8DZJ

Cryo-EM structure of Acidibacillus sulfuroxidans Cas12f in complex with sgRNA and target DNA

Summary for 8DZJ

• Entry DOI: 10.2210/pdb8dzj/pdb
• EMDB information: 27801
• Descriptor: OrfB_Zn_ribbon domain-containing protein, target DNA strand, Non-target DNA strand, ... (5 entities in total)
• Functional Keywords: crispr, cas12, gene editing, rna binding protein-rna-dna complex, rna binding protein/rna/dna
• Biological source: Acidibacillus sulfuroxidans; More
• Total number of polymer chains: 5
• Total formula weight: 191064.53

Authors

Liu, C.,Zhao, M. (deposition date: 2022-08-07, release date: 2023-07-12, Last modification date: 2023-11-08)

Primary citation

Wu, T.,Liu, C.,Zou, S.,Lyu, R.,Yang, B.,Yan, H.,Zhao, M.,Tang, W.
An engineered hypercompact CRISPR-Cas12f system with boosted gene-editing activity.
Nat.Chem.Biol., 19:1384-1393, 2023

PubMed Abstract: Compact CRISPR-Cas systems offer versatile treatment options for genetic disorders, but their application is often limited by modest gene-editing activity. Here we present enAsCas12f, an engineered RNA-guided DNA endonuclease up to 11.3-fold more potent than its parent protein, AsCas12f, and one-third of the size of SpCas9. enAsCas12f shows higher DNA cleavage activity than wild-type AsCas12f in vitro and functions broadly in human cells, delivering up to 69.8% insertions and deletions at user-specified genomic loci. Minimal off-target editing is observed with enAsCas12f, suggesting that boosted on-target activity does not impair genome-wide specificity. We determine the cryo-electron microscopy (cryo-EM) structure of the AsCas12f-sgRNA-DNA complex at a resolution of 2.9 Å, which reveals dimerization-mediated substrate recognition and cleavage. Structure-guided single guide RNA (sgRNA) engineering leads to sgRNA-v2, which is 33% shorter than the full-length sgRNA, but with on par activity. Together, the engineered hypercompact AsCas12f system enables robust and faithful gene editing in mammalian cells.

PubMed: 37400536
DOI: 10.1038/s41589-023-01380-9

Experimental method

ELECTRON MICROSCOPY (2.9 Å)