9SJO

Type I-F_HNH variant Cascade target-free RNP, HNH domain in middle position

これはPDB形式変換不可エントリーです。

9SJO の概要

• エントリーDOI: 10.2210/pdb9sjo/pdb
• EMDBエントリー: 54950
• 分子名称: Cas8f fusion with HNH, Cas5f, Cas6f, ... (5 entities in total)
• 機能のキーワード: crispr-cas type i-f hnh nuclease, rna binding protein
• 由来する生物種: Selenomonas sp.; 詳細
• タンパク質・核酸の鎖数: 10
• 化学式量合計: 340420.39

構造登録者

Fuglsang, A.,Montoya, G. (登録日: 2025-08-31, 公開日: 2026-02-04, 最終更新日: 2026-02-18)

主引用文献

Fuglsang, A.,Rout, S.S.,Koutna, E.B.,Sofos, N.,Gallego, A.R.,Montoya, G.
Conformational dynamics of CRISPR-Cas type I-F-HNH inform nickase engineering in a cascade scaffold.
Nucleic Acids Res., 54:-, 2026

PubMed Abstract: The type I-FHNH CRISPR-Cas system is a non-canonical Class 1 effector complex distinguished by the replacement of the Cas3 recruitment domain with a catalytic HNH domain in Cas8, enabling autonomous DNA cleavage without accessory nucleases. Using cryo-EM, we determined high-resolution structures of the effector complex in three catalytic states-precatalytic, NTS-cleaved, and post-catalytic-revealing a dynamic trajectory of the HNH domain through inward, middle, and outward conformations. Biochemical assays demonstrated that the complex cleaves the nontarget strand (NTS) prior to the target strand (TS), consistent with a sequential cleavage mechanism similar to Cas12 effectors but notably lacking trans-cleavage activity on single-stranded DNA. Structural comparisons confirmed a minimal PAM requirement (5'-CN) and a constrained HNH catalytic site poised for precise strand scission. We engineered a ΔLinker variant of Cas8 that repositions the HNH domain, selectively abolishing TS cleavage and converting the system into a programmable NTS-specific nickase. Importantly, we validated the functionality of both wild-type and mutant complexes in human cells. While the wild-type system induced indels and base substitutions, the ΔLinker variant triggered targeted single-strand nicks without double-stranded breaks. Together, our work establishes type I-FHNH as a compact and precise genome editing platform with in vivo efficacy.

PubMed: 41603736
DOI: 10.1093/nar/gkag053

実験手法

ELECTRON MICROSCOPY (3.07 Å)