Clustered, regularly interspaced, short palindromic repeats (CRISPR)/ CRISPR-associated (Cas) systems provide adaptive immunity against viruses and plasmids in bacteria and archaea. The silencing of invading nucleic acids is executed by ribonucleoprotein complexes preloaded with small, interfering CRISPR RNAs (crRNAs) that act as guides for targeting and degradation of foreign nucleic acid. Here, we demonstrate that the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system introduces in vitro a doublestrand break at a specific site in DNA containing a sequence complementary to crRNA. DNA cleavage is executed by Cas9, which uses two distinct active sites, RuvC and HNH, to generate sitespecific nicks on opposite DNA strands. Results demonstrate that the Cas9-crRNA complex functions as an RNA-guided endonuclease with RNA-directed target sequence recognition and proteinmediated DNA cleavage. These findings pave the way for engineering of universal programmable RNA-guided DNA endonucleases.nuclease | site-directed mutagenesis | RNA interference | DNA interference

The number and diversity of CRISPR-Cas systems has substantially increased in recent years. Here, we provide an updated evolutionary classification of CRISPR-Cas systems and cas genes, with an emphasis on the major developments that occurred since the publication of the latest classification in 2015. The new classification includes 2 classes, 6 types and 33 subtypes compared to 5 types and 16 subtypes in 2015. A key development is the ongoing discovery of multiple, novel class 2 CRISPR-Cas systems that now include 3 types and 17 subtypes. A second major novelty is the discovery of numerous derived CRISPR-Cas variants, often associated with mobile genetic elements that lack the nucleases required for interference. Some of these variants are involved in RNA-guided transposition whereas others are predicted to perform functions distinct from adaptive immunity that remain to be characterized experimentally. The third highlight is the discovery of numerous families of ancillary CRISPRlinked genes, often implicated in signal transduction. Together, these findings substantially clarify the functional diversity and evolutionary history of CRISPR-Cas. This work complements Ref. 34 by experimentally validating the prediction made in Ref. 33, that interference-deficient subtype IF CRISPR-Cas systems encoded in Tn7like transposons enable crRNA-dependent transposition.

The CRISPR/Cas adaptive immune system provides resistance against phages and plasmids in Archaea and Bacteria. CRISPR loci integrate short DNA sequences from invading genetic elements that provide small RNA-mediated interference in subsequent exposure to matching nucleic acids. In Streptococcus thermophilus, it was previously shown that the CRISPR1/Cas system can provide adaptive immunity against phages and plasmids by integrating novel spacers following exposure to these foreign genetic elements that subsequently direct the specific cleavage of invasive homologous DNA sequences. Here, we show that the S. thermophilus CRISPR3/Cas system can be transferred into Escherichia coli and provide heterologous protection against plasmid transformation and phage infection. We show that interference is sequence-specific, and that mutations in the vicinity or within the proto-spacer adjacent motif (PAM) allow plasmids to escape CRISPR-encoded immunity. We also establish that cas9 is the sole cas gene necessary for CRISPR-encoded interference. Furthermore, mutation analysis revealed that interference relies on the Cas9 McrA/HNH- and RuvC/RNaseH-motifs. Altogether, our results show that active CRISPR/Cas systems can be transferred across distant genera and provide heterologous interference against invasive nucleic acids. This can be leveraged to develop strains more robust against phage attack, and safer organisms less likely to uptake and disseminate plasmid-encoded undesirable genetic elements.

Clustered, regularly interspaced, short palindromic repeats (CRISPR)/ CRISPR-associated (Cas) systems protect bacteria and archaea from infection by viruses and plasmids. Central to this defense is a ribonucleoprotein complex that produces RNA-guided cleavage of foreign nucleic acids. In DNA-targeting CRISPR-Cas systems, the RNA component of the complex encodes target recognition by forming a sitespecific hybrid (R-loop) with its complement (protospacer) on an invading DNA while displacing the noncomplementary strand. Subsequently, the R-loop structure triggers DNA degradation. Although these reactions have been reconstituted, the exact mechanism of Rloop formation has not been fully resolved. Here, we use singlemolecule DNA supercoiling to directly observe and quantify the dynamics of torque-dependent R-loop formation and dissociation for both Cascade-and Cas9-based CRISPR-Cas systems. We find that the protospacer adjacent motif (PAM) affects primarily the Rloop association rates, whereas protospacer elements distal to the PAM affect primarily R-loop stability. Furthermore, Cascade has higher torque stability than Cas9 by using a conformational locking step. Our data provide direct evidence for directional R-loop formation, starting from PAM recognition and expanding toward the distal protospacer end. Moreover, we introduce DNA supercoiling as a quantitative tool to explore the sequence requirements and promiscuities of orthogonal CRISPR-Cas systems in rapidly emerging gene-targeting applications.magnetic tweezers | genome engineering | crRNA C lustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems constitute an adaptable immune system that protects bacteria and archaea against foreign nucleic acids. The defense is initiated by a ribonucleoprotein (RNP) complex that mediates cleavage of dsDNA (1) or RNA (2, 3). The RNA component (crRNA) of the complex is derived by transcription and posttranscriptional processing from a locus containing CRISPRs (2, 4, 5) in which short spacer fragments were integrated from foreign nucleic acids (6-8). Each transcribed crRNA spacer sequence encodes the recognition of the targets. In DNA-targeting CRISPR-Cas systems, the crRNAs form a hybrid with a matching complement (protospacer) on an invading DNA, which leads to the displacement of the noncomplementary strand. The resulting structure is called an R-loop and constitutes the signal for subsequent DNA degradation. R-loop formation is additionally dependent on a short protospacer adjacent motif (PAM) (Fig. 1A), which provides discrimination between self and nonself DNA in CRISPR systems; it is absolutely required for recognition of the invading DNA but is absent from the host CRISPR array (9).On the basis of sequence homology, different CRISPR-Cas families have been identified (10). We investigate here a type IE and a type II system from Streptococcus thermophilus St-CRISPR4 and St-CRISPR3, respectively. The Cas proteins of type IE systems (4, 11, 12) associate with a crRNA into a mult...

Type III CRISPR-Cas systems in prokaryotes provide immunity against invading nucleic acids through the coordinated degradation of transcriptionally active DNA and its transcripts by the Csm effector complex. The Cas10 subunit of the complex contains an HD nuclease domain that is responsible for DNA degradation and two Palm domains with elusive functions. In addition, Csm6, a ribonuclease that is not part of the complex, is also required to provide full immunity. We show here that target RNA binding by the Csm effector complex of triggers Cas10 to synthesize cyclic oligoadenylates (cA ; = 2 to 6) by means of the Palm domains. Acting as signaling molecules, cyclic oligoadenylates bind Csm6 to activate its nonspecific RNA degradation. This cyclic oligoadenylate-based signaling pathway coordinates different components of CRISPR-Cas to prevent phage infection and propagation.