EMD-45633

Single-particle
3.5 Å
EMD-45633 Deposition: 06/07/2024
Map released: 08/01/2025
Last modified: 08/01/2025
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links

EMD-45633

Cas12a:gRNA:DNA (Acidaminococcus sp.) with 0 RNA:DNA base pairs, structure 3

EMD-45633

Single-particle
3.5 Å
EMD-45633 Deposition: 06/07/2024
Map released: 08/01/2025
Last modified: 08/01/2025
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links
Sample Organism: synthetic construct, Acidaminococcus sp. BV3L6
Sample: Cas12a:gRNA:DNA (Acidaminococcus sp.) with 0 RNA:DNA base pairs, structure 3
Fitted models: 9cjj (Avg. Q-score: 0.324)

Deposition Authors: Soczek KM, Doudna JA
CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.
Soczek KM, Cofsky JC, Tuck OT, Shi H , Doudna JA
(2024) Nucleic Acids Res
PUBMED: 39698811
DOI: doi:10.1093/nar/gkae1192
ISSN: 1362-4962
ASTM: NARHAD
Abstract:
RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA interference begins with local helix distortion by transient CRISPR-Cas protein binding.