RNA interference is widely distributed in eukaryotes and has a variety of functions, including antiviral defence and gene regulation1,2. All RNA interference pathways use small single-stranded RNA (ssRNA) molecules that guide proteins of the Argonaute (Ago) family to complementary ssRNA targets: RNA-guided RNA interference1,2. The role of prokaryotic Ago variants has remained elusive, although bioinformatics analysis has suggested their involvement in host defence3. Here we demonstrate that Ago of the bacterium Thermus thermophilus (TtAgo) acts as a barrier for the uptake and propagation of foreign DNA. In vivo, TtAgo is loaded with 5′-phosphorylated DNA guides, 13–25 nucleotides in length, that are mostly plasmid derived and have a strong bias for a 5′-end deoxycytidine. These small interfering DNAs guide TtAgo to cleave complementary DNA strands. Hence, despite structural homology to its eukaryotic counterparts, TtAgo functions in host defence by DNA-guided DNA interference.

CRISPR-Cas is a prokaryotic adaptive immune system that provides sequence-specific defense against foreign nucleic acids. Here we report the structure and function of the effector complex of the Type III-A CRISPR-Cas system of Thermus thermophilus: the Csm complex (TtCsm). TtCsm is composed of five different protein subunits (Csm1-Csm5) with an uneven stoichiometry and a single crRNA of variable size (35-53 nt). The TtCsm crRNA content is similar to the Type III-B Cmr complex, indicating that crRNAs are shared among different subtypes. A negative stain EM structure of the TtCsm complex exhibits the characteristic architecture of Type I and Type III CRISPR-associated ribonucleoprotein complexes. crRNA-protein crosslinking studies show extensive contacts between the Csm3 backbone and the bound crRNA. We show that, like TtCmr, TtCsm cleaves complementary target RNAs at multiple sites. Unlike Type I complexes, interference by TtCsm does not proceed via initial base pairing by a seed sequence.

Summary The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex co-purifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5’ ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a ‘sea worm’ and is composed of a Cmr2-3 heterodimer ‘tail’, a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled ‘head’ containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes.

Several prokaryotic Argonaute proteins (pAgos) utilize small DNA guides to mediate host defense by targeting invading DNA complementary to the DNA guide. It is unknown how these DNA guides are being generated and loaded onto pAgo. Here, we demonstrate that guide-free Argonaute from Thermus thermophilus (TtAgo) can degrade double-stranded DNA (dsDNA), thereby generating small dsDNA fragments that subsequently are loaded onto TtAgo. Combining single-molecule fluorescence, molecular dynamic simulations, and structural studies, we show that TtAgo loads dsDNA molecules with a preference toward a deoxyguanosine on the passenger strand at the position opposite to the 5' end of the guide strand. This explains why in vivo TtAgo is preferentially loaded with guides with a 5' end deoxycytidine. Our data demonstrate that TtAgo can independently generate and selectively load functional DNA guides.

Adaptive immunity in bacteria involves RNA-guided surveillance complexes that use CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target invasive nucleic acids for degradation. While Type I and Type II CRISPR-Cas surveillance complexes target double-stranded DNA, Type III complexes target single-stranded RNA. Near-atomic resolution cryo-electron microscopy (cryo-EM) reconstructions of native Type III Cmr (CRISPR RAMP module) complexes in the absence and presence of target RNA reveal a helical protein arrangement that positions the crRNA for substrate binding. Thumb-like β-hairpins intercalate between segments of duplexed crRNA:target RNA to facilitate cleavage of the target at 6-nt intervals. The Cmr complex is architecturally similar to the Type I CRISPR-Cascade complex, suggesting divergent evolution of these immune systems from a common ancestor.Bacteria and archaea defend themselves against infection using adaptive immune systems comprising CRISPR (clustered regularly interspaced short palindromic repeats) arrays and CRISPR-associated (Cas) genes (1). A defining feature of CRISPR-Cas systems is the use of Cas proteins in complex with small CRISPR RNAs (crRNAs) to identify and cleave The effector complex of the Type III system from T. thermophilus (Cmr) is a 12-subunit assembly composed of six Cmr subunits (Cmr1-6) and a crRNA with a stoichiometry of Cmr1 1 2 1 3 1 4 4 5 3 6 1 :crRNA 1 (7). The Cmr complex binds to target RNA that is complementary to the bound 40 or 46-nt crRNA and cleaves the target at 6-nt intervals measured from the 5' end of the crRNA sequence (7,8). Although low-resolution structural studies revealed an overall capsule-like architecture of the Cmr complex (7), the molecular basis of subunit assembly, crRNA binding and ssRNA target recognition and cleavage by the intact surveillance complex remains unknown.We performed cryo-electron microscopy (cryo-EM) of the intact ~350-kDa Cmr complex in the absence and presence of target ssRNA. We purified endogenous apo-Cmr (containing a crRNA) and used this sample for step-wise assembly with a 56-nt biotinylated ssRNA target followed by purification using streptavidin affinity chromatography. Frozen-hydrated samples of both apo-Cmr and target-bound Cmr were visualized using an FEI Titan Krios microscope equipped with a Gatan K2 Summit direct electron detector. Cryo-EM micrographs of both apo-Cmr and the ssRNA-bound complex showed mono-disperse, easily identifiable particles with sea worm-like features ( fig. S1). Using LEGINON (9), we acquired ~7,000 and ~4,000 micrographs and automatically picked ~700,000 and ~300,000 apo-and target-bound Cmr particles, respectively, using Appion (10). After 3D classification and single-particle reconstruction (Supplementary Material and Methods) in RELION (11), we obtained structures of intact apo-Cmr and target-bound Cmr at ~4.1 and 4.4-Å resolution ( fig. S1, S2) (using the 0.143 gold standard Fourier Shell Correlationcalc...