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

Soczek, Katarzyna M.; Cofsky, Joshua C.; Tuck, Owen T.; Shi, Honglue; Doudna, Jennifer A.

doi:10.1093/nar/gkae1192

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

Journal Article · 18 December 2024 · Nucleic Acids Research

DOI: https://doi.org/10.1093/nar/gkae1192 · OSTI ID:2483361

Soczek, Katarzyna M. ^[1]; Cofsky, Joshua C. ^[1]; Tuck, Owen T. ^[1];

^[1];

^[2]

University of California, Berkeley, CA (United States)
University of California, Berkeley, CA (United States); Univ. of California, San Francisco, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

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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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE; USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2483361

Journal Information:: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 2 Vol. 53; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: English

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