Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA

Abstract

SUMMARY The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA, at 2.5 Å resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively-charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and non-complementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies.

Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry

“…The ''gold standard'' for characterizing molecular interactions of RBDs with their cognate RNA molecules by structure determination is co-crystallization [4,5]; others include NMR of the complex [6], or high-resolution EM of entire RNPs, as performed for the ribosome [7]. Although the number of co-structures of RBPs has been steadily increasing with more than 200 co-structures of protein-RNA complexes available in the PDB, most RBPs are still crystallized without RNA.…”

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

“…The length of sgRNA for Cas9 without the guide sequence (donated as sgRNA-∆guide) is roughly 3.5-4 times longer than the direct repeat region of crRNA for Cpf1. The sgRNA-∆guide contains multiple structural modules and adopts an extended conformation, forming extensive interactions with a large surface of Cas9 protein [13,14,17,18]. The interaction between sgRNA-∆guide and Cas9 stabilizes the conformation of several domains in Cas9 (Supplementary information, Figure S6A).…”

“…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].…”

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).…”