{"doi":"10.1101/2020.12.06.413757","title":"A slow-exchange conformational switch regulates off-target cleavage by high-fidelity Cas9","abstract":"Abstract The Cas9 endonuclease is broadly used for genome engineering applications by programming its single-guide RNA, where high specificity is required. Although mechanistic understanding of DNA cleavage in the CRISPR-Cas9 system has been guided by crystallographic structures and single-molecule FRET experiments, the role of conformational plasticity within its DNA recognition lobe (REC) for target accuracy remains elusive. Through NMR analysis of milliseconds-timescale dynamics, we show that the REC3 domain exchanges between a major, closed and a minor, open conformation. We find that a single mutation in the HiFi Cas9 variant (R691A) increases conformational dynamics in REC3, eliciting a global transition to the open conformation which is regulated by an intramolecular salt-bridge network. These observations suggest a mechanism for reduced off-target recognition by switching on a global conformational exchange process to allow REC3 scanning for proper base pairing. Our data establish a framework for rational engineering Cas9 variants with improved target discrimination.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2020,"id":124281,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9484,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":407357,"name":"Abhinav Dubey","orcid":"0000-0002-2392-8050","position":1,"is_corresponding":false},{"id":218700,"name":"Haribabu Arthanari","orcid":"0000-0002-7281-1289","position":2,"is_corresponding":false},{"id":314926,"name":"Nikolaos G. Sgourakis","orcid":"0000-0003-3655-3902","position":3,"is_corresponding":false},{"id":392206,"name":"Viviane S. De Paula","orcid":"0000-0003-3171-6477","position":0,"is_corresponding":true}],"reference_count":41,"raw_metadata":null,"created_at":"2026-07-18T23:15:07.789881Z","pmid":null,"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":[]}