8T77

SpRY-Cas9:gRNA complex bound to non-target DNA with 6 bp R-loop

8T77 の概要

• エントリーDOI: 10.2210/pdb8t77/pdb
• EMDBエントリー: 41086
• 分子名称: CRISPR-associated endonuclease Cas9/Csn1, gRNA, TS, ... (4 entities in total)
• 機能のキーワード: spry-cas9, crispr, cas9, immune system, r-loop
• 由来する生物種: Streptococcus pyogenes; 詳細
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 195501.52

構造登録者

Hibshman, G.N.,Bravo, J.P.K.,Taylor, D.W. (登録日: 2023-06-19, 公開日: 2024-05-01, 最終更新日: 2024-05-15)

主引用文献

Hibshman, G.N.,Bravo, J.P.K.,Hooper, M.M.,Dangerfield, T.L.,Zhang, H.,Finkelstein, I.J.,Johnson, K.A.,Taylor, D.W.
Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9.
Nat Commun, 15:3663-3663, 2024

PubMed Abstract: CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable genes. Recently developed Cas9 variants have been engineered with relaxed PAM requirements, including SpG-Cas9 (SpG) and the nearly PAM-less SpRY-Cas9 (SpRY). However, the molecular mechanisms of how SpRY recognizes all potential PAM sequences remains unclear. Here, we combine structural and biochemical approaches to determine how SpRY interrogates DNA and recognizes target sites. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region, which facilitates tight binding to off-target DNA sequences. Nuclease activation occurs ~1000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Experiments with SpG position it as an intermediate enzyme between Cas9 and SpRY. Our findings shed light on the molecular mechanisms of PAMless genome editing.

PubMed: 38688943
DOI: 10.1038/s41467-024-47830-3

実験手法

ELECTRON MICROSCOPY (3.25 Å)