{"doi":"10.1101/2025.09.25.678665","title":"Sensitive, direct detection of non-coding off-target base editor unwinding and editing in primary cells","abstract":"Abstract Base editors create precise nucleotide changes in DNA, but their off-target activity remains challenging to quantify. Here, we develop and deploy a direct, in cellulo sequencing assay that simultaneously measures both Cas9-mediated unwinding and deaminase editing of genomic DNA (beCasKAS). Our strategy nominates >460-fold more potential off-target sites than other methods by enriching for Cas9-dependent R-loops immediately preceding editing. Using beCasKAS in primary human T-cells, we observe that mRNA-encoded ABE8e and PAMless ABE8e-SpRY base editors have distinct off-target profiles that can be mitigated by optimizing mRNA dose. Finally, we combine beCasKAS with base-resolution deep learning models to risk-stratify off-target edits by their likelihood of epigenetic dysregulation. Collectively, beCasKAS offers a sensitive and facile tool to optimize the balance between base editor on- and off-target activity.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":575915,"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":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9522,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":246704,"name":"Selin Jessa","orcid":"0000-0003-4192-6523","position":1,"is_corresponding":false},{"id":11727,"name":"Georgi K. Marinov","orcid":"0000-0003-1822-7273","position":2,"is_corresponding":false},{"id":278291,"name":"Sandy Klemm","orcid":"0000-0001-7556-6795","position":3,"is_corresponding":false},{"id":360,"name":"Anshul Kundaje","orcid":"0000-0003-3084-2287","position":4,"is_corresponding":false},{"id":15493,"name":"William J. Greenleaf","orcid":"0000-0003-1409-3095","position":5,"is_corresponding":false},{"id":354522,"name":"Tong Wang","orcid":"0000-0003-4208-5578","position":0,"is_corresponding":true}],"reference_count":73,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:57:52.712371Z","pmid":"41040263","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":[]}