Prokaryotes acquire virus resistance by integrating short fragments of viral nucleic acid into clusters of regularly interspaced short palindromic repeats (CRISPRs). Here we show how virus-derived sequences contained in CRISPRs are used by CRISPR-associated (Cas) proteins from the host to mediate an antiviral response that counteracts infection. After transcription of the CRISPR, a complex of Cas proteins termed Cascade cleaves a CRISPR RNA precursor in each repeat and retains the cleavage products containing the virus-derived sequence. Assisted by the helicase Cas3, these mature CRISPR RNAs then serve as small guide RNAs that enable Cascade to interfere with virus proliferation. Our results demonstrate that the formation of mature guide RNAs by the CRISPR RNA endonuclease subunit of Cascade is a mechanistic requirement for antiviral defense.

Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)/Cas (CRISPR-associated sequences) systems provide adaptive immunity against viruses when a spacer sequence of small CRISPR RNA (crRNA) matches a protospacer sequence in the viral genome. Viruses that escape CRISPR/Cas resistance carry point mutations in protospacers, though not all protospacer mutations lead to escape. Here, we show that in the case of Escherichia coli subtype CRISPR/Cas system, the requirements for crRNA matching are strict only for a seven-nucleotide seed region of a protospacer immediately following the essential protospacer-adjacent motif. Mutations in the seed region abolish CRISPR/Cas mediated immunity by reducing the binding affinity of the crRNA-guided Cascade complex to protospacer DNA. We propose that the crRNA seed sequence plays a role in the initial scanning of invader DNA for a match, before base pairing of the full-length spacer occurs, which may enhance the protospacer locating efficiency of the E. coli Cascade complex. In agreement with this proposal, single or multiple mutations within the protospacer but outside the seed region do not lead to escape. The relaxed specificity of the CRISPR/ Cas system limits escape possibilities and allows a single crRNA to effectively target numerous related viruses.bacteriophage | RNA interference | small RNA C RISPR (clustered regularly interspaced short palindromic repeats) cassettes are present in virtually every archaeon and in approximately 40% of bacteria (1-3). A CRISPR cassette consists of almost identical direct repeats that are regularly interspersed with spacers (4). In any given cassette, the length of spacers is similar, whereas their sequences vary. CRISPR cassettes are often flanked by a diverse set of CRISPR-associated (cas) genes (2,5,6).CRISPR/Cas (CRISPR-associated sequences) functions as an adaptive immunity system by excluding viruses and other mobile genetic elements that contain sequences matching CRISPR cassette spacers (6-9). Bacterial and archaeal CRISPR/Cas systems generally target DNA (10-13), although one archaeal system has been demonstrated in vitro to interfere at the level of RNA (14). Transcription of a CRISPR cassette, followed by processing with the help of dedicated endoribonucleases, creates small CRISPR RNAs (crRNAs) that guide the Cas machinery to the target, eventually resulting in target cleavage (11,(15)(16)(17)(18)(19)(20).Although a match between a single CRISPR spacer and a foreign DNA sequence called the protospacer can provide immunity to the entry of that DNA into the host, it is not sufficient. Mutations in the conserved protospacer-adjacent motif (PAM, ref. 21) abolish CRISPR-mediated immunity even in the presence of a perfect spacer-protospacer match. Likewise, some point mutations in protospacer that introduce single mismatches with the spacer abolish CRISPR/Cas function even when the PAM is intact (22). Thus, a PAM and a match between a spacer and protospacer are both required for CRISPR/Cas function.Recently, how...

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA(1)B(2)C(6)D(1)E(1)) and a 61-nucleotide CRISPR RNA (crRNA) with 5'-hydroxyl and 2',3'-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.