8IP0

Cryo-EM structure of type I-B Cascade bound to a PAM-containing dsDNA target at 3.6 angstrom resolution

Summary for 8IP0

• Entry DOI: 10.2210/pdb8ip0/pdb
• EMDB information: 35629
• Descriptor: Type I-MYXAN CRISPR-associated protein Cmx8, Type I-MYXAN CRISPR-associated protein Cas5/Cmx5/DevS, Fruiting body developmental protein R-like protein, ... (8 entities in total)
• Functional Keywords: type i-b, crispr-cas, cascade, rna binding protein/rna/dna, rna binding protein-rna-dna complex
• Biological source: Synechocystis sp. PCC 6714 (Aphanocapsa sp. (strain PCC 6714)); More
• Total number of polymer chains: 16
• Total formula weight: 404717.28

Authors

Xiao, Y.,Lu, M.,Yu, C.,Zhang, Y. (deposition date: 2023-03-13, release date: 2024-03-20, Last modification date: 2025-07-23)

Primary citation

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

Experimental method

ELECTRON MICROSCOPY (3.6 Å)