The CRISPR-Cas adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference. We characterize the Class 2 type VI-A CRISPR-Cas effector C2c2 and demonstrate its RNA-guided RNase function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage. In vitro biochemical analysis show that C2c2 is guided by a single crRNA and can be programmed to cleave ssRNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved HEPN domains, mutations in which generate catalytically inactive RNA-binding proteins. These results broaden our understanding of CRISPR-Cas systems and suggest that C2c2 can be used to develop new RNA-targeting tools.

Microbial CRISPR-Cas systems are divided into Class 1, with multisubunit effector complexes, and Class 2, with single protein effectors. Currently, only two Class 2 effectors, Cas9 and Cpf1, are known. We describe here three distinct Class 2 CRISPR-Cas systems. The effectors of two of the identified systems, C2c1 and C2c3, contain RuvC-like endonuclease domains distantly related to Cpf1. The third system, C2c2, contains an effector with two predicted HEPN RNase domains. Whereas production of mature CRISPR RNA (crRNA) by C2c1 depends on tracrRNA, C2c2 crRNA maturation is tracrRNA-independent. We found that C2c1 systems can mediate DNA interference in a 5’-PAM-dependent fashion analogous to Cpf1. However, unlike Cpf1, which is a single-RNA-guided nuclease, C2c1 depends on both crRNA and tracrRNA for DNA cleavage. Finally, comparative analysis indicates that Class 2 CRISPR-Cas systems evolved on multiple occasions through recombination of Class 1 adaptation modules with effector proteins acquired from distinct mobile elements.

The CRISPR-Cas adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference. We characterize the Class 2 type VI-A CRISPR-Cas effector C2c2 and demonstrate its RNA-guided RNase function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage. In vitro biochemical analysis show that C2c2 is guided by a single crRNA and can be programmed to cleave ssRNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific † Correspondence should be addressed to zhang@broadinstitute.org (F.Z.) and koonin@ncbi.nlm.nih.gov (E.V.K.). * These authors contributed equally to this work. Almost all archaea and about half of bacteria possess Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes (CRISPR-Cas) adaptive immune systems (1, 2), which protect microbes from viruses and other invading DNA through three steps: (i) adaptation, i.e., insertion of foreign nucleic acid segments (spacers) into the CRISPR array in between pairs of direct repeats (DRs), (ii) transcription and processing of the CRISPR array to produce mature CRISPR RNAs (crRNAs), and (iii) interference, whereby Cas enzymes are guided by the crRNAs to target and cleave cognate sequences in the respective invader genomes (3-5). All CRISPR-Cas systems characterized to date follow these three steps, although the mechanistic implementation and proteins involved in these processes display extensive diversity. Supplementary Materials Supplementary Materials and MethodsThe CRISPR-Cas systems are broadly divided into two classes on the basis of the architecture of the interference module: Class 1 systems rely on multi-subunit protein complexes whereas Class 2 systems utilize single effector proteins (1). Within these two classes, types and subtypes are delineated according to the presence of distinct signature genes, protein sequence conservation, and organization of the respective genomic loci. (Fig. 1A). This resulted in a library of 3,473 spacer sequences (along with 490 non-targeting guides designed to have a Levenshtein distance of ≥8 with respect to the MS2 and E. coli genomes) which we inserted between pLshC2c2 direct repeats (DRs). After transformation in of this construct into E. coli, we infected cells with varying dilutions of MS2 (10 −1 , 10 −3 , and 10 −5 ) and analyzed surviving cells to determine the spacer sequences carried by cells that survived the infection. Cells carrying spacers that confer robust interference against MS2 are expected to proliferate faster than those that lack such sequences. Following growth for 16 hours, we identified a number of spacers that were consistently enriched across three independent infection replicas in both the 10 −1 and 10 −3 dilution conditions, suggesting that they enabled interference against MS2. Specifically, 147 and 150 spacers showed >1.25 log 2 -fold enrichment in all three replicates for the 10 −1 and 10 −3 phage dilutions, respectively; of these two groups of...

The X-ray crystal structure of Thermus aquaticus core RNA polymerase reveals a "crab claw"-shaped molecule with a 27 A wide internal channel. Located on the back wall of the channel is a Mg2+ ion required for catalytic activity, which is chelated by an absolutely conserved motif from all bacterial and eukaryotic cellular RNA polymerases. The structure places key functional sites, defined by mutational and cross-linking analysis, on the inner walls of the channel in close proximity to the active center Mg2+. Further out from the catalytic center, structural features are found that may be involved in maintaining the melted transcription bubble, clamping onto the RNA product and/or DNA template to assure processivity, and delivering nucleotide substrates to the active center.

Pseudomonas aeruginosa bacteriophage KZ is the type representative of the giant phage genus, which is characterized by unusually large virions and genomes. By unraveling the transcriptional map of the ϳ280-kb KZ genome to single-nucleotide resolution, we combine 369 KZ genes into 134 operons. Early transcription is initiated from highly conserved AT-rich promoters distributed across the KZ genome and located on the same strand of the genome. Early transcription does not require phage or host protein synthesis. Transcription of middle and late genes is dependent on protein synthesis and mediated by poorly conserved middle and late promoters. Unique to KZ is its ability to complete its infection in the absence of bacterial RNA polymerase (RNAP) enzyme activity. We propose that transcription of the KZ genome is performed by the consecutive action of two KZ-encoded, noncanonical multisubunit RNAPs, one of which is packed within the virion, another being the product of early genes. This unique, rifampin-resistant transcriptional machinery is conserved within the diverse giant phage genus. IMPORTANCEThe data presented in this paper offer, for the first time, insight into the complex transcriptional scheme of giant bacteriophages. We show that Pseudomonas aeruginosa giant phage KZ is able to infect and lyse its host cell and produce phage progeny in the absence of functional bacterial transcriptional machinery. This unique property can be attributed to two phage-encoded putative RNAP enzymes, which contain very distant homologues of bacterial ␤ and ␤=-like RNAP subunits. T ranscription is driven by DNA-dependent RNA polymerases (RNAPs), which synthesize RNA from DNA templates (1). RNAPs can be classified into two unrelated families: small singlesubunit enzymes (ssRNAPs), encoded by some bacteriophages and also found in mitochondria and chloroplasts, and large multisubunit cellular enzymes (msRNAPs), transcribing genes in bacterial, archaeal, and eukaryal genomes. The catalytic activity of enzymes from both families is accomplished through a common two-metal-ion mechanism. The canonical bacterial msRNAP is a 400-kDa complex consisting of five core subunits (␣ 2 ␤␤=) which are directed to specific promoter sequences by a variety of factors (2). The two largest RNAP subunits, ␤ and ␤=, contain conserved double-psi beta-barrel (DPBB) domains that together form the active center (3-5). Members of the ssRNAP family rely on different catalytic domains and amino acid motifs for catalysis of the RNA polymerization reaction and are related to DNA polymerases and reverse transcriptases (6, 7). Bacterial RNAPs are inactivated by the antibiotic rifampin (Rif), which acts by binding to the ␤-subunit pocket deep inside the active-site channel, preventing synthesis of RNA sequences longer than 3 to 4 nucleotides (nt) (8).Bacterial RNAPs play a key role during the infection of bacterial cells by bacteriophages. Most known phages do not encode their own RNAP but redirect the host transcription machinery to viral promoters by rel...