{"doi":"10.1093/nar/gkaf1316","title":"Gene-sized DNA insertion at genomic safe harbors in human cells using a site-directed transposase","abstract":"Achieving precise and efficient integration of gene-sized DNA sequences into the human genome remains a major obstacle to gene therapy. Existing approaches depend on double-strand DNA breaks, which can lead to unintended genome alterations. Many monogenic diseases arise from diverse patient-specific mutations, making individualized correction impractical and underscoring the need for universal full-gene replacement strategies. We developed INsertion by Targeted Anchoring and Conditional Transposition (INTACT) to enable targeted insertion at genomic safe harbor loci. We engineered a mammalian transposase with mutations in its DNA-binding domain to reduce off-target integration. Site specificity was then restored by linking programmable sequence-specific DNA-binding proteins to the transposase. Systematic optimization of INTACT revealed key determinants of precision, including noncovalent linkage between the transposase and DNA-binding protein, strict spacing between the binding site and the TTAA insertion sequence, and linkage of the DNA-binding protein to an internal position within the transposase. On-target insertion was achieved across multiple loci, with optimized INTACT averaging 1.2 targeted insertions per cell. An off-target assay confirmed that DNA-binding domain mutations substantially reduced unwanted integration events to near-background levels. Our site-directed transposase enables precise, efficient genomic insertion of >4 kb DNA without double-strand breaks, offering a powerful new tool for genome engineering.","journal":"Nucleic Acids Research","year":2025,"id":584755,"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":0.9506,"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":593860,"name":"Lisa Sharek","orcid":null,"position":1,"is_corresponding":false},{"id":1498256,"name":"Joshua F. Meckler","orcid":null,"position":2,"is_corresponding":false},{"id":1215465,"name":"Ilko Stoytchev","orcid":null,"position":3,"is_corresponding":false},{"id":693558,"name":"Christopher Tran","orcid":"0000-0003-4860-3794","position":4,"is_corresponding":false},{"id":1498257,"name":"Clara Errard","orcid":null,"position":5,"is_corresponding":false},{"id":1215461,"name":"Brian E Hew","orcid":null,"position":6,"is_corresponding":false},{"id":176119,"name":"Brandon E. Johnson","orcid":null,"position":7,"is_corresponding":false},{"id":1215464,"name":"David F Waller","orcid":null,"position":8,"is_corresponding":false},{"id":1498258,"name":"Shanfu Xie","orcid":null,"position":9,"is_corresponding":false},{"id":1067524,"name":"Joseph Higgins","orcid":"0000-0003-3454-5773","position":10,"is_corresponding":false},{"id":1498259,"name":"Raymond Tabibiazar","orcid":null,"position":11,"is_corresponding":false},{"id":917596,"name":"Jesse B. Owens","orcid":"0000-0003-4741-9661","position":12,"is_corresponding":false},{"id":1214967,"name":"James E. Short","orcid":"0000-0002-4591-4290","position":0,"is_corresponding":true}],"reference_count":81,"raw_metadata":null,"created_at":"2026-07-19T02:59:16.166424Z","pmid":"41359386","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":[]}