9UDI

Cryo-EM structure of the RdCas12n-sgRNA-DNA ternary complex, Conformation 2

9UDI の概要

• エントリーDOI: 10.2210/pdb9udi/pdb
• EMDBエントリー: 64070
• 分子名称: Transposase, DNA (40-MER), DNA (5'-D(P*GP*CP*TP*GP*TP*GP*AP*GP*AP*AP*AP*CP*CP*G)-3'), ... (4 entities in total)
• 機能のキーワード: crispr-cas, complex, dna binding protein, dna binding protein-dna-rna complex, dna binding protein/dna/rna
• 由来する生物種: Rothia dentocariosa; 詳細
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 147127.96

構造登録者

Fu, W.,Ji, Q. (登録日: 2025-04-06, 公開日: 2025-06-04, 最終更新日: 2025-07-23)

主引用文献

Fu, W.,Ma, J.,Wang, Z.,Tang, N.,Pan, D.,Su, M.,Wu, Z.,Gan, J.,Ji, Q.
Mechanisms and engineering of a miniature type V-N CRISPR-Cas12 effector enzyme.
Nat Commun, 16:5667-5667, 2025

PubMed Abstract: Type V CRISPR-Cas12 systems are highly diverse in their functionality and molecular compositions, including miniature Cas12f1 and Cas12n genome editors that provide advantages for efficient in vivo therapeutic delivery due to their small size. In contrast to Cas12f1 nucleases that utilize a homodimer structure for DNA targeting and cleavage with a preference for T- or C-rich PAMs, Cas12n nucleases are likely monomeric proteins and uniquely recognize rare A-rich PAMs. However, the molecular mechanisms behind RNA-guided genome targeting and cleavage by Cas12n remain unclear. Here, we present the cryo-electron microscopy (cryo-EM) structure of Rothia dentocariosa Cas12n (RdCas12n) bound to a single guide RNA (sgRNA) and target DNA, illuminating the intricate molecular architecture of Cas12n and its sgRNA, as well as PAM recognition and nucleic-acid binding mechanisms. Through structural comparisons with other Cas12 nucleases and the ancestral precursor TnpB, we provide insights into the evolutionary significance of Cas12n in the progression from TnpB to various Cas12 nucleases. Additionally, we extensively modify the sgRNA and convert RdCas12n into an effective genome editor in human cells. Our findings enhance the understanding of the evolutionary mechanisms of type V CRISPR-Cas12 systems and offer a molecular foundation for engineering Cas12n genome editors.

PubMed: 40595633
DOI: 10.1038/s41467-025-61290-3

実験手法

ELECTRON MICROSCOPY (3.01 Å)