9K31

Cryo-EM structure of the YnpsCas9-guide RNA-target DNA complex

9K31 の概要

• エントリーDOI: 10.2210/pdb9k31/pdb
• EMDBエントリー: 62003
• 分子名称: YnpsCas9, Guide RNA, Target DNA strand, ... (7 entities in total)
• 機能のキーワード: rna-guided dna nuclease, rna binding protein
• 由来する生物種: metagenome; 詳細
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 251687.85

構造登録者

Nagahata, N.,Yamada, S.,Yamashita, K.,Nishimasu, H. (登録日: 2024-10-18, 公開日: 2026-02-04, 最終更新日: 2026-03-04)

主引用文献

Nagahata, N.,Kato, K.,Yamada, S.,Kannan, S.,Okazaki, S.,Isayama, Y.,Hiraizumi, M.,Yamashita, K.,Koonin, E.V.,Zhang, F.,Nishimasu, H.
Structural visualization of the molecular evolution of CRISPR-Cas9.
Nat.Struct.Mol.Biol., 33:304-317, 2026

PubMed Abstract: RNA-guided DNA nucleases Cas9 and IscB (insertion sequences Cas9-like OrfB) are components of type II CRISPR-Cas adaptive immune systems and transposon-associated OMEGA (obligate mobile element-guided activity) systems, respectively. Sequence and structural comparisons indicate that IscB (~500 residues) evolved into Cas9 (~700-1,600 residues) through protein expansion coupled with guide RNA miniaturization. However, the specific sequence of events in this evolutionary transition remains unknown. Here, we report cryo-electron microscopy structures of four phylogenetically diverse RNA-guided nucleases-two IscBs and two Cas9s-each in complex with its cognate guide RNA and target DNA. Comparisons of these four complex structures to previously reported IscB and Cas9 structures indicate that evolution from IscB to Cas9 involved the loss of the N-terminal PLMP domain and the acquisition of the zinc-finger-containing REC3 domain, followed by bridge helix extension and REC1 domain acquisition. These structural changes led to expansion of the REC lobe, increasing the target DNA cleavage specificity. Additionally, the structural conservation of the RNA scaffolds indicates that the dual CRISPR RNA (crRNA) and trans-activating crRNA guides of CRISPR-Cas9 evolved from the single ωRNA guides of OMEGA systems. Our findings provide insights into the succession of structural changes involved in the exaptation of transposon-associated RNA-guided nucleases for the role of effector nucleases in adaptive immune systems.

PubMed: 41617991
DOI: 10.1038/s41594-025-01743-x

実験手法

ELECTRON MICROSCOPY (2.38 Å)