In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus

Clustered regularly interspaced short palindromic repeats (CRISPR)-encoded immunity in Type I systems relies on the Cascade (CRISPR-associated complex for antiviral defence) ribonucleoprotein complex, which triggers foreign DNA degradation by an accessory Cas3 protein. To establish the mechanism for adaptive immunity provided by the Streptococcus thermophilus CRISPR4-Cas (CRISPR-associated) system (St-CRISPR4-Cas), we isolated an effector complex (St-Cascade) containing 61-nucleotide CRISPR RNA (crRNA). We show that St-Cascade, guided by crRNA, binds in vitro to a matching proto-spacer if a proto-spacer adjacent motif (PAM) is present. Surprisingly, the PAM sequence determined from binding analysis is promiscuous and limited to a single nucleotide (A or T) immediately upstream (-1 position) of the proto-spacer. In the presence of a correct PAM, St-Cascade binding to the target DNA generates an R-loop that serves as a landing site for the Cas3 ATPase/nuclease. We show that Cas3 binding to the displaced strand in the R-loop triggers DNA cleavage, and if ATP is present, Cas3 further degrades DNA in a unidirectional manner. These findings establish a molecular basis for CRISPR immunity in St-CRISPR4-Cas and other Type I systems.