{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,1]],"date-time":"2026-02-01T09:27:08Z","timestamp":1769938028995,"version":"3.49.0"},"reference-count":21,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2019,1,17]],"date-time":"2019-01-17T00:00:00Z","timestamp":1547683200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2019,1,17]],"date-time":"2019-01-17T00:00:00Z","timestamp":1547683200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"name":"Liverpool-Mahidol Partnership Scholarship"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>CRISPR\/Cas9 causes double-stranded DNA breaks that can undergo DNA repair either via non-homologous end joining (NHEJ) or, in the presence of a template, homology-directed repair (HDR). HDR is typically used to insert a specific genetic modification into the genome but has low efficiency compared to NHEJ, which is lowered even further when trying to create a homozygous change. In this study we devised a novel approach for homozygous single base editing based on utilising simultaneously two donor DNA templates cloned in plasmids with different antibiotic resistant genes. The donor templates were co-transfected alongside the CRISPR\/Cas9 machinery into cells and a double antibiotic selection was optimised and allowed the isolation of viable desired clones. We applied the method for obtaining isogenic cells homozygous for variant B cystatin C, a recessive risk factor for age-related macular degeneration and Alzheimer\u2019s disease, in both induced Pluripotent Stem Cells (iPSCs) and a human RPE cell line. Bi-allelic gene edited clones were validated by sequencing, demonstrating that the double antibiotic templates approach worked efficiently for both iPSCs and human differentiated cells. We propose that this one step gene editing approach can be used to improve the specificity and frequency of introducing homozygous modifications in mammalian cells.<\/jats:p>","DOI":"10.1038\/s41598-018-36740-2","type":"journal-article","created":{"date-parts":[[2019,1,12]],"date-time":"2019-01-12T07:18:52Z","timestamp":1547277532000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":34,"title":["CRISPR\/Cas9-mediated one step bi-allelic change of genomic DNA in iPSCs and human RPE cells in vitro with dual antibiotic selection"],"prefix":"10.1038","volume":"9","author":[{"given":"Wasu","family":"Supharattanasitthi","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Emil","family":"Carlsson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Umar","family":"Sharif","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Luminita","family":"Paraoan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2019,1,17]]},"reference":[{"key":"36740_CR1","doi-asserted-by":"publisher","first-page":"933","DOI":"10.1038\/nbt.3659","volume":"34","author":"R Barrangou","year":"2016","unstructured":"Barrangou, R. & Doudna, J. A. Applications of CRISPR technologies in research and beyond. Nat. Biotechnol. 34, 933\u2013941 (2016).","journal-title":"Nat. Biotechnol."},{"key":"36740_CR2","doi-asserted-by":"publisher","first-page":"3222","DOI":"10.1111\/febs.13763","volume":"283","author":"J Lin","year":"2016","unstructured":"Lin, J. & Musunuru, K. Genome engineering tools for building cellular models of disease. FEBS J. 283, 3222\u20133231 (2016).","journal-title":"FEBS J."},{"key":"36740_CR3","doi-asserted-by":"publisher","unstructured":"Komor, A. C., Badran, A. H. & Liu, D. R. Review CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes. Cell 1\u201317, https:\/\/doi.org\/10.1016\/j.cell.2016.10.044 (2017).","DOI":"10.1016\/j.cell.2016.10.044"},{"key":"36740_CR4","doi-asserted-by":"publisher","first-page":"1041","DOI":"10.1007\/s00439-016-1691-5","volume":"135","author":"Y Young","year":"2016","unstructured":"Young, Y. & Zhaohui, J. 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