecular basis of its enhanced specificity remains unknown. Here, we uncover the mechanism of FnCas9 specificity through kinetic characterization in concert with structural determination. We identify a novel region within the REC3 domain, the REC3 clamp, as critical for mismatch discrimination through interaction with the PAM-distal heteroduplex. Mutational analysis confirmed the role of the REC3 clamp in reducing cleavage of off-target sequences. Further, a small CRISPR-associated RNA (scaRNA) within the F. novicida CRISPR locus has been shown to guide transcriptional repression of an endogeneous gene without the consequence of cleavage. This secondary function of FnCas9 enables host subversion and heightened virulence, yet the mechanism distinguishing target repression versus cleavage is unclear. Through experimental and simulated structural analysis, we identify the REC3 clamp as the key to differentiating DNA sequences targeted for repression versus cleavage, ultimately bolstering the virulence of F. novicida . The conservation of the REC3 clamp across type II-B CRISPR systems highlights its evolutionary significance, and potential as a target for developing specific genome editors and novel antibacterial effectors.