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bioRxiv · 10.1101/2024.07.31.606079

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

Soczek, K. M.·Cofsky, J. C.·Tuck, O. T.·Shi, H.·Doudna, J. A.
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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 engineering begins with local helix distortion by transient CRISPR-Cas protein binding.

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Soczek, K. M., Cofsky, J. C., Tuck, O. T., Shi, H., Doudna, J. A.. 2024-07-31. CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation. https://doi.org/10.1101/2024.07.31.606079

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