{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,17]],"date-time":"2025-10-17T14:06:25Z","timestamp":1760709985218,"version":"3.41.2"},"reference-count":28,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2019,8,12]],"date-time":"2019-08-12T00:00:00Z","timestamp":1565568000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/501100000925","name":"National Health and Medical Research Council","doi-asserted-by":"publisher","award":["1061868","1120443"],"award-info":[{"award-number":["1061868","1120443"]}],"id":[{"id":"10.13039\/501100000925","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000901","name":"Juvenile Diabetes Research Foundation International","doi-asserted-by":"publisher","award":["3\u2010SRA\u20102017\u2010366\u2010S\u2010B"],"award-info":[{"award-number":["3\u2010SRA\u20102017\u2010366\u2010S\u2010B"]}],"id":[{"id":"10.13039\/100000901","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Xenotransplantation"],"published-print":{"date-parts":[[2020,1]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Gene editing using clustered regularly interspaced short palindromic repeats\/Cas9 has great potential for improving the compatibility of porcine organs with human recipients. However, the risk of detrimental off\u2010target mutations in gene\u2010edited pigs remains largely undefined. We have previously generated <jats:italic>GGTA1<\/jats:italic> knock\u2010in pigs for xenotransplantation using <jats:italic>Fok<\/jats:italic>I\u2010dCas9, a variant of Cas9 that is reported to reduce the frequency of off\u2010target mutagenesis. In this study, we used whole genome sequencing (WGS) and optimized bioinformatic analysis to assess the fidelity of <jats:italic>Fok<\/jats:italic>I\u2010dCas9 editing in the generation of these pigs. Genomic DNA was isolated from porcine cells before and after gene editing and sequenced by WGS. The genomic sequences were analyzed using <jats:italic>GRIDSS<\/jats:italic> variant\u2010calling software to detect putative structural variations (SVs), which were validated by PCR of DNA from knock\u2010in and wild\u2010type pigs. <jats:italic>Platypus<\/jats:italic> variant\u2010calling software was used to detect single\u2010nucleotide variations (SNVs) and small insertions\/deletions (indels). <jats:italic>GRIDSS<\/jats:italic> analysis confirmed the precise integration of one copy of the knock\u2010in construct in the gene\u2010edited cells. Three additional SVs were detected by <jats:italic>GRIDSS<\/jats:italic>: deletions in intergenic regions in chromosome 6 and the X chromosome and a duplication of part of the <jats:italic>CALD1<\/jats:italic> gene on chromosome 18. These mutations were not associated with plausible off\u2010target sites, and were not detected in a second line of knock\u2010in pigs generated using the same pair of guide RNAs, suggesting that they were the result of background mutation rather than off\u2010target activity. <jats:italic>Platypus<\/jats:italic> identified 1375 SNVs\/indels after quality filtering, but none of these were located in proximity to potential off\u2010target sites, indicating that they were probably also spontaneous mutations. This is the first WGS analysis of pigs generated from <jats:italic>Fok<\/jats:italic>I\u2010dCas9\u2010edited cells. Our results demonstrate that <jats:italic>Fok<\/jats:italic>I\u2010dCas9 is capable of high\u2010fidelity gene editing with negligible off\u2010target or undesired on\u2010target mutagenesis.<\/jats:p>","DOI":"10.1111\/xen.12551","type":"journal-article","created":{"date-parts":[[2019,8,13]],"date-time":"2019-08-13T05:48:45Z","timestamp":1565675325000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["<i>Fok<\/i>I\u2010dCas9 mediates high\u2010fidelity genome editing in pigs"],"prefix":"10.1111","volume":"27","author":[{"given":"Nella","family":"Fisicaro","sequence":"first","affiliation":[{"name":"Immunology Research Centre St Vincent\u2019s Hospital Melbourne Melbourne Vic. Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Evelyn J.","family":"Salvaris","sequence":"additional","affiliation":[{"name":"Immunology Research Centre St Vincent\u2019s Hospital Melbourne Melbourne Vic. Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gayle K.","family":"Philip","sequence":"additional","affiliation":[{"name":"Melbourne Bioinformatics University of Melbourne Melbourne Vic. Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Matthew J.","family":"Wakefield","sequence":"additional","affiliation":[{"name":"Melbourne Bioinformatics University of Melbourne Melbourne Vic. Australia"},{"name":"Walter and Eliza Hall Institute Melbourne Vic. Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mark B.","family":"Nottle","sequence":"additional","affiliation":[{"name":"Robinson Research Institute &amp; Adelaide Medical School University of Adelaide Adelaide SA Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wayne J.","family":"Hawthorne","sequence":"additional","affiliation":[{"name":"Department of Surgery, Westmead Clinical School Westmead Hospital, University of Sydney Sydney NSW Australia"},{"name":"The Centre for Transplant and Renal Research Westmead Institute for Medical Research Westmead NSW Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9016-4954","authenticated-orcid":false,"given":"Peter J.","family":"Cowan","sequence":"additional","affiliation":[{"name":"Immunology Research Centre St Vincent\u2019s Hospital Melbourne Melbourne Vic. Australia"},{"name":"Department of Medicine University of Melbourne Melbourne Vic. Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2019,8,12]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1097\/MOT.0000000000000589"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2013.08.022"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.npep.2016.11.003"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41419-018-1146-0"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms17101668"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.18632\/oncotarget.17154"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/srep16623"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-018-1610-2"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1007503"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/srep20620"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.4192"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1097\/MOT.0000000000000347"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.2909"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-017-09030-6"},{"key":"e_1_2_8_16_1","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btu170"},{"key":"e_1_2_8_17_1","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btp324"},{"key":"e_1_2_8_18_1","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btp352"},{"key":"e_1_2_8_19_1","doi-asserted-by":"publisher","DOI":"10.1101\/gr.222109.117"},{"key":"e_1_2_8_20_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-019-1720-5"},{"key":"e_1_2_8_21_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-016-0974-4"},{"key":"e_1_2_8_22_1","doi-asserted-by":"publisher","DOI":"10.1038\/ng.3036"},{"key":"e_1_2_8_23_1","doi-asserted-by":"publisher","DOI":"10.1038\/srep43169"},{"key":"e_1_2_8_24_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkh103"},{"key":"e_1_2_8_25_1","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btq033"},{"key":"e_1_2_8_26_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms15183"},{"key":"e_1_2_8_27_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0168-9525(00)02024-2"},{"key":"e_1_2_8_28_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1007994"},{"key":"e_1_2_8_29_1","doi-asserted-by":"publisher","DOI":"10.1385\/CBB:35:3:275"}],"container-title":["Xenotransplantation"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/api.wiley.com\/onlinelibrary\/tdm\/v1\/articles\/10.1111%2Fxen.12551","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/xen.12551","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1111\/xen.12551","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/xen.12551","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,9,8]],"date-time":"2023-09-08T10:06:20Z","timestamp":1694167580000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1111\/xen.12551"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,8,12]]},"references-count":28,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2020,1]]}},"alternative-id":["10.1111\/xen.12551"],"URL":"https:\/\/doi.org\/10.1111\/xen.12551","archive":["Portico"],"relation":{},"ISSN":["0908-665X","1399-3089"],"issn-type":[{"type":"print","value":"0908-665X"},{"type":"electronic","value":"1399-3089"}],"subject":[],"published":{"date-parts":[[2019,8,12]]},"assertion":[{"value":"2019-04-14","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2019-07-24","order":1,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2019-08-12","order":2,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e12551"}}