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

By T. Yamano, H. Nishimasu, Bernd Zetsche, H. Hirano, I. Slaymaker, Yinqing Li, I. Fedorova, Takanori Nakane, Kira S. Makarova, Eugene V. Koonin and 3 more. 2014

2,037 citations recorded by the source corpus.

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.

Subfields: Molecular Biology

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Citations in the corpus

The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells (2016, neutral): “To this end, although the crystal structures of both Spy and Sau Cas9 proteins 38,39 have been determined, the crystal structure of Nme Cas9 protein has not yet been reported.”
CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity (2014, background): “Recent advances in understanding the biochemical nature of Cas9 targeting (Sternberg et al., 2014), together with structural insights into Cas9:sgRNA complex formation (Jinek et al., 2014; Nishimasu et al., 2014), will lead to the development of improvedmolecular biology tools.”
Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells (2014, positive): “While this paper was under review, a pair of Cas9 structural studies were published 44,45 , including a crystal structure of dCas9 in complex with sgRNA and target DNA, which not only supports our observation of a PAM-proximal 5-nucleotide seed but also suggests a large conformation change during…”
Repurposing the CRISPR-Cas9 system for targeted DNA methylation (2016, background): “The predicted structure of the fusion product based on the Cas9 crystal structure (42) with the DNMT3A catalytic domain protruding at the PAM–interacting (PI) domain side of Cas9 is depicted in Figure 1A.”
Target specificity of the CRISPR-Cas9 system (2014, positive): “The PAM is recognized by an individual domain in the Cas9 protein [56], and the PAM sequence varies across bacteria species [57,58].”