{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T19:24:58Z","timestamp":1791487498511,"version":"4.3.4"},"reference-count":49,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,6,4]],"date-time":"2025-06-04T00:00:00Z","timestamp":1748995200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Genome Ed."],"abstract":"<jats:p>\n                    Over the last decade CRISPR gene editing has been successfully used to streamline the generation of animal models for biomedical research purposes. However, one limitation to its use is the potential occurrence of on-target mutations that may be detrimental or otherwise unintended. These bystander mutations are often undetected using conventional genotyping methods. The use of Adeno-Associated Viruses (AAVs) to bring donor templates in zygotes is currently being deployed by transgenic cores around the world to generate knock-ins with large transgenes (i.e., 1\u20134\u00a0kb payloads). Thanks to a high level of efficiency and the relative ease to establish this technique, it recently became a method of choice for transgenic laboratories. However, a thorough analysis of the editing outcomes following this method is yet to be developed. To this end, we generated three different types of integration using AAVs in two different murine genes (i.e.,\n                    <jats:italic>Ace2<\/jats:italic>\n                    and\n                    <jats:italic>Foxg1<\/jats:italic>\n                    ) and employed Oxford Nanopore Technologies long read sequencing to analyze the outcomes. Using a workflow that includes Cas9 enrichment and adaptive sampling, we showed that unintended on-target mutations, including duplication events and integration of viral sequences (sometimes reported using other workflows) can occur when using AAVs. This work highlights the importance of in-depth validation of the mutant lines generated and informs the uptake of this new method.\n                  <\/jats:p>","DOI":"10.3389\/fgeed.2025.1582097","type":"journal-article","created":{"date-parts":[[2025,6,4]],"date-time":"2025-06-04T01:36:38Z","timestamp":1749000998000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":8,"title":["Long read sequencing reveals transgene concatemerization and vector sequences integration following AAV-driven electroporation of CRISPR RNP complexes in mouse zygotes"],"prefix":"10.3389","volume":"7","author":[{"given":"Muhammad W.","family":"Luqman","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Piroon","family":"Jenjaroenpun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jessica","family":"Spathos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nikhil","family":"Shingte","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mitchell","family":"Cummins","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pattaraporn","family":"Nimsamer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lars M.","family":"Ittner","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thidathip","family":"Wongsurawat","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fabien","family":"Delerue","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,6,4]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"596931","DOI":"10.1101\/2024.06.17.596931","article-title":"Prevalence of errors in lab-made plasmids across the globe","volume":"17","author":"Bai","year":"2024","journal-title":"bioRxiv"},{"key":"B2","doi-asserted-by":"publisher","first-page":"163","DOI":"10.1089\/crispr.2022.0099","article-title":"CRISPR-Cas9 long-read sequencing for mapping transgenes in the mouse genome","volume":"6","author":"Bryant","year":"2023","journal-title":"CRISPR J."},{"key":"B3","doi-asserted-by":"publisher","first-page":"69","DOI":"10.1177\/00236772211016922","article-title":"Screening and validation of genome-edited animals","volume":"56","author":"Bunton-Stasyshyn","year":"2022","journal-title":"Lab. Anim."},{"key":"B4","doi-asserted-by":"publisher","first-page":"3780","DOI":"10.1016\/j.celrep.2019.05.103","article-title":"CRISPR-READI: efficient generation of knockin mice by CRISPR RNP electroporation and AAV donor infection","volume":"27","author":"Chen","year":"2019","journal-title":"Cell Rep."},{"key":"B5","doi-asserted-by":"publisher","first-page":"70","DOI":"10.1186\/s12915-018-0530-7","article-title":"Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants","volume":"16","author":"Codner","year":"2018","journal-title":"BMC Biol."},{"key":"B6","doi-asserted-by":"publisher","first-page":"55765","DOI":"10.3791\/55765","article-title":"Generation of genetically modified mice through the microinjection of oocytes","author":"Delerue","year":"2017","journal-title":"J. Vis. Exp."},{"key":"B7","doi-asserted-by":"publisher","first-page":"1028","DOI":"10.1038\/nmeth.2641","article-title":"Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination","volume":"10","author":"Dickinson","year":"2013","journal-title":"Nat. Methods"},{"key":"B8","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":"B9","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":"B10","doi-asserted-by":"publisher","first-page":"401","DOI":"10.3390\/genes14020401","article-title":"Increased on-target rate and risk of concatemerization after CRISPR-enhanced targeting in ES cells","volume":"14","author":"Erbs","year":"2023","journal-title":"Genes (Basel)"},{"key":"B11","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":"B12","doi-asserted-by":"publisher","first-page":"100065","DOI":"10.1016\/j.jhepr.2019.100065","article-title":"Efficient in vivo editing of OTC-deficient patient-derived primary human hepatocytes","volume":"2","author":"Ginn","year":"2020","journal-title":"JHEP Rep."},{"key":"B13","doi-asserted-by":"publisher","first-page":"e39468","DOI":"10.7554\/eLife.39468","article-title":"Efficient single-copy HDR by 5\u2019 modified long dsDNA donors","volume":"7","author":"Gutierrez-Triana","year":"2018","journal-title":"Elife"},{"key":"B14","doi-asserted-by":"publisher","first-page":"4439","DOI":"10.1038\/s41467-019-12449-2","article-title":"High levels of AAV vector integration into CRISPR-induced DNA breaks","volume":"10","author":"Hanlon","year":"2019","journal-title":"Nat. Commun."},{"key":"B15","doi-asserted-by":"publisher","first-page":"105","DOI":"10.1016\/j.omtn.2022.12.006","article-title":"Multiplex HDR for disease and correction modeling of SCID by CRISPR genome editing in human HSPCs","volume":"31","author":"Iancu","year":"2023","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"B16","doi-asserted-by":"publisher","first-page":"479","DOI":"10.1038\/nmeth.3408","article-title":"Off-target mutations are rare in Cas9-modified mice","volume":"12","author":"Iyer","year":"2015","journal-title":"Nat. Methods"},{"key":"B17","doi-asserted-by":"publisher","first-page":"e0142755","DOI":"10.1371\/journal.pone.0142755","article-title":"Simple genome editing of rodent intact embryos by electroporation","volume":"10","author":"Kaneko","year":"2015","journal-title":"PLoS One"},{"key":"B18","doi-asserted-by":"publisher","first-page":"1972","DOI":"10.1007\/s11064-022-03582-4","article-title":"Developmental delay and late onset HBSL pathology in hypomorphic Dars1 M256L mice","volume":"47","author":"Klugmann","year":"2022","journal-title":"Neurochem. Res."},{"key":"B19","doi-asserted-by":"publisher","first-page":"439","DOI":"10.2144\/btn-2021-0051","article-title":"A simple approach for effective shearing and reliable concentration measurement of ultra-high-molecular-weight DNA","volume":"71","author":"Koetsier","year":"2021","journal-title":"Biotechniques"},{"key":"B20","doi-asserted-by":"publisher","first-page":"W272","DOI":"10.1093\/nar\/gkw398","article-title":"CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering","volume":"44","author":"Labun","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"B21","doi-asserted-by":"publisher","first-page":"53","DOI":"10.1007\/978-1-0716-2990-1_3","article-title":"Genetic and molecular quality control of genetically engineered mice","volume":"2631","author":"Lintott","year":"2023","journal-title":"Methods Mol. Biol."},{"key":"B22","doi-asserted-by":"publisher","first-page":"5424","DOI":"10.1038\/s41598-022-09445-w","article-title":"Efficient targeted transgenesis of large donor DNA into multiple mouse genetic backgrounds using bacteriophage Bxb1 integrase","volume":"12","author":"Low","year":"2022","journal-title":"Sci. Rep."},{"key":"B23","doi-asserted-by":"publisher","first-page":"11","DOI":"10.1186\/s13059-021-02582-x","article-title":"Nanopore adaptive sampling: a tool for enrichment of low abundance species in metagenomic samples","volume":"23","author":"Martin","year":"2022","journal-title":"Genome Biol."},{"key":"B24","doi-asserted-by":"publisher","first-page":"e1011187","DOI":"10.1371\/journal.pgen.1011187","article-title":"Long-read sequencing for fast and robust identification of correct genome-edited alleles: PCR-based and Cas9 capture methods","volume":"20","author":"McCabe","year":"2024","journal-title":"PLoS Genet."},{"key":"B25","doi-asserted-by":"publisher","first-page":"e14","DOI":"10.1093\/nar\/gkac1150","article-title":"Efficient single copy integration via homology-directed repair (scHDR) by 5\u2032modification of large DNA donor fragments in mice","volume":"51","author":"Medert","year":"2023","journal-title":"Nucleic Acids Res."},{"key":"B26","doi-asserted-by":"publisher","first-page":"318","DOI":"10.1186\/s12864-018-4713-y","article-title":"CLICK: one-step generation of conditional knockout mice","volume":"19","author":"Miyasaka","year":"2018","journal-title":"BMC Genomics"},{"key":"B27","doi-asserted-by":"publisher","first-page":"286","DOI":"10.1016\/j.isci.2018.10.030","article-title":"Intra-embryo gene cassette knockin by CRISPR\/Cas9-Mediated genome editing with adeno-associated viral vector","volume":"9","author":"Mizuno","year":"2018","journal-title":"iScience"},{"key":"B28","doi-asserted-by":"publisher","first-page":"eadd2577","DOI":"10.1126\/sciadv.add2577","article-title":"Treatment of epilepsy using a targeted p38\u03b3 kinase gene therapy","volume":"8","author":"Morey","year":"2022","journal-title":"Sci. Adv."},{"key":"B29","doi-asserted-by":"publisher","first-page":"34703","DOI":"10.1038\/srep34703","article-title":"Exome sequencing in the knockin mice generated using the CRISPR\/Cas system","volume":"6","author":"Nakajima","year":"2016","journal-title":"Sci. Rep."},{"key":"B30","doi-asserted-by":"publisher","first-page":"163","DOI":"10.1038\/s41587-019-0394-6","article-title":"Template plasmid integration in germline genome-edited cattle","volume":"38","author":"Norris","year":"2020","journal-title":"Nat. Biotechnol."},{"key":"B31","doi-asserted-by":"publisher","first-page":"W267","DOI":"10.1093\/nar\/gkw407","article-title":"Breaking-Cas-interactive design of guide RNAs for CRISPR-Cas experiments for ENSEMBL genomes","volume":"44","author":"Oliveros","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"B32","doi-asserted-by":"publisher","first-page":"35","DOI":"10.1186\/1471-2199-11-35","article-title":"Analysis of illegitimate genomic integration mediated by zinc-finger nucleases: implications for specificity of targeted gene correction","volume":"11","author":"Olsen","year":"2010","journal-title":"BMC Mol. Biol."},{"key":"B33","doi-asserted-by":"publisher","first-page":"442","DOI":"10.1038\/s41587-020-00746-x","article-title":"Readfish enables targeted nanopore sequencing of gigabase-sized genomes","volume":"39","author":"Payne","year":"2020","journal-title":"Nat. Biotechnol."},{"key":"B34","doi-asserted-by":"publisher","first-page":"626","DOI":"10.1038\/s42003-023-04974-0","article-title":"Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity","volume":"6","author":"Peterson","year":"2023","journal-title":"Commun. Biol."},{"key":"B35","doi-asserted-by":"publisher","first-page":"423","DOI":"10.1534\/genetics.115.176594","article-title":"Efficient CRISPR\/cas9-mediated genome editing in mice by zygote electroporation of nuclease","volume":"200","author":"Qin","year":"2015","journal-title":"Genetics"},{"key":"B36","doi-asserted-by":"publisher","first-page":"743","DOI":"10.1038\/mt.2009.304","article-title":"Zinc-finger nuclease-induced gene repair with oligodeoxynucleotides: wanted and unwanted target locus modifications","volume":"18","author":"Radecke","year":"2010","journal-title":"Mol. Ther."},{"key":"B37","doi-asserted-by":"publisher","first-page":"85","DOI":"10.1016\/j.bbrc.2020.03.026","article-title":"AAV diffuses across zona pellucida for effortless gene delivery to fertilized eggs: AAV diffuses across mouse ZP","volume":"526","author":"Romeo","year":"2020","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"B38","doi-asserted-by":"publisher","first-page":"e1010176","DOI":"10.1371\/journal.pgen.1010176","article-title":"Cas9 targeted nanopore sequencing with enhanced variant calling improves CYP2D6-CYP2D7 hybrid allele genotyping","volume":"18","author":"Rubben","year":"2022","journal-title":"PLoS Genet."},{"key":"B39","doi-asserted-by":"publisher","first-page":"54","DOI":"10.1186\/s13578-021-00566-9","article-title":"When the genome bluffs: a tandem duplication event during generation of a novel Agmo knockout mouse model fools routine genotyping","volume":"11","author":"Sailer","year":"2021","journal-title":"Cell Biosci."},{"key":"B40","doi-asserted-by":"publisher","first-page":"267","DOI":"10.1128\/mcb.17.1.267","article-title":"Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination","volume":"17","author":"Sargent","year":"1997","journal-title":"Mol. Cell Biol."},{"key":"B41","doi-asserted-by":"publisher","first-page":"622099","DOI":"10.1101\/2024.11.05.622099","article-title":"Parallel in-depth analysis of repeat expansions: an updated Clin-CATS workflow for nanopore R10 flow cells","volume":"11","author":"Scholz","year":"2024","journal-title":"bioRxiv"},{"key":"B42","doi-asserted-by":"publisher","first-page":"993","DOI":"10.1016\/j.stemcr.2022.02.008","article-title":"Homozygous might be hemizygous: CRISPR\/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls","volume":"17","author":"Simkin","year":"2022","journal-title":"Stem Cell Rep."},{"key":"B43","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":"B44","doi-asserted-by":"publisher","first-page":"eaax2941","DOI":"10.1126\/sciadv.aax2941","article-title":"Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9-mediated genome editing events","volume":"6","author":"Skryabin","year":"2020","journal-title":"Sci. Adv."},{"key":"B45","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":"B46","doi-asserted-by":"publisher","first-page":"124","DOI":"10.1016\/j.chom.2020.05.020","article-title":"A mouse model of SARS-CoV-2 infection and pathogenesis","volume":"28","author":"Sun","year":"2020","journal-title":"Cell Host Microbe"},{"key":"B47","doi-asserted-by":"publisher","first-page":"87","DOI":"10.1186\/s40478-020-00963-0","article-title":"A novel Cas9-targeted long-read assay for simultaneous detection of IDH1\/2 mutations and clinically relevant MGMT methylation in fresh biopsies of diffuse glioma","volume":"8","author":"Wongsurawat","year":"2020","journal-title":"Acta Neuropathol. Commun."},{"key":"B48","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 adenoassociated viruses","volume":"9","author":"Yoon","year":"2018","journal-title":"Nat. Commun."},{"key":"B49","doi-asserted-by":"publisher","first-page":"927296","DOI":"10.3389\/fphar.2022.927296","article-title":"Behavioral phenotypes of Foxg1 heterozygous mice","volume":"13","author":"Younger","year":"2022","journal-title":"Front. Pharmacol."}],"container-title":["Frontiers in Genome Editing"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fgeed.2025.1582097\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,6,4]],"date-time":"2025-06-04T01:36:39Z","timestamp":1749000999000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fgeed.2025.1582097\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,6,4]]},"references-count":49,"alternative-id":["10.3389\/fgeed.2025.1582097"],"URL":"https:\/\/doi.org\/10.3389\/fgeed.2025.1582097","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2024.02.18.580906","asserted-by":"object"}]},"ISSN":["2673-3439"],"issn-type":[{"value":"2673-3439","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,6,4]]},"article-number":"1582097"}}