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

• Published in: Cell 2014-02, Vol.156 (5), p.935-949
• Main Authors: Nishimasu, Hiroshi, Ran, F. Ann, Hsu, Patrick D., Konermann, Silvana, Shehata, Soraya I., Dohmae, Naoshi, Ishitani, Ryuichiro, Zhang, Feng, Nureki, Osamu
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacteria - enzymology; CRISPR-Associated Proteins - chemistry; CRISPR-Associated Proteins - metabolism; Crystallography, X-Ray; DNA, Bacterial - chemistry; DNA, Bacterial - metabolism; Endonucleases - chemistry; Endonucleases - metabolism; Models, Molecular; Molecular Sequence Data; Protein Structure, Tertiary; RNA, Bacterial - chemistry; RNA, Bacterial - metabolism; RNA, Guide - chemistry; RNA, Guide - metabolism; Sequence Alignment; Streptococcus pyogenes; Streptococcus pyogenes - chemistry; Streptococcus pyogenes - enzymology; Streptococcus pyogenes - metabolism
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/j.cell.2014.02.001

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 noncomplementary 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. [Display omitted] •Crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and target DNA•Cas9 has a bilobed architecture consisting of target recognition and nuclease lobes•Cas9 extensively interacts with the sgRNA•The C-terminal region of Cas9 participates in PAM recognition Crystal structure of Cas9 in complex with single guide RNA and target DNA elucidates the molecular mechanism of RNA-guided DNA targeting by Cas9.