CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.

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 engineering begins with local helix distortion by transient CRISPR-Cas protein binding.
Competing Interests: CONFLICT OF INTEREST STATEMENT J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine, Intellia Therapeutics, Mammoth Biosciences and Scribe Therapeutics, and a director of Altos, Johnson & Johnson and Tempus. J.A.D. is a scientific advisor to Caribou Biosciences, Intellia Therapeutics, Mammoth Biosciences, Inari, Scribe Therapeutics, Felix Biosciences and Algen. J.A.D. also serves as Chief Science Advisor to Sixth Street and a Scientific Advisory Board member at The Column Group. J.A.D. conducts academic research projects sponsored by Roche and Apple Tree Partners.