7Z4D

Crystal structure of SpCas9 bound to a 10 nucleotide complementary DNA substrate

7Z4D の概要

• エントリーDOI: 10.2210/pdb7z4d/pdb
• 分子名称: sgRNA, CRISPR-associated endonuclease Cas9/Csn1, Target strand of 10 nucleotide complementary DNA substrate, ... (6 entities in total)
• 機能のキーワード: crispr, cas9, r-loop, substrate binding, off-target, hydrolase
• 由来する生物種: Streptococcus pyogenes; 詳細
• タンパク質・核酸の鎖数: 8
• 化学式量合計: 409031.44

構造登録者

Pacesa, M.,Jinek, M. (登録日: 2022-03-03, 公開日: 2022-08-31, 最終更新日: 2024-01-31)

主引用文献

Pacesa, M.,Loeff, L.,Querques, I.,Muckenfuss, L.M.,Sawicka, M.,Jinek, M.
R-loop formation and conformational activation mechanisms of Cas9.
Nature, 609:191-196, 2022

PubMed Abstract: Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications, yet its precise mechanisms of target DNA binding and off-target discrimination remain incompletely understood. Here we report a series of cryo-electron microscopy structures of Streptococcus pyogenes Cas9 capturing the directional process of target DNA hybridization. In the early phase of R-loop formation, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the distal end of the target DNA duplex. Guide-target hybridization past the seed region induces rearrangements of the REC2 and REC3 domains and relocation of the HNH nuclease domain to assume a catalytically incompetent checkpoint conformation. Completion of the guide-target heteroduplex triggers conformational activation of the HNH nuclease domain, enabled by distortion of the guide-target heteroduplex, and complementary REC2 and REC3 domain rearrangements. Together, these results establish a structural framework for target DNA-dependent activation of Cas9 that sheds light on its conformational checkpoint mechanism and may facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.

PubMed: 36002571
DOI: 10.1038/s41586-022-05114-0

実験手法

X-RAY DIFFRACTION (3.1 Å)