Crystal structure of Cas9 in complex with guide RNA and target DNA.
Abstract
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.
REC lobe and PI domain
(A) Structure of the REC lobe. The REC2 domain and the Bridge helix are colored dark gray and green, respectively. The REC1 domain is colored gray, with the repeat-interacting and anti-repeat-interacting regions colored pale blue and pink, respectively. The bound sgRNA:DNA is shown as a semi-transparent ribbon representation.
(B) Mutational analysis of the REC lobe. Schematics show the truncation mutants. The bar graph shows indel mutations generated by the truncation mutants, measured by the SURVEYOR assay (n = 3, error bars show mean ± S.E.M., N.D., not detectable).
(C) Western blot showing the expression of the truncation mutants in HEK 293FT cells.
(D) Structure of the PI domain. The bound sgRNA is shown as a semi-transparent ribbon representation.
(E) Mutational analysis of the PI domain. Schematics show wild-type SpCas9 and St3Cas9, chimeric Sp-St3Cas9 and St3-SpCas9, and the SpCas9 PI domain truncation mutant. Cas9s were assayed for indel generation at target sites upstream of either NGG (left bar graph) or NGGNG (right bar graph) PAMs (n = 3, error bars show mean ± S.E.M., N.D., not detectable).
