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.

Summary Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two cryo-EM snapshots of the Thermobifida fusca Type I-E Cascade: 1) unwinding 11-bp of dsDNA at the seed-sequence region to scout for sequence complementarity, and 2) further unwinding of the entire protospacer to form a full R-loop. These structures provide the much-needed temporal and spatial resolution to resolve key mechanistic steps leading to Cas3 recruitment. In the early steps, PAM recognition causes severe DNA bending, leading to spontaneous DNA unwinding to form a seed-bubble. The full R-loop formation triggers conformational changes in Cascade, licensing Cas3 to bind. The same process also generates a bulge in the non-target DNA strand, enabling its handover to Cas3 for cleavage. The combination of both negative and positive checkpoints ensures stringent yet efficient target degradation in Type I CRISPR-Cas systems.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and the nearby cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes1–5. Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer6–9. Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical7–9. The conserved Cas1 and Cas2 proteins form an integrase complex consisting two distal Cas1 dimers bridged by a Cas2 dimer in the middle6,10. The prespacer is bound by Cas1-Cas2 as a dual forked DNA, and the terminal 3′-OH of each 3′-overhang serves as an attacking nucleophile during integration11–14. Importantly, the prespacer is preferentially integrated into the leader-proximal region of the CRISPR array1,7,10,15, guided by the leader sequence and a pair of inverted repeats (IRs) inside the CRISPR repeat7,15–20. Spacer integration in the most well-studied Escherichia coli Type I-E CRISPR system further relies on the bacterial Integration Host Factor (IHF)21,22. In Type II-A CRISPR, however, Cas1-Cas2 alone integrates spacer efficiently in vitro18; other Cas proteins (Cas9 and Csn2) play accessory roles in prespacer biogenesis17,23. Focusing on the Enterococcus faecalis Type II-A system24, here we report four structure snapshots of Cas1-Cas2 during spacer integration. EfaCas1-Cas2 selectively binds to a splayed 30-bp prespacer bearing 4-nt 3′-overhangs. Three molecular events take place upon encountering a target: Cas1-Cas2/prespacer first searches for half-sites stochastically, then preferentially interacts with the leader-side CRISPR repeat and catalyzes a nucleophilic attack that connects one strand of the leader-proximal repeat to the prespacer 3′-overhang. Recognition of the spacer half-site requires DNA bending and leads to full integration. We derive a mechanistic framework explaining the stepwise spacer integration process and the leader-proximal preference.

CRISPR-Cas systems enable microbial adaptive immunity and provide eukaryotic genome editing tools. These tools employ a single effector enzyme of type II or V CRISPR to generate RNA-guided, precise genome breaks. Here we demonstrate the feasibility of using type I CRISPR-Cas to effectively introduce a spectrum of long-range chromosomal deletions with a single RNA guide in human embryonic stem cells and HAP1 cells. Type I CRISPR systems rely on the multi-subunit ribonucleoprotein (RNP) complex Cascade to identify DNA targets and on the helicasenuclease enzyme Cas3 to degrade DNA processively. With RNP delivery of T. fusca Cascade and Cas3, we obtained 13%-60% editing efficiency. Long-range PCR-based and high-throughput-sequencing-based lesion analyses reveal that a variety of deletions, ranging from a few hundred base pairs to 100 kilobases, are created upstream of the target site. These results highlight the potential utility of type I CRISPR-Cas for long-range genome manipulations and deletion screens in eukaryotes.