Type II CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA–induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.

Eukaryotic Argonaute proteins induce gene silencing by small RNAguided recognition and cleavage of mRNA targets. Although structural similarities between human and prokaryotic Argonautes are consistent with shared mechanistic properties, sequence and structure-based alignments suggested that Argonautes encoded within CRISPR-cas [clustered regularly interspaced short palindromic repeats (CRISPR)-associated] bacterial immunity operons have divergent activities. We show here that the CRISPR-associated Marinitoga piezophila Argonaute (MpAgo) protein cleaves single-stranded target sequences using 5′-hydroxylated guide RNAs rather than the 5′-phosphorylated guides used by all known Argonautes. The 2.0-Å resolution crystal structure of an MpAgo-RNA complex reveals a guide strand binding site comprising residues that block 5′ phosphate interactions. Using structure-based sequence alignment, we were able to identify other putative MpAgo-like proteins, all of which are encoded within CRISPRcas loci. Taken together, our data suggest the evolution of an Argonaute subclass with noncanonical specificity for a 5′-hydroxylated guide.Argonaute | small noncoding RNA | RNA interference A rgonaute (Ago) proteins bind small RNA or DNA guides, which provide base-pairing specificity for recognition and cleavage of complementary nucleic acid targets. Members of this protein family are present in all three domains of life (1). In eukaryotes, Argonautes are the key effectors of RNA interference (RNAi) pathways that regulate posttranscriptional gene expression (2-4). However, the role of Argonaute proteins in bacteria and archaea, which lack RNAi pathways, remains poorly understood (5).Recent studies suggested that DNA-guided bacterial and archaeal Argonaute proteins are directly involved in host defense by cleaving foreign DNA elements, such as DNA viruses and plasmids (6, 7). In addition, a catalytically inactive Argonaute protein in Rhodobacter sphaeroides (RsAgo) was demonstrated to use RNA guides and possibly recruits an associated nuclease for subsequent target cleavage (8). Despite these divergent modes of action, bacterial and archaeal Argonaute proteins adopt a highly conserved bilobed architecture. Herein, an N-terminal and a PIWI-ArgonauteZwille (PAZ) domain constitute one lobe, whereas the other lobe consists of the middle (MID) domain and the catalytic RNase H-like P element-induced wimpy testis (PIWI) domain (9-15). Molecular structures of a eukaryotic Argonaute MID domain and an Archaeoglobus fulgidus Piwi (AfPiwi) enzyme bound to a guide RNA showed the importance of the 5′-terminal base identity, as well as the 5′ phosphate in guide strand binding, to Ago (10,[13][14][15][16][17]. Notably, recognition of the 5′ end of the guide in the MID domain and guide strand preorganization for target interaction are conserved across the entire Argonaute superfamily (1).The nucleic acid-guided binding and cleavage activities of Argonaute proteins are reminiscent of the activities of RNA-guided proteins within CRISPR-Cas systems [cluste...

Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo. We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. These results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.

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