8H67

type I-B Cascade bound to a PAM-containing dsDNA target at 3.8 angstrom resolution.

8H67 の概要

• エントリーDOI: 10.2210/pdb8h67/pdb
• EMDBエントリー: 34495
• 分子名称: CRISPR RNA, Target DNA, Non target DNA, ... (7 entities in total)
• 機能のキーワード: type i-b, crispr-cas, cascade, rna binding protein
• 由来する生物種: Synechocystis sp. PCC 6714; 詳細
• タンパク質・核酸の鎖数: 15
• 化学式量合計: 404761.13

構造登録者

Xiao, Y.,Lu, M.,Yu, C.,Zhang, Y. (登録日: 2022-10-15, 公開日: 2024-05-01, 最終更新日: 2024-05-22)

主引用文献

Lu, M.,Yu, C.,Zhang, Y.,Ju, W.,Ye, Z.,Hua, C.,Mao, J.,Hu, C.,Yang, Z.,Xiao, Y.
Structure and genome editing of type I-B CRISPR-Cas.
Nat Commun, 15:4126-4126, 2024

PubMed Abstract: Type I CRISPR-Cas systems employ multi-subunit effector Cascade and helicase-nuclease Cas3 to target and degrade foreign nucleic acids, representing the most abundant RNA-guided adaptive immune systems in prokaryotes. Their ability to cause long fragment deletions have led to increasing interests in eukaryotic genome editing. While the Cascade structures of all other six type I systems have been determined, the structure of the most evolutionarily conserved type I-B Cascade is still missing. Here, we present two cryo-EM structures of the Synechocystis sp. PCC 6714 (Syn) type I-B Cascade, revealing the molecular mechanisms that underlie RNA-directed Cascade assembly, target DNA recognition, and local conformational changes of the effector complex upon R-loop formation. Remarkably, a loop of Cas5 directly intercalated into the major groove of the PAM and facilitated PAM recognition. We further characterized the genome editing profiles of this I-B Cascade-Cas3 in human CD3 T cells using mRNA-mediated delivery, which led to unidirectional 4.5 kb deletion in TRAC locus and achieved an editing efficiency up to 41.2%. Our study provides the structural basis for understanding target DNA recognition by type I-B Cascade and lays foundation for harnessing this system for long range genome editing in human T cells.

PubMed: 38750051
DOI: 10.1038/s41467-024-48598-2

実験手法

ELECTRON MICROSCOPY (3.8 Å)