Abstract

Towards Innovative Genome-editing Tool Based on the New Structure of CRISPR-Cas9 Complex

Osamu Nureki, Professor, University of Tokyo

The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we solved the crystal structure of Streptococcus pyrogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A resolution. The structure revealed a bilobed architecture consisting of target recognition and nuclease lobes (Rec and Nuc lobes, respectively), accommodating the sgRNA:DNA heteroduplex in a positively-charged groove at their interface. While Rec lobe is essential for binding sgRNA and DNA, Nuc lobe contains the HNH and RuvC nuclease domains, which are properly located for cleavage of the complementary and noncomplemantary strands of the target DNA, respectively. Nuc lobe also contains a C-terminal domain responsible for the recognition of the protospacer adjacent motif (PAM). This high-resolution structure combined with functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9. Recently, we solved crystal structures of quaternary complexes of several Cas9 orthologues, which uncovered the distinct mechanisms of PAM recognition. On the basis of the structures, we succeeded in changing the specificity of PAM recognition, which paves the way for rational design of new, versatile genome-editing technologies.