{"doi":"10.1101/2021.08.11.455876","title":"Whole genome analysis for 163 guide RNAs in Cas9 edited mice reveals minimal off-target activity","abstract":"Introductory Paragraph The Knockout Mouse Phenotyping Program (KOMP 2 ) uses CRISRPR/Cas9 for high-throughput mouse line production to generate null alleles in the inbred C57BL/6N strain for broad-based in vivo phenotyping. In order to assess the risk of spurious S. pyogenes Cas9-induced off-target mutagenesis, we applied whole genome sequencing to compare the genomes of 50 Cas9-derived founder mice representing 163 different gRNAs to 28 untreated inbred control mice. Our analysis pipeline detected 28 off-target sequence variants associated with 21 guides. These potential off-targets were identified in 18/50 (36%) founders with 9/28 (32%) independently validated corresponding to 8 founder animals. In total, only 4.9% (8/163) of all guides exhibited off-target activity resulting in a rate of 0.16 Cas9 off-target mutations per founder analyzed. In comparison, we observed ~1225 unique variants in each mouse regardless of whether or not it was exposed to Cas9. These findings indicate that Cas9-mediated off-target mutagenesis is rare in founder knockout mice generated using guide RNAs designed to minimize off-target risk. Overall, bona fide off-target variants comprise a small fraction of the genetic heterogeneity found in carefully maintained colonies of inbred strains.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2021,"id":217757,"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":0.8915,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":272955,"name":"Sam Khalouei","orcid":null,"position":1,"is_corresponding":false},{"id":414920,"name":"Joshua A. Wood","orcid":null,"position":2,"is_corresponding":false},{"id":266981,"name":"Denise G. Lanza","orcid":"0000-0001-8750-6933","position":3,"is_corresponding":false},{"id":815030,"name":"Lauri G. Lintott","orcid":"0000-0003-1119-590X","position":4,"is_corresponding":false},{"id":414918,"name":"Brandon Willis","orcid":null,"position":5,"is_corresponding":false},{"id":406301,"name":"John R. Seavitt","orcid":"0000-0003-3209-3187","position":6,"is_corresponding":false},{"id":815031,"name":"Nour Hanafi","orcid":"0000-0003-2922-705X","position":7,"is_corresponding":false},{"id":266992,"name":"Robert E. Braun","orcid":"0000-0003-3856-9465","position":8,"is_corresponding":false},{"id":266972,"name":"Mary E. Dickinson","orcid":"0000-0003-4372-9638","position":9,"is_corresponding":false},{"id":399269,"name":"Jacqueline K. White","orcid":"0000-0001-6268-2826","position":10,"is_corresponding":false},{"id":30197,"name":"K. C. Kent Lloyd","orcid":"0000-0002-5318-4144","position":11,"is_corresponding":false},{"id":266982,"name":"Jason D. Heaney","orcid":"0000-0001-8475-8828","position":12,"is_corresponding":false},{"id":266971,"name":"Stephen A. Murray","orcid":"0000-0002-0594-1702","position":13,"is_corresponding":false},{"id":271385,"name":"Arun Ramani","orcid":"0000-0002-8162-8523","position":14,"is_corresponding":false},{"id":266973,"name":"Lauryl M. J. Nutter","orcid":"0000-0001-9619-146X","position":15,"is_corresponding":false},{"id":266974,"name":"Kevin A. Peterson","orcid":"0000-0001-8353-3694","position":0,"is_corresponding":true}],"reference_count":42,"raw_metadata":null,"created_at":"2026-07-18T23:53:24.683898Z","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":[]}