Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System

Abstract

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.

Real-Time Observation of Target Search by the CRISPR Surveillance Complex Cascade

“…This model suggests that PAM recognition during target searching may be accompanied by a DNA bending event that enables local melting of the dsDNA helix. A recent Cascade/seed-bubble structure also suggests that PAM recognition is coupled with DNA bending, which drives the melting of dsDNA at the seed region (Xiao et al, 2017). However, this model does not account for how Cascade may interact with DNA prior to helical bending.…”

“…Recent structures of Cascade bound to dsDNA have revealed how Cascade engages the PAM and target upon R-loop formation (Hayes et al, 2016; Xiao et al, 2017). However, it remains unclear how Cascade recognizes the PAM during target searching.…”

“…Consistent with this hypothesis, our data implicate this motif in target sampling, revealing that interactions between the lysine vise and dsDNA are initiated prior to duplex unwinding and maintained following R-loop formation. T. fusca Cascade (type I–E) contains similar Lys-rich loops in its Cas7 subunits (K-vise and K-Rim) indicating that type I–E Cascade uses a common mechanism to engage dsDNA (Xiao et al, 2017). Although no corresponding domain exists in Cas7 subunits of the Csy complex (type I–F), it has been proposed that the Cas8f “hook” domain clamps onto dsDNA and holds it in a secure vise (Chowdhury et al, 2017; Guo et al, 2017).…”

“…The Cse1 PAM recognition motif identifies PAMs during target searching and maintains contact following target binding, an interaction that is thought to be important for stabilizing Cse1 in a conformation required for Cas3 recruitment and target degradation (Hayes et al, 2016; Xiao et al, 2017; Xue et al, 2016). In addition, our data also implicate the Cse1 lysine-rich β hairpin in the PAM searching process, although this motif does not contact DNA following target binding.…”

“…Similar to the Type II surveillance complex Cas9, Cascade searches for targets by first recognizing a short sequence called the protospacer-adjacent motif (PAM) (Mojica et al, 2009; Redding et al, 2015; Rollins et al, 2015; Sashital et al, 2012; Semenova et al, 2011; Sternberg et al, 2014). PAM recognition destabilizes the DNA duplex by inserting a glutamine wedge located in the large Cse1 (Cas8e) subunit into the dsDNA adjacent to the PAM, enabling strand invasion and formation of an RNA-DNA heteroduplex in the seed region (positions 1–5 and 7–8 of the crRNA spacer) (Hayes et al, 2016; Xiao et al, 2017). Complementary base pairing in the seed region efficiently promotes further R-loop formation (Semenova et al, 2011; Szczelkun et al, 2014).…”