Summary The prokaryotic CRISPR/Cas immune system is based on genomic loci that contain incorporated sequence tags from viruses and plasmids. Using small guide RNA molecules, these sequences act as a memory to reject returning invaders. Both the Cascade ribonucleoprotein complex and the Cas3 nuclease/helicase are required for CRISPR-interference in Escherichia coli, but it is unknown how natural target DNA molecules are recognized and neutralized by their combined action. Here we show that Cascade efficiently locates target sequences in negatively supercoiled DNA, but only if these are flanked by a Protospacer Adjacent Motif (PAM). PAM recognition by Cascade exclusively involves the crRNA-complementary DNA strand. After Cascade-mediated R-loop formation, the Cse1 subunit recruits Cas3, which catalyzes nicking of target DNA through its HD-nuclease domain. The target is then progressively unwound and cleaved by the joint ATP-dependent helicase activity and Mg2+-dependent HD-nuclease activity of Cas3, leading to complete target DNA degradation and invader neutralization.

Clustered regularly interspaced short palindromic repeats (CRISPRs) are essential components of RNA-guided adaptive immune systems that protect bacteria and archaea from viruses and plasmids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble into a 405 kDa multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Here we present the 3.24 Å resolution x-ray crystal structure of Cascade. Eleven proteins and a 61-nucleotide crRNA assemble into a sea-horse-shaped architecture that binds double-stranded DNA targets complementary to the crRNA-guide sequence. Conserved sequences on the 3′- and 5′-ends of the crRNA are anchored by proteins at opposite ends of the complex, while the guide sequence is displayed along a helical assembly of six interwoven subunits that present 5-nucleotide segments of the crRNA in pseudo A-form configuration. The structure of Cascade suggests a mechanism for assembly and provides insights into the mechanisms of target recognition.

Clusters of regularly interspaced short palindromic repeats (CRISPRs) and cas (CRISPR-associated) operon form an RNA-based adaptive immune system against foreign genetic elements in prokaryotes1. Type I account for 95% of CRISPR systems, and have been utilized to control gene expression and cell fate2,3. During CRISPR RNA (crRNA)-guided interference, Cascade (CRISPR-associated complex for antiviral defense) facilitates crRNA-guided invasion of double-stranded DNA (dsDNA) for complementary base-pairing with the target DNA strand, while displacing the non-target strand, forming an R-loop4,5. Cas3 nuclease/helicase is recruited subsequently to degrade two DNA strands4,6,7. Protospacer adjacent motif (PAM) flanking target DNA is crucial for self vs. foreign discrimination4,8–16. Here we present a 2.45 Å crystal structure of E. coli Cascade bound to a foreign dsDNA target. The 5′-ATG PAM is recognized in double-stranded form, from the minor groove side, by three structural features in Cse1. The promiscuity inherent to minor groove DNA recognition rationalizes the puzzling observation that a single Cascade can respond to several distinct PAM sequences. Optimal PAM recognition coincides with a wedge insertion, initiating the directional target DNA strand unwinding for segmented base-pairing with crRNA. The non-target strand is guided along a parallel path 25 Å apart, and the R-loop structure is further stabilized by locking this strand behind Cse2 dimer. These observations provide the structure basis for understanding the PAM-dependent directional R-loop formation process17,18.

In bacteria and archaea, short fragments of foreign DNA are integrated into Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci, providing a molecular memory of previous encounters with foreign genetic elements. In Escherichia coli, short CRISPR-derived RNAs are incorporated into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Recent structures of Cascade capture snapshots of this seahorse-shaped RNA-guided surveillance complex before and after binding to a DNA target. Here we determine a 3.2 Å x-ray crystal structure of Cascade in a new crystal form that provides insight into the mechanism of double-stranded DNA binding. Molecular dynamic simulations performed using available structures reveal functional roles for residues in the tail, backbone and belly subunits of Cascade that are critical for binding double-stranded DNA. Structural comparisons are used to make functional predictions and these predictions are tested in vivo and in vitro. Collectively, the results in this study reveal underlying mechanisms involved in target-induced conformational changes and highlight residues important in DNA binding and protospacer adjacent motif recognition.

Bacteria and archaea rely on CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided adaptive immune systems for sequence specific elimination of foreign nucleic acids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble with Cas (CRISPR-associated) proteins into a 405-kilodalton multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade binds foreign DNA complementary to the crRNA guide and recruits Cas3, a trans-acting nuclease-helicase required for target degradation. Structural models of Cascade have captured static snapshots of the complex in distinct conformational states, but conformational dynamics of the 11-subunit surveillance complex have not been measured. Here we use hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to map conformational dynamics of Cascade onto the three-dimensional structure. New insights from structural dynamics are used to make functional predictions about the mechanisms of the R-loop coordination and Cas3 recruitment. We test these predictions in vivo and in vitro. Collectively, we show how mapping conformational dynamics onto static 3D-structures adds an additional dimension to the functional understanding of this biological machine.

Adaptive immune systems in bacteria and archaea rely on small CRISPR-derived RNAs (crRNAs) to guide specialized nucleases to foreign nucleic acids. The activation of these nucleases is controlled by a series of molecular checkpoints that ensure precise cleavage of nucleic acid targets, while minimizing toxic off-target cleavage events. In this review, we highlight recent advances in understanding regulatory mechanisms responsible for controlling the activation of these nucleases and identify emerging regulatory themes conserved across diverse CRISPR systems.

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The interface between viruses and their hosts’ are hot spots for biological and biotechnological innovation. Bacteria use restriction endonucleases to destroy invading DNA, and industry has exploited these enzymes for molecular cut-and-paste reactions that are central to many recombinant DNA technologies. Today, another class of nucleases central to adaptive immune systems that protect bacteria and archaea from invading viruses and plasmids are blazing a similar path from basic science to profound biomedical and industrial applications.