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TitleCRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.
Journal, issue, pagesNucleic Acids Res, Vol. 53, Issue 2, Year 2025
Publish dateJan 11, 2025
AuthorsKatarzyna M Soczek / Joshua C Cofsky / Owen T Tuck / Honglue Shi / Jennifer A Doudna /
PubMed AbstractRNA-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 ...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.
External linksNucleic Acids Res / PubMed:39698811 / PubMed Central
MethodsEM (single particle)
Resolution3.4 - 3.6 Å
Structure data

45631

9cjh

EMDB-45631, PDB-9cjh:
Cas12a:gRNA:DNA (Acidaminococcus sp.) with 0 RNA:DNA base pairs, structure 1
Method: EM (single particle) / Resolution: 3.6 Å

45632

9cji

EMDB-45632, PDB-9cji:
Cas12a:gRNA:DNA (Acidaminococcus sp.) with 0 RNA:DNA base pairs, structure 2
Method: EM (single particle) / Resolution: 3.4 Å

45633

9cjj

EMDB-45633, PDB-9cjj:
Cas12a:gRNA:DNA (Acidaminococcus sp.) with 0 RNA:DNA base pairs, structure 3
Method: EM (single particle) / Resolution: 3.5 Å

Source
  • acidaminococcus sp. bv3l6 (bacteria)
  • synthetic construct (others)
KeywordsHYDROLASE/RNA/DNA / DNase / complex / ribonucleoprotein / genome editor / HYDROLASE-RNA complex / HYDROLASE / HYDROLASE-RNA-DNA complex

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