{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T21:17:45Z","timestamp":1777670265320,"version":"3.51.4"},"reference-count":66,"publisher":"SAGE Publications","issue":"17-18","license":[{"start":{"date-parts":[[2022,9,1]],"date-time":"2022-09-01T00:00:00Z","timestamp":1661990400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.sagepub.com\/page\/policies\/text-and-data-mining-license"}],"content-domain":{"domain":["journals.sagepub.com"],"crossmark-restriction":true},"short-container-title":["Human Gene Therapy"],"published-print":{"date-parts":[[2022,9]]},"abstract":"<jats:p>\n                    Recombinant adenovirus vectors enable highly efficient gene delivery\n                    <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                    and\n                    <jats:italic toggle=\"yes\">in vivo.<\/jats:italic>\n                    As a result, they are widely used in gene therapy, vaccination, and anticancer applications. We have previously developed the AdZ vector system, which uses recombineering to permit high-throughput cloning of transgenes into Adenovirus vectors, simplifies alteration of the vector backbone, and enables rapid recovery of infectious virus, even if a transgene is incompatible with vector replication. In this study, we adapt this vector system to enable high-throughput cloning of sequences for CRISPR\/Cas9 editing. Vectors were optimized to ensure efficient cloning, and high editing efficiency using spCas9 and single guide RNA (sgRNA) sequences in a single vector. Using a multiplicity of infection of 50, knockout efficiencies of up to 80% could be achieved with a single sgRNA. Vectors were further enhanced by altering the spCas9 sequence to match that of SniperCas9, which has reduced off-target activity, but maintains on-target efficiency, and by applying modifications to the sgRNA sequence that significantly enhance editing efficiency. Thus, the AdZ-CRISPR vectors offer highly efficient knockout, even in hard to transfect cells, and enables large-scale CRISPR\/Cas9 projects to be undertaken easily and quickly.\n                  <\/jats:p>","DOI":"10.1089\/hum.2021.120","type":"journal-article","created":{"date-parts":[[2022,2,24]],"date-time":"2022-02-24T04:22:46Z","timestamp":1645676566000},"page":"990-1001","update-policy":"https:\/\/doi.org\/10.1177\/sage-journals-update-policy","source":"Crossref","is-referenced-by-count":11,"title":["An Optimized CRISPR\/Cas9 Adenovirus Vector (AdZ-CRISPR) for High-Throughput Cloning of sgRNA, Using Enhanced sgRNA and Cas9 Variants"],"prefix":"10.1177","volume":"33","author":[{"given":"Evelina","family":"Statkute","sequence":"first","affiliation":[{"name":"Infection and Immunity, School of Medicine, Cardiff University, Cardiff, United Kingdom."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Eddie C.Y.","family":"Wang","sequence":"additional","affiliation":[{"name":"Infection and Immunity, School of Medicine, Cardiff University, Cardiff, United Kingdom."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6799-1182","authenticated-orcid":false,"given":"Richard J.","family":"Stanton","sequence":"additional","affiliation":[{"name":"Infection and Immunity, School of Medicine, Cardiff University, Cardiff, United Kingdom."}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"179","published-online":{"date-parts":[[2022,9,16]]},"reference":[{"key":"e_1_3_5_2_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-018-04252-2"},{"key":"e_1_3_5_3_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1225829"},{"key":"e_1_3_5_4_2","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.00471"},{"key":"e_1_3_5_5_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1231143"},{"key":"e_1_3_5_6_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1232033"},{"key":"e_1_3_5_7_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymthe.2020.01.001"},{"key":"e_1_3_5_8_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-24454-5"},{"key":"e_1_3_5_9_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-26518-y"},{"key":"e_1_3_5_10_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jconrel.2020.09.003"},{"key":"e_1_3_5_11_2","doi-asserted-by":"publisher","DOI":"10.1089\/hum.2015.087"},{"key":"e_1_3_5_12_2","doi-asserted-by":"publisher","DOI":"10.3390\/ijms22052417"},{"key":"e_1_3_5_13_2","doi-asserted-by":"publisher","DOI":"10.1099\/vir.0.19497-0"},{"key":"e_1_3_5_14_2","doi-asserted-by":"publisher","DOI":"10.1099\/0022-1317-36-1-59"},{"key":"e_1_3_5_15_2","doi-asserted-by":"publisher","DOI":"10.1089\/hum.1998.9.13-1909"},{"key":"e_1_3_5_16_2","doi-asserted-by":"publisher","DOI":"10.1016\/0042-6822(88)90302-9"},{"key":"e_1_3_5_17_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.91.19.8802"},{"key":"e_1_3_5_18_2","doi-asserted-by":"publisher","DOI":"10.2144\/00293st04"},{"key":"e_1_3_5_19_2","doi-asserted-by":"publisher","DOI":"10.1089\/10430340050015590"},{"key":"e_1_3_5_20_2","doi-asserted-by":"publisher","DOI":"10.2144\/000112993"},{"key":"e_1_3_5_21_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.100127597"},{"key":"e_1_3_5_22_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gni035"},{"key":"e_1_3_5_23_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1004058"},{"key":"e_1_3_5_24_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2014.07.005"},{"key":"e_1_3_5_25_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1004811"},{"key":"e_1_3_5_26_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1720950115"},{"key":"e_1_3_5_27_2","doi-asserted-by":"publisher","DOI":"10.1172\/JCI139296"},{"key":"e_1_3_5_28_2","doi-asserted-by":"publisher","DOI":"10.1038\/gt.2014.52"},{"key":"e_1_3_5_29_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.omto.2018.01.003"},{"key":"e_1_3_5_30_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.05759-11"},{"key":"e_1_3_5_31_2","doi-asserted-by":"publisher","DOI":"10.1038\/cgt.2013.85"},{"key":"e_1_3_5_32_2","article-title":"Adenovirus serotype 5 vectors with Tat-PTD modified hexon and serotype 35 fiber show greatly enhanced transduction capacity of primary cell cultures","volume":"8","author":"Yu D","year":"2013","unstructured":"Yu D, , Jin C, , Ramachandran M, et al. Adenovirus serotype 5 vectors with Tat-PTD modified hexon and serotype 35 fiber show greatly enhanced transduction capacity of primary cell cultures. 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