Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage

Abstract

Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)–associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding to form an RNA-DNA hybrid and a displaced DNA strand inside the protein. The role of this R-loop structure in positioning each DNA strand for cleavage by the two Cas9 nuclease domains is unknown. We determine molecular structures of the catalytically active Streptococcus pyogenes Cas9 R-loop that show the displaced DNA strand located near the RuvC nuclease domain active site. These protein-DNA interactions, in turn, position the HNH nuclease domain adjacent to the target DNA strand cleavage site in a conformation essential for concerted DNA cutting. Cas9 bends the DNA helix by 30°, providing the structural distortion needed for R-loop formation.

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

“…Since the HNH domain does not directly contact nucleic acids at the PAM-distal end 13,17–19 , it is likely that a separate domain of Cas9 senses target complementarity to govern HNH domain mobility. Structural studies suggested that a domain within the Cas9 recognition (REC) lobe (REC3) interacts with the RNA/DNA heteroduplex and undergoes conformational changes upon target binding (Extended Data Figure 2e–f) 13,14,17–19 .…”

“…Structural studies suggested that a domain within the Cas9 recognition (REC) lobe (REC3) interacts with the RNA/DNA heteroduplex and undergoes conformational changes upon target binding (Extended Data Figure 2e–f) 13,14,17–19 . Because the function of this non-catalytic domain was previously unknown, we labeled SpCas9 with Cy3/Cy5 dyes at positions S701C (within the “mobile” REC3 domain) and S960C (within the “stationary” RuvC domain) to generate SpCas9 REC3 and observed that the conformational states of REC3 become more heterogeneous as PAM-distal mismatches increase (Extended Data Figure 4a–c).…”

“…Structural studies suggested that REC2 occludes the HNH domain from the scissile phosphate in the sgRNA-bound state 19 , and undergoes a large outward rotation upon binding to double-stranded DNA (dsDNA) 13,14 (Figure 2e). To test whether the REC2 domain regulates access of HNH to the target strand scissile phosphate, we labeled SpCas9 with Cy3/Cy5 dyes at positions E60C (within the “stationary” Arginine-rich helix) and D273C (within the “mobile” REC2 domain) to generate SpCas9 REC2 in order to detect REC2 conformational changes (Extended Data Figure 1b–c).…”

Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition

“…CRISPR-Cas9 has been extensively used for genome editing in various cell types and organisms [11,12]. A series of structural studies of Streptococcus pyogenes Cas9 (SpyCas9) and its orthologs have revealed the detailed intermolecular interactions, as well as the conformational changes among different substrate-bound states [13][14][15][16][17][18].…”

LncRNAs: Bridging environmental sensing and gene expression

“…67,68 These structures can form in cis 60 or trans 69 in vivo, either by "threading back" of nascent RNAs during transcription by RNA Pol II, through the action of the homologous recombination machinery, or associated RNA-binding proteins as in the case of CRISPR. 60,70,71 Given that RNP assembly defects cause R-loops, 60,70 we speculated that loss of DBP2 might promote formation of these structures between the GAL lncRNAs and GAL gene promoters. To test this, we asked if ectopic expression of human RNAse H1 in dbp2D cells would prevent rapid induction of the GAL genes by the GAL lncRNAs.…”