Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes

Abstract

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...

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

“…Our findings provide direct evidence to support previous speculation that Cas9 relies on protospacer sensing to enable accurate targeting 21,22 . In particular, we propose that REC3 binding to the RNA-DNA duplex is necessary for re-orienting REC2, which enables HNH docking at the active site (Extended Data Figure 4h–i).…”

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

“…Upon subsequent encounters, the complex of Cas proteins with CRISPR RNA (crRNA) bearing the spacer sequence binds and cleaves foreign DNA containing the matching sequence (1-3), thus providing the host organism with adaptive hereditable immunity. The DNA endonuclease Cas9 of type II CRISPR-Cas systems is targeted to specific DNA sequences by a 20-base crRNA spacer that binds to the complementary strand of protospacer DNA, displacing the noncomplementary strand to form an R-loop (4,5). Both binding and cleavage of target DNA by the Cas9/crRNA complex require a short protospacer adjacent motif (PAM) located immediately downstream of the targeted sequence (6).…”

Highly efficient primed spacer acquisition from targets destroyed by the Escherichia coli type I-E CRISPR-Cas interfering complex

“…In DNA-targeting type I and type II CRISPR-Cas systems, target recognition requires, in addition to crRNA spacer-target protospacer complementarity, a protospacer adjacent motif (PAM) (8,9) recognized by effector proteins (10)(11)(12). Upon target DNA recognition, a stable R-loop containing locally melted protospacer DNA and an RNA-DNA heteroduplex is formed (13,14). R-loop formation is followed by target DNA destruction either by the effector complex alone (type II) or through recruitment of an additional endonuclease Cas3 (type I systems) (4).…”