{"doi":"10.1101/160036","title":"Enhanced proofreading governs CRISPR-Cas9 targeting accuracy","abstract":"<jats:p>\n                  The RNA-guided CRISPR-Cas9 nuclease from\n                  <jats:italic>Streptococcus pyogenes</jats:italic>\n                  (SpCas9) has been widely repurposed for genome editing\n                  <jats:sup>1-4</jats:sup>\n                  . High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown\n                  <jats:sup>5-9</jats:sup>\n                  . Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state\n                  <jats:sup>10</jats:sup>\n                  when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target mismatches and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we identified residues within REC3 involved in mismatch sensing and designed a new hyper-accurate Cas9 variant (HypaCas9) that retains robust on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.\n                </jats:p>","journal":null,"year":null,"id":636588,"datarank":0.4636563680037475,"base_score":3.091042453358316,"endowment":3.091042453358316,"self_citation_contribution":0.4636563680037475,"citation_network_contribution":0.0,"self_endowment_contribution":0.4636563680037475,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":21,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1652330,"name":"Yavuz S. Dagdas","orcid":null,"position":1,"is_corresponding":false},{"id":44843,"name":"Benjamin P. Kleinstiver","orcid":"0000-0002-5469-0655","position":2,"is_corresponding":false},{"id":1652331,"name":"Moira M. Welch","orcid":null,"position":3,"is_corresponding":false},{"id":303437,"name":"Lucas B. Harrington","orcid":null,"position":4,"is_corresponding":false},{"id":561678,"name":"Samuel H. Sternberg","orcid":"0000-0001-8240-9114","position":5,"is_corresponding":false},{"id":23566,"name":"J. Keith Joung","orcid":"0000-0001-5630-5645","position":6,"is_corresponding":false},{"id":203751,"name":"Ahmet Yildiz","orcid":null,"position":7,"is_corresponding":false},{"id":2407,"name":"Jennifer A. Doudna","orcid":"0000-0001-9161-999X","position":8,"is_corresponding":false},{"id":104244,"name":"Janice S. Chen","orcid":"0000-0003-4797-3056","position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Enhanced proofreading governs CRISPR-Cas9 targeting accuracy","abstract":"<jats:p>\n                  The RNA-guided CRISPR-Cas9 nuclease from\n                  <jats:italic>Streptococcus pyogenes</jats:italic>\n                  (SpCas9) has been widely repurposed for genome editing\n                  <jats:sup>1-4</jats:sup>\n                  . High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown\n                  <jats:sup>5-9</jats:sup>\n                  . Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state\n                  <jats:sup>10</jats:sup>\n                  when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target mismatches and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we identified residues within REC3 involved in mismatch sensing and designed a new hyper-accurate Cas9 variant (HypaCas9) that retains robust on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.\n                </jats:p>","is_dataset_classified":null,"base_score":3.091042453358316,"endowment":3.091042453358316,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"19767382","pmcid":null,"openalex_id":"https://openalex.org/W2951560455","authors":[],"funders":[{"funder_name":"Natural Sciences and Engineering Research Council of Canada","grant_id":"unidentified","title":"unidentified"},{"funder_name":"National Institutes of Health","grant_id":"5R35GM118158-03","title":"Evolution, Optimization, and Application of Genome Editing Technologies"},{"funder_name":"National Science Foundation","grant_id":"1244557","title":"Mechanisms of Acquired Immunity in Bacteria"},{"funder_name":"National Institutes of Health","grant_id":"5R01GM118773-02","title":"Structural and Functional Characterization of Telomere Protection and Maintenance"}],"total_grants":4,"fwci":null,"citation_percentile":null,"influential_citations":0,"citation_trend":[{"year":2017,"count":3},{"year":2018,"count":6},{"year":2019,"count":5},{"year":2020,"count":1},{"year":2022,"count":5},{"year":2023,"count":1}],"oa_status":"green","license":"https://www.biorxiv.org/about/FAQ#license","oa_locations":[{"url":"https://www.biorxiv.org/content/biorxiv/early/2017/08/12/160036.full.pdf","host_type":"repository"},{"url":"https://www.biorxiv.org/content/biorxiv/early/2017/08/12/160036.full.pdf","host_type":"repository"},{"url":"https://syndication.highwire.org/content/doi/10.1101/160036","host_type":"publisher"},{"url":"https://doi.org/10.1101/160036","host_type":"repository"},{"url":"https://europepmc.org/articles/pmc5918688?pdf=render","host_type":""},{"url":"https://dx.doi.org/10.1101/160036","host_type":""},{"url":"http://dx.doi.org/10.1101/160036","host_type":""}],"fields_of_study":["CRISPR and Genetic Engineering","RNA and protein synthesis mechanisms","Advanced biosensing and bioanalysis techniques","0301 basic medicine","03 medical and health sciences"],"mesh_terms":[],"keywords":["Cas9","Nuclease","Proofreading","CRISPR","Genome editing","Computational biology","Förster resonance energy transfer","Guide RNA","Fidelity","Biology","Polymerase","Genetics","DNA","Computer science","Gene","Physics"],"sdg_mappings":[{"sdg_number":0,"sdg_label":"Peace, Justice and strong institutions"}],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-08-06T17:21:22.498000Z","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":[]}