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

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…