{"doi":"10.1101/2024.07.31.606079","title":"CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation","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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2024,"id":491545,"datarank":0.10397207708399181,"base_score":0.6931471805599453,"endowment":0.6931471805599453,"self_citation_contribution":0.10397207708399181,"citation_network_contribution":0.0,"self_endowment_contribution":0.10397207708399181,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9611,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":267755,"name":"Joshua C. Cofsky","orcid":"0000-0001-5403-8555","position":1,"is_corresponding":false},{"id":348596,"name":"Owen T. Tuck","orcid":"0000-0002-0348-6910","position":2,"is_corresponding":false},{"id":259596,"name":"Honglue Shi","orcid":"0000-0003-3847-1652","position":3,"is_corresponding":false},{"id":2407,"name":"Jennifer A. Doudna","orcid":"0000-0001-9161-999X","position":4,"is_corresponding":false},{"id":580679,"name":"Katarzyna M. Soczek","orcid":"0000-0003-3803-6079","position":0,"is_corresponding":true}],"reference_count":37,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:08:45.247225Z","pmid":"39131396","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}