{"doi":"10.1242/dev.204571","title":"Identifying optimal conditions for precise knock-in of exogenous DNA into the zebrafish genome","abstract":"CRISPR nucleases can be used to insert exogenous DNA into the zebrafish genome by homology-dependent repair (HDR), although germline transmission rates for precise edits remain quite low. Comparative studies to optimize HDR parameters for introducing base pair changes using short-read deep sequencing have been successful, but similar analysis for insertions is challenging due to read-length constraints. Here, we quantified editing outcomes using long-read sequencing to identify optimal template and CRISPR parameters for precise targeted insertion in zebrafish. Through side-by-side comparisons, we found that chemically modified templates out-perform those released in vivo from a plasmid, while Cas9 and Cas12a nucleases performed similarly for targeted insertion. Consistent with previous studies, precise editing rates were dependent on the distance between a double-strand break and the inserted sequence. We further found that non-homologous base pairs in homology templates significantly reduced precise editing rates. Using optimized parameters, we consistently achieved germline founder rates of greater than 20% for precise insertions across four loci. Together, our quantitative analyses identified optimal conditions for precise insertion of exogenous DNA into the zebrafish genome.","journal":"Development","year":2025,"id":523495,"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":4,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9524,"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":582412,"name":"Kai Hu","orcid":"0000-0002-6555-2581","position":1,"is_corresponding":false},{"id":649394,"name":"Masahiro Shin","orcid":"0000-0002-0231-894X","position":2,"is_corresponding":false},{"id":453068,"name":"Feston Idrizi","orcid":"0000-0002-8035-3951","position":3,"is_corresponding":false},{"id":1395931,"name":"Aliece Goodman‐Khan","orcid":"0009-0006-9645-5924","position":4,"is_corresponding":false},{"id":694420,"name":"Amy Kolb","orcid":null,"position":5,"is_corresponding":false},{"id":563089,"name":"Krishna S. Ghanta","orcid":"0000-0001-7502-3141","position":6,"is_corresponding":false},{"id":558787,"name":"Jonathan Lee","orcid":"0000-0001-7065-8041","position":7,"is_corresponding":false},{"id":1072096,"name":"Atish A. Wagh","orcid":"0000-0002-1275-1341","position":8,"is_corresponding":false},{"id":588037,"name":"Scot A. Wolfe","orcid":"0000-0002-7042-201X","position":9,"is_corresponding":false},{"id":246163,"name":"Lihua Julie Zhu","orcid":"0000-0001-7416-0590","position":10,"is_corresponding":false},{"id":348431,"name":"Jonathan K. Watts","orcid":"0000-0001-5706-1734","position":11,"is_corresponding":false},{"id":304178,"name":"Nathan D. Lawson","orcid":"0000-0001-7788-9619","position":12,"is_corresponding":false},{"id":1026121,"name":"Sarah Oikemus","orcid":null,"position":0,"is_corresponding":true}],"reference_count":61,"raw_metadata":null,"created_at":"2026-07-19T02:50:03.004175Z","pmid":"40446205","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":[]}