Structural basis of a dual-function type II-B CRISPR–Cas9

Cas9 from Streptococcus pyogenes (SpCas9) revolutionized genome editing by enabling programmable DNA cleavage guided by an RNA. However, SpCas9 tolerates mismatches in the DNA-RNA duplex, which can lead to deleterious off-target editing. Here, we reveal that Cas9 from Francisella novicida (FnCas9) possesses a unique structural feature-the REC3 clamp-that underlies its intrinsic high-fidelity DNA targeting. Through kinetic and structural analyses, we show that the REC3 clamp forms critical contacts with the PAM-distal region of the R-loop, thereby imposing a novel checkpoint during enzyme activation. Notably, F. novicida encodes a noncanonical small CRISPR-associated RNA (scaRNA) that enables FnCas9 to repress an endogenous bacterial lipoprotein gene, subverting host immune detection. Structures of FnCas9 with scaRNA illustrate how partial R-loop complementarity hinders REC3 clamp docking and prevents cleavage in favor of transcriptional repression. The REC3 clamp is conserved across type II-B CRISPR-Cas9 systems, pointing to a potential path for engineering precise genome editors or developing novel antibacterial strategies. These findings reveal the molecular basis of heightened specificity and virulence enabled by FnCas9, with broad implications for biotechnology and therapeutic development.