8YNY

Structure of Cas9-sgRNA ribonucleoprotein bound to nucleosome

8YNY の概要

• エントリーDOI: 10.2210/pdb8yny/pdb
• EMDBエントリー: 39431
• 分子名称: Histone H3.1, Histone H4, Histone H2A type 1-B/E, ... (9 entities in total)
• 機能のキーワード: cas9, nucleosome, complex, nuclear protein
• 由来する生物種: Homo sapiens (human); 詳細
• タンパク質・核酸の鎖数: 13
• 化学式量合計: 415667.83

構造登録者

Nagamura, R.,Kujirai, T.,Kusakizako, T.,Hirano, H.,Kurumizaka, H.,Nureki, O. (登録日: 2024-03-12, 公開日: 2025-01-22)

主引用文献

Nagamura, R.,Kujirai, T.,Kato, J.,Shuto, Y.,Kusakizako, T.,Hirano, H.,Endo, M.,Toki, S.,Saika, H.,Kurumizaka, H.,Nureki, O.
Structural insights into how Cas9 targets nucleosomes.
Nat Commun, 15:10744-10744, 2024

PubMed Abstract: 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.

PubMed: 39737984
DOI: 10.1038/s41467-024-54768-z

実験手法

ELECTRON MICROSCOPY (4.52 Å)