{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T23:59:08Z","timestamp":1780617548436,"version":"3.54.1"},"reference-count":39,"publisher":"Oxford University Press (OUP)","issue":"6","license":[{"start":{"date-parts":[[2026,4,7]],"date-time":"2026-04-07T00:00:00Z","timestamp":1775520000000},"content-version":"vor","delay-in-days":19,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100014571","name":"Alvin J. Siteman Cancer Center","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100014571","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,3,19]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Multi-kilobase knock-ins (KIs) are a necessary, yet challenging type of genome editing to create and characterize in cell lines and animals. The combination of rAAV donor transduction and electroporation of single-cell mouse embryos with Cas9\/gRNA ribonucleoprotein complex enables highly efficient KI, but the insert size is limited by the viral packaging capacity. Here, we report the creation of up to 6.7 kb precise KI achieved in one step by using three rAAVs designed to insert one after the other. To fully characterize the edited genome with large KIs, we developed LOCK-seq (LOng-read sequencing of Captured Kilo-base targets), where relevant genomic regions are enriched via hybridization, achieving over 100-fold greater coverage compared with other long-read methods with enrichment. LOCK-seq simultaneously detects the presence of precise KI alleles, imprecision in the insert and donor concatenation, genotypes of non-KI alleles, and more importantly, uniquely identifies and localizes random integration of the full or partial donor(s). Additionally, the multi-rAAV donor approach is successfully applied to cell lines, including lines intolerant of plasmid DNA, whereas LOCK-seq reliably and efficiently screens for KI clones. Together, the two approaches significantly improve the creation and precision of knock-in models.<\/jats:p>","DOI":"10.1093\/nar\/gkag237","type":"journal-article","created":{"date-parts":[[2026,4,9]],"date-time":"2026-04-09T08:54:10Z","timestamp":1775724850000},"source":"Crossref","is-referenced-by-count":1,"title":["Efficient multi-kilobase knock-ins in mice and cell lines using CRISPR\/Cas9 and rAAV donors with unbiased whole-genome characterization by LOCK-seq"],"prefix":"10.1093","volume":"54","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-7084-5705","authenticated-orcid":false,"given":"Monica F","family":"Sentmanat","sequence":"first","affiliation":[{"name":"Genome Engineering & Stem Cell Center, McDonnell Genome Institute, Dept. of Genetics, Washington University in St. Louis School of Medicine , 660 S. Euclid Ave, St. Louis, MO 63110 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zi Teng","family":"Wang","sequence":"additional","affiliation":[{"name":"Genome Engineering & Stem Cell Center, McDonnell Genome Institute, Dept. of Genetics, Washington University in St. Louis School of Medicine , 660 S. Euclid Ave, St. Louis, MO 63110 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Evguenia","family":"Kouranova","sequence":"additional","affiliation":[{"name":"Genome Engineering & Stem Cell Center, McDonnell Genome Institute, Dept. of Genetics, Washington University in St. Louis School of Medicine , 660 S. Euclid Ave, St. Louis, MO 63110 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Samuel T","family":"Peters","sequence":"additional","affiliation":[{"name":"Genome Engineering & Stem Cell Center, McDonnell Genome Institute, Dept. of Genetics, Washington University in St. Louis School of Medicine , 660 S. Euclid Ave, St. Louis, MO 63110 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wan\u00a0Ching","family":"Chan","sequence":"additional","affiliation":[{"name":"Genome Engineering & Stem Cell Center, McDonnell Genome Institute, Dept. of Genetics, Washington University in St. Louis School of Medicine , 660 S. Euclid Ave, St. Louis, MO 63110 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jed","family":"Lin","sequence":"additional","affiliation":[{"name":"Genome Engineering & Stem Cell Center, McDonnell Genome Institute, Dept. of Genetics, Washington University in St. Louis School of Medicine , 660 S. Euclid Ave, St. Louis, MO 63110 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-6261-7595","authenticated-orcid":false,"given":"Yong","family":"Miao","sequence":"additional","affiliation":[{"name":"Genome Engineering & Stem Cell Center, McDonnell Genome Institute, Dept. of Genetics, Washington University in St. Louis School of Medicine , 660 S. Euclid Ave, St. Louis, MO 63110 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J Michael","family":"White","sequence":"additional","affiliation":[{"name":"Transgenic, Knockout and Microinjection Core, Department of Pathology & Immunology, Washington University in St. Louis School of Medicine , 660 S. Euclid Ave, St. Louis, MO 63110 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mia","family":"Wallace","sequence":"additional","affiliation":[{"name":"Mouse Genetics Core, Department of Pediatrics, Washington University in St. Louis School of Medicine , 660 S. Euclid Ave, St. Louis, MO 63110 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3073-7332","authenticated-orcid":false,"given":"Xiaoxia","family":"Cui","sequence":"additional","affiliation":[{"name":"Genome Engineering & Stem Cell Center, McDonnell Genome Institute, Dept. of Genetics, Washington University in St. Louis School of Medicine , 660 S. Euclid Ave, St. Louis, MO 63110 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,4,7]]},"reference":[{"key":"2026040904540690200_B1","doi-asserted-by":"publisher","first-page":"816","DOI":"10.1126\/science.1225829","article-title":"A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity","volume":"337","author":"Jinek","year":"2012","journal-title":"Science"},{"key":"2026040904540690200_B2","doi-asserted-by":"publisher","first-page":"31","DOI":"10.1186\/s12915-021-01223-w","article-title":"Highly reliable creation of floxed alleles by electroporating single-cell embryos","volume":"20","author":"Sentmanat","year":"2022","journal-title":"BMC Biol"},{"key":"2026040904540690200_B3","doi-asserted-by":"publisher","first-page":"14457","DOI":"10.1074\/jbc.M116.733154","article-title":"Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes","volume":"291","author":"Chen","year":"2016","journal-title":"J Biol Chem"},{"key":"2026040904540690200_B4","doi-asserted-by":"publisher","first-page":"412","DOI":"10.1038\/s41467-017-02706-7","article-title":"Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses","volume":"9","author":"Yoon","year":"2018","journal-title":"Nat Commun"},{"key":"2026040904540690200_B5","doi-asserted-by":"publisher","first-page":"3780","DOI":"10.1016\/j.celrep.2019.05.103","article-title":"CRISPR-READI: efficient generation of knock-in mice by CRISPR RNP electroporation and AAV donor infection","volume":"27","author":"Chen","year":"2019","journal-title":"Cell Rep"},{"key":"2026040904540690200_B6","doi-asserted-by":"publisher","first-page":"e98","DOI":"10.1093\/nar\/gkx154","article-title":"Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery","volume":"45","author":"Gaj","year":"2017","journal-title":"Nucleic Acids Res"},{"key":"2026040904540690200_B7","doi-asserted-by":"publisher","first-page":"750","DOI":"10.1016\/j.celrep.2017.06.064","article-title":"CRISPR-mediated integration of large gene cassettes using AAV donor vectors","volume":"20","author":"Bak","year":"2017","journal-title":"Cell Rep"},{"key":"2026040904540690200_B8","doi-asserted-by":"publisher","first-page":"191","DOI":"10.1007\/s10577-010-9111-5","article-title":"Integration of exogenous DNA into mouse embryonic stem cell chromosomes shows preference into genes and frequent modification at junctions","volume":"18","author":"Suzuki","year":"2010","journal-title":"Chromosome Res"},{"key":"2026040904540690200_B9","doi-asserted-by":"publisher","first-page":"279","DOI":"10.1016\/0378-1119(86)90332-X","article-title":"Patterns of integration of exogenous DNA sequences transfected into mammalian cells of primate and rodent origin","volume":"50","author":"Colb\u00e8re-Garapin","year":"1986","journal-title":"Gene"},{"key":"2026040904540690200_B10","doi-asserted-by":"publisher","first-page":"6333","DOI":"10.1093\/nar\/gkn649","article-title":"Impact of non-homologous end-joining deficiency on random and targeted DNA integration: implications for gene targeting","volume":"36","author":"Iiizumi","year":"2008","journal-title":"Nucleic Acids Res"},{"key":"2026040904540690200_B11","doi-asserted-by":"crossref","first-page":"1791","DOI":"10.1038\/sj.gt.3302074","article-title":"Illegitimate DNA integration in mammalian cells","volume":"10","author":"W\u00fcrtele","year":"2003","journal-title":"Gene Ther"},{"key":"2026040904540690200_B12","doi-asserted-by":"publisher","first-page":"433","DOI":"10.1038\/s41587-020-0407-5","article-title":"Targeted nanopore sequencing with Cas9-guided adapter ligation","volume":"38","author":"Gilpatrick","year":"2020","journal-title":"Nat Biotechnol"},{"key":"2026040904540690200_B13","doi-asserted-by":"publisher","first-page":"760","DOI":"10.1016\/j.ymthe.2023.01.004","article-title":"Targeted long-read sequencing captures CRISPR editing and AAV integration outcomes in brain","volume":"31","author":"Simpson","year":"2023","journal-title":"Mol Ther"},{"key":"2026040904540690200_B14","doi-asserted-by":"publisher","first-page":"e291","DOI":"10.1038\/mtna.2016.4","article-title":"Overcoming the specific toxicity of large plasmids electrotransfer in primary cells in vitro","volume":"5","author":"Lesueur","year":"2016","journal-title":"Mol Ther Nucleic Acids"},{"key":"2026040904540690200_B15","doi-asserted-by":"publisher","first-page":"e72216","DOI":"10.7554\/eLife.72216","article-title":"5\u2032-Modifications improve potency and efficacy of DNA donors for precision genome editing","volume":"10","author":"Ghanta","year":"2021","journal-title":"eLife"},{"key":"2026040904540690200_B16","doi-asserted-by":"publisher","first-page":"827","DOI":"10.1038\/nbt.2647","article-title":"DNA targeting specificity of RNA-guided Cas9 nucleases","volume":"31","author":"Hsu","year":"2013","journal-title":"Nat Biotechnol"},{"key":"2026040904540690200_B17","doi-asserted-by":"publisher","first-page":"4194","DOI":"10.1038\/s41598-019-40896-w","article-title":"CRIS.py: a versatile and high-throughput analysis program for CRISPR-based genome editing","volume":"9","author":"Connelly","year":"2019","journal-title":"Sci Rep"},{"key":"2026040904540690200_B18","doi-asserted-by":"publisher","first-page":"2033","DOI":"10.1101\/gr.177881.114","article-title":"Tn5 transposase and tagmentation procedures for massively scaled sequencing projects","volume":"24","author":"Picelli","year":"2014","journal-title":"Genome Res"},{"key":"2026040904540690200_B19","doi-asserted-by":"publisher","first-page":"26","DOI":"10.1186\/s12915-024-01834-z","article-title":"Donor template delivery by recombinant adeno-associated virus for the production of knock-in mice","volume":"22","author":"Duddy","year":"2024","journal-title":"BMC Biol"},{"key":"2026040904540690200_B20","doi-asserted-by":"publisher","first-page":"646","DOI":"10.1038\/nature03556","article-title":"Highly efficient endogenous human gene correction using designed zinc-finger nucleases","volume":"435","author":"Urnov","year":"2005","journal-title":"Nature"},{"key":"2026040904540690200_B21","doi-asserted-by":"publisher","first-page":"8105","DOI":"10.1093\/nar\/gkx497","article-title":"The contribution of homology arms to nuclease-assisted genome engineering","volume":"45","author":"Baker","year":"2017","journal-title":"Nucleic Acids Res"},{"key":"2026040904540690200_B22","doi-asserted-by":"publisher","first-page":"192","DOI":"10.1016\/j.omtm.2016.12.008","article-title":"Homology-directed recombination for enhanced engineering of chimeric antigen receptor T cells","volume":"4","author":"Hale","year":"2017","journal-title":"Mol Ther Methods Clin Dev"},{"key":"2026040904540690200_B23","doi-asserted-by":"publisher","first-page":"376","DOI":"10.1007\/s00335-024-10099-4","article-title":"Genome engineering with Cas9 and AAV repair templates, successes and pitfalls","volume":"36","author":"Birling","year":"2025","journal-title":"Mamm Genome"},{"key":"2026040904540690200_B24","doi-asserted-by":"publisher","first-page":"204","DOI":"10.1038\/s41587-024-02171-w","article-title":"Genome engineering with Cas9 and AAV repair templates generates frequent concatemeric insertions of viral vectors","volume":"43","author":"Suchy","year":"2024","journal-title":"Nat Biotechnol"},{"key":"2026040904540690200_B25","doi-asserted-by":"crossref","first-page":"1582097","DOI":"10.3389\/fgeed.2025.1582097","article-title":"Long read sequencing reveals transgene concatemerization and backbone integration following AAV-driven electroporation of CRISPR RNP complexes in mouse zygotes","volume":"7","author":"Luqman","year":"2025","journal-title":"Front Genome Ed"},{"key":"2026040904540690200_B26","doi-asserted-by":"publisher","first-page":"163","DOI":"10.1089\/crispr.2022.0099","article-title":"CRISPR\u2013Cas9 long-read sequencing for mapping transgenes in the mouse genome","volume":"6","author":"Bryant","year":"2023","journal-title":"CRISPR J"},{"key":"2026040904540690200_B27","doi-asserted-by":"publisher","first-page":"290","DOI":"10.1186\/s13059-020-02206-w","article-title":"Amplification-free long-read sequencing reveals unforeseen CRISPR\u2013Cas9 off-target activity","volume":"21","author":"Hoijer","year":"2020","journal-title":"Genome Biol"},{"key":"2026040904540690200_B28","doi-asserted-by":"publisher","first-page":"631","DOI":"10.12688\/f1000research.11547.1","article-title":"Investigation of chimeric reads using the MinION","volume":"6","author":"White","year":"2017","journal-title":"F1000Research"},{"key":"2026040904540690200_B29","doi-asserted-by":"publisher","first-page":"35","DOI":"10.1186\/s13059-017-1164-8","article-title":"Efficient precise knockin with a double cut HDR donor after CRISPR\/Cas9-mediated double-stranded DNA cleavage","volume":"18","author":"Zhang","year":"2017","journal-title":"Genome Biol"},{"key":"2026040904540690200_B30","doi-asserted-by":"crossref","first-page":"632","DOI":"10.1038\/nbt.4166","article-title":"Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos","volume":"6","author":"Gu","year":"2018","journal-title":"Nat Biotechnol"},{"key":"2026040904540690200_B31","doi-asserted-by":"publisher","first-page":"8568","DOI":"10.1128\/JVI.72.11.8568-8577.1998","article-title":"Circular intermediates of recombinant adeno-associated virus have defined structural characteristics responsible for long-term episomal persistence in muscle tissue","volume":"72","author":"Duan","year":"1998","journal-title":"J Virol"},{"key":"2026040904540690200_B32","doi-asserted-by":"publisher","first-page":"7966","DOI":"10.1073\/pnas.93.15.7966","article-title":"The recombination signals for adeno-associated virus site-specific integration","volume":"93","author":"Linden","year":"1996","journal-title":"Proc Natl Acad Sci"},{"key":"2026040904540690200_B33","doi-asserted-by":"publisher","first-page":"9451","DOI":"10.1128\/JVI.74.20.9451-9463.2000","article-title":"Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo","volume":"74","author":"Nakai","year":"2000","journal-title":"J Virol"},{"key":"2026040904540690200_B34","doi-asserted-by":"publisher","first-page":"9468","DOI":"10.1128\/JVI.73.11.9468-9477.1999","article-title":"Concatamerization of adeno-associated virus circular genomes occurs through intermolecular recombination","volume":"73","author":"Yang","year":"1999","journal-title":"J Virol"},{"key":"2026040904540690200_B35","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1038\/s41587-025-02565-4","article-title":"Spatial genomics of AAVs reveals mechanism of transcriptional crosstalk that enables targeted delivery of large genetic cargo","volume":"44","author":"Coughlin","year":"2026","journal-title":"Nat Biotechnol"},{"key":"2026040904540690200_B36","doi-asserted-by":"publisher","first-page":"lqac061","DOI":"10.1093\/nargab\/lqac061","article-title":"Measuring transcription factor binding and gene expression using barcoded self-reporting transposon calling cards and transcriptomes","volume":"4","author":"Lalli","year":"2022","journal-title":"NAR Genom Bioinform"},{"key":"2026040904540690200_B37","doi-asserted-by":"publisher","first-page":"992","DOI":"10.1016\/j.cell.2020.06.037","article-title":"Self-reporting transposons enable simultaneous readout of gene expression and transcription factor binding in single cells","volume":"182","author":"Moudgil","year":"2020","journal-title":"Cell"},{"key":"2026040904540690200_B38","doi-asserted-by":"publisher","first-page":"187","DOI":"10.1038\/nbt.3117","article-title":"GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR\u2013Cas nucleases","volume":"33","author":"Tsai","year":"2015","journal-title":"Nat Biotechnol"},{"key":"2026040904540690200_B39","doi-asserted-by":"publisher","first-page":"286","DOI":"10.1126\/science.aav9023","article-title":"Unbiased detection of CRISPR off-targets in vivo using DISCOVER-seq","volume":"364","author":"Wienert","year":"2019","journal-title":"Science"}],"container-title":["Nucleic Acids Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/nar\/article-pdf\/54\/6\/gkag237\/67930401\/gkag237.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/nar\/article-pdf\/54\/6\/gkag237\/67930401\/gkag237.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,9]],"date-time":"2026-04-09T08:54:12Z","timestamp":1775724852000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/nar\/article\/doi\/10.1093\/nar\/gkag237\/8625898"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,19]]},"references-count":39,"journal-issue":{"issue":"6","published-print":{"date-parts":[[2026,3,19]]}},"URL":"https:\/\/doi.org\/10.1093\/nar\/gkag237","relation":{},"ISSN":["0305-1048","1362-4962"],"issn-type":[{"value":"0305-1048","type":"print"},{"value":"1362-4962","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2026,4,13]]},"published":{"date-parts":[[2026,3,19]]},"article-number":"gkag237"}}