{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T02:55:30Z","timestamp":1760237730726,"version":"build-2065373602"},"reference-count":66,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2020,6,18]],"date-time":"2020-06-18T00:00:00Z","timestamp":1592438400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Fondo de Investigaci\u00f3n Sanitaria (FIS). Instituto de Salud Carlos III","award":["PI18\/00337"],"award-info":[{"award-number":["PI18\/00337"]}]},{"name":"Consejer\u00eda de Salud","award":["salud-2016000073332-TRA"],"award-info":[{"award-number":["salud-2016000073332-TRA"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>In spite of the enormous potential of CRISPR\/Cas in basic and applied science, the levels of undesired genomic modifications cells still remain mostly unknown and controversial. Nowadays, the efficiency and specificity of the cuts generated by CRISPR\/Cas is the main concern. However, there are also other potential drawbacks when DNA donors are used for gene repair or gene knock-ins. These GE strategies should take into account not only the specificity of the nucleases, but also the fidelity of the DNA donor to carry out their function. The current methods to quantify the fidelity of DNA donor are costly and lack sensitivity to detect illegitimate DNA donor integrations. In this work, we have engineered two reporter cell lines (K562_SEWAS84 and K562GWP) that efficiently quantify both the on-target and the illegitimate DNA donor integrations in a WAS-locus targeting setting. K562_SEWAS84 cells allow the detection of both HDR-and HITI-based donor integration, while K562GWP cells only report HDR-based GE. To the best of our knowledge, these are the first reporter systems that allow the use of gRNAs targeting a relevant locus to measure efficacy and specificity of DNA donor-based GE strategies. By using these models, we have found that the specificity of HDR is independent of the delivery method and that the insertion of the target sequence into the DNA donor enhances efficiency but do not affect specificity. Finally, we have also shown that the higher the number of the target sites is, the higher the specificity and efficacy of GE will be.<\/jats:p>","DOI":"10.3390\/cells9061492","type":"journal-article","created":{"date-parts":[[2020,6,18]],"date-time":"2020-06-18T12:21:46Z","timestamp":1592482906000},"page":"1492","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Development of Cellular Models to Study Efficiency and Safety of Gene Edition by Homologous Directed Recombination Using the CRISPR\/Cas9 System"],"prefix":"10.3390","volume":"9","author":[{"given":"Sabina","family":"S\u00e1nchez-Hern\u00e1ndez","sequence":"first","affiliation":[{"name":"Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalusian Regional Government, Parque Tecnol\u00f3gico Ciencias de la Salud, Av. de la Ilustraci\u00f3n 114, 18016 Granada, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2960-8672","authenticated-orcid":false,"given":"Araceli","family":"Aguilar-Gonz\u00e1lez","sequence":"additional","affiliation":[{"name":"Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalusian Regional Government, Parque Tecnol\u00f3gico Ciencias de la Salud, Av. de la Ilustraci\u00f3n 114, 18016 Granada, Spain"},{"name":"Department of Medicinal &amp; Organic Chemistry and Excellence Research Unit of \u201cChemistry Applied to Biomedicine and the Environment\u201d, Faculty of Pharmacy, University of Granada, Campus de Cartuja s\/n, 18071, Granada, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Beatriz","family":"Guijarro-Albaladejo","sequence":"additional","affiliation":[{"name":"Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalusian Regional Government, Parque Tecnol\u00f3gico Ciencias de la Salud, Av. de la Ilustraci\u00f3n 114, 18016 Granada, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5975-0488","authenticated-orcid":false,"given":"Noelia","family":"Maldonado-P\u00e9rez","sequence":"additional","affiliation":[{"name":"Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalusian Regional Government, Parque Tecnol\u00f3gico Ciencias de la Salud, Av. de la Ilustraci\u00f3n 114, 18016 Granada, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Iris","family":"Ramos-Hern\u00e1ndez","sequence":"additional","affiliation":[{"name":"Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalusian Regional Government, Parque Tecnol\u00f3gico Ciencias de la Salud, Av. de la Ilustraci\u00f3n 114, 18016 Granada, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marina","family":"Cortijo-Guti\u00e9rrez","sequence":"additional","affiliation":[{"name":"Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalusian Regional Government, Parque Tecnol\u00f3gico Ciencias de la Salud, Av. de la Ilustraci\u00f3n 114, 18016 Granada, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8912-9799","authenticated-orcid":false,"given":"Rosario Mar\u00eda","family":"S\u00e1nchez Mart\u00edn","sequence":"additional","affiliation":[{"name":"Department of Medicinal &amp; Organic Chemistry and Excellence Research Unit of \u201cChemistry Applied to Biomedicine and the Environment\u201d, Faculty of Pharmacy, University of Granada, Campus de Cartuja s\/n, 18071, Granada, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4673-2111","authenticated-orcid":false,"given":"Karim","family":"Benabdellah","sequence":"additional","affiliation":[{"name":"Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalusian Regional Government, Parque Tecnol\u00f3gico Ciencias de la Salud, Av. de la Ilustraci\u00f3n 114, 18016 Granada, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1961-8612","authenticated-orcid":false,"given":"Francisco","family":"Martin","sequence":"additional","affiliation":[{"name":"Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalusian Regional Government, Parque Tecnol\u00f3gico Ciencias de la Salud, Av. de la Ilustraci\u00f3n 114, 18016 Granada, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2020,6,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"100789","DOI":"10.1016\/j.isci.2019.100789","article-title":"Sharpening the Molecular Scissors: Advances in Gene-Editing Technology","volume":"23","author":"Broeders","year":"2020","journal-title":"Iscience"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"674","DOI":"10.1002\/sctm.19-0338","article-title":"Genome-edited adult stem cells: Next-generation advanced therapy medicinal products (ATMPs)","volume":"9","author":"Benabdellah","year":"2020","journal-title":"Stem Cells Transl. 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