Structural insights into how Cas9 targets nucleosomes

Abstract The CRISPR-associated endonuclease Cas9 is used as a genome editing, which targets specific genomic loci by single guide RNAs (sgRNAs). In eukaryotes, genome DNA is stored in chromatin, in which the nucleosome is a basic unit. Despite previous structural analyses focusing on Cas9 cleaving free DNA, structural insights into Cas9 targeting of DNA within nucleosomes are limited, leading to uncertainties in understanding how Cas9 operates in the eukaryotic genome. In the present study, we found that Cas9 targets the linker DNA and the entry-exit DNA region of the nucleosome but does not the DNA tightly wrapped around the histone octamer. We determined cryo-electron microscopy (cryo-EM) structure of the Cas9-sgRNA-nucleosome ternary complex that targets linker DNA in nucleosomes. The structure suggested interactions between Cas9 and nucleosomes at multiple sites, and mutants that reduced the interaction between nucleosomal DNA and Cas9 improved nucleosomal DNA cleavage activity both in vitro and in vivo. These findings will contribute to the development of novel genome editing tools in chromatin.