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CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation
Nucleic Acids Research ( IF 16.6 ) Pub Date : 2024-12-19 , DOI: 10.1093/nar/gkae1192 Katarzyna M Soczek, Joshua C Cofsky, Owen T Tuck, Honglue Shi, Jennifer A Doudna
Nucleic Acids Research ( IF 16.6 ) Pub Date : 2024-12-19 , DOI: 10.1093/nar/gkae1192 Katarzyna M Soczek, Joshua C Cofsky, Owen T Tuck, Honglue Shi, Jennifer A Doudna
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.
更新日期：2024-12-19
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