Nagamura Reina, Kujirai Tomoya, Kato Junko, Shuto Yutaro, Kusakizako Tsukasa, Hirano Hisato, Endo Masaki, Toki Seiichi, Saika Hiroaki, Kurumizaka Hitoshi, Nureki Osamu
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan.
Institute for Quantitative Biosciences, Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan.
Nat Commun. 2024 Dec 30;15(1):10744. doi: 10.1038/s41467-024-54768-z.
The CRISPR-associated endonuclease Cas9 derived from prokaryotes is used as a genome editing, which targets specific genomic loci by single guide RNAs (sgRNAs). The eukaryotes, the target of genome editing, store their genome DNA 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 perform native-polyacrylamide gel electrophoresis (PAGE) analyses and find that Cas9 targets the linker DNA and the entry-exit DNA region of the nucleosome but not the DNA tightly wrapped around the histone octamer. We further determine cryo-electron microscopy (cryo-EM) structure of the Cas9-sgRNA-nucleosome ternary complex that targets linker DNA in nucleosomes. The structure suggests interactions between Cas9 and nucleosomes at multiple sites. Mutants that reduce the interaction between nucleosomal DNA and Cas9 improve nucleosomal DNA cleavage activity in vitro, although inhibition by the interaction between Cas9 and nucleosomes is limited in vivo. These findings will contribute to the development of novel genome editing tools in chromatin.
源自原核生物的CRISPR相关核酸内切酶Cas9被用作基因组编辑工具，它通过单导向RNA（sgRNA）靶向特定的基因组位点。作为基因组编辑的目标，真核生物将其基因组DNA存储在染色质中，其中核小体是基本单位。尽管先前的结构分析聚焦于Cas9切割游离DNA，但对于Cas9靶向核小体内DNA的结构见解有限，这导致在理解Cas9如何在真核基因组中发挥作用方面存在不确定性。在本研究中，我们进行了非变性聚丙烯酰胺凝胶电泳（PAGE）分析，发现Cas9靶向核小体的连接区DNA和进出DNA区域，而不是紧密缠绕在组蛋白八聚体周围的DNA。我们进一步确定了Cas9-sgRNA-核小体三元复合物的冷冻电子显微镜（cryo-EM）结构，该复合物靶向核小体中的连接区DNA。该结构表明Cas9与核小体在多个位点存在相互作用。减少核小体DNA与Cas9之间相互作用的突变体在体外提高了核小体DNA切割活性，尽管在体内Cas9与核小体之间的相互作用对其抑制作用有限。这些发现将有助于开发新型的染色质基因组编辑工具。
