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DOE OSTI · 2483361

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

Soczek, Katarzyna M. [University of California, Berkeley, CA (United States)]·Cofsky, Joshua C. [University of California, Berkeley, CA (United States)]·Tuck, Owen T. [University of California, Berkeley, CA (United States)]·Shi, Honglue [University of California, Berkeley, CA (United States)] (ORCID:0000000338471652)·Doudna, Jennifer A. [University of California, Berkeley, CA (United States); Univ. of California, San Francisco, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:000000019161999X)
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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 interference begins with local helix distortion by transient CRISPR-Cas protein binding.

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Soczek, Katarzyna M. [University of California, Berkeley, CA (United States)], Cofsky, Joshua C. [University of California, Berkeley, CA (United States)], Tuck, Owen T. [University of California, Berkeley, CA (United States)], Shi, Honglue [University of California, Berkeley, CA (United States)] (ORCID:0000000338471652), Doudna, Jennifer A. [University of California, Berkeley, CA (United States); Univ. of California, San Francisco, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)] (ORCID:000000019161999X). 2024-12-19. CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation. https://doi.org/10.1093/nar%2Fgkae1192

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