Research Articles

Bacteria and archaea target invasive DNA using RNA-guided adaptive immune systems encoded by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) genomic loci (1– 4). Following integration of short fragments of invader-derived DNA into a CRISPR array within the host chromosome (5), enzymatic pro-cessing of CRISPR transcripts produces mature CRISPR RNAs (crR-NAs) that direct Cas protein-mediated targeting of DNA bearing complementary sequences (protospacers) to foreign nucleic acids (6). While Type I and III CRISPR–Cas systems rely on multi-protein com-plexes for crRNA-guided DNA targeting (1, 4, 7), Type II systems em-ploy a single RNA-guided endonuclease, Cas9, that requires both a mature crRNA and a trans-activating crRNA (tracrRNA) for target DNA recognition and cleavage (8, 9). Both a seed sequence in the crRNA and conserved protospacer adjacent motif (PAM) sequence in the target are crucial for Cas9-mediated cleavage (8, 10). Cas9 proteins are abundant across the bacterial kingdom, but vary widely in both sequence and size. All known Cas9 enzymes contain an HNH domain that cleaves the DNA strand comple-mentary to the guide RNA sequence (target strand), and a RuvC nuclease domain required for cleaving the non-complementary strand (non-target strand), yielding double-strand DNA breaks (DSBs) (8, 10). In addition, Cas9 enzymes contain a highly con-served arginine-rich (Arg-rich) region previously suggested to mediate nucle-ic acid binding (11). Based on CRISPR-Cas locus architecture and protein sequence phylogeny, Cas9 genes cluster into three subfamilies