{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T04:41:15Z","timestamp":1760244075274,"version":"build-2065373602"},"reference-count":42,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2022,10,1]],"date-time":"2022-10-01T00:00:00Z","timestamp":1664582400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Hiege Foundation\u2014the German Skin Cancer Foundation"},{"name":"Forschungsf\u00f6rderung der Universit\u00e4tsmedizin Rostock FORUN"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Defects in DNA repair pathways have been associated with an improved response to immune checkpoint inhibition (ICI). In particular, patients with the nucleotide excision repair (NER) defect disease Xeroderma pigmentosum (XP) responded impressively well to ICI treatment. Recently, in melanoma patients, pretherapeutic XP gene expression was predictive for anti-programmed cell death-1 (PD-1) ICI response. The underlying mechanisms of this finding are still to be revealed. Therefore, we used CRISPR\/Cas9 to disrupt XPA in A375 melanoma cells. The resulting subclonal cell lines were investigated by Sanger sequencing. Based on their genetic sequence, candidates from XPA exon 1 and 2 were selected and further analyzed by immunoblotting, immunofluorescence, HCR and MTT assays. In XPA exon 1, we established a homozygous (c.19delG; p.A7Lfs*8) and a compound heterozygous (c.19delG\/c.19_20insG; p.A7Lfs*8\/p.A7Gfs*55) cell line. In XPA exon 2, we generated a compound heterozygous mutated cell line (c.206_208delTTG\/c.208_209delGA; p.I69_D70delinsN\/p.D70Hfs*31). The better performance of the homozygous than the heterozygous mutated exon 1 cells in DNA damage repair (HCR) and post-UV-C cell survival (MTT), was associated with the expression of a novel XPA protein variant. The results of our study serve as the fundamental basis for the investigation of the immunological consequences of XPA disruption in melanoma.<\/jats:p>","DOI":"10.3390\/ijms231911649","type":"journal-article","created":{"date-parts":[[2022,10,8]],"date-time":"2022-10-08T22:19:27Z","timestamp":1665267567000},"page":"11649","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Homozygous CRISPR\/Cas9 Knockout Generated a Novel Functionally Active Exon 1 Skipping XPA Variant in Melanoma Cells"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7545-5400","authenticated-orcid":false,"given":"Veronika","family":"Banicka","sequence":"first","affiliation":[{"name":"Clinic and Policlinic for Dermatology and Venereology, University Medical Center Rostock, 18057 Rostock, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marie Christine","family":"Martens","sequence":"additional","affiliation":[{"name":"Clinic and Policlinic for Dermatology and Venereology, University Medical Center Rostock, 18057 Rostock, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"R\u00fcdiger","family":"Panzer","sequence":"additional","affiliation":[{"name":"Clinic and Policlinic for Dermatology and Venereology, University Medical Center Rostock, 18057 Rostock, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6931-8194","authenticated-orcid":false,"given":"David","family":"Schrama","sequence":"additional","affiliation":[{"name":"Department of Dermatology, Venereology and Allergology, University Hospital W\u00fcrzburg, 97080 W\u00fcrzburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Steffen","family":"Emmert","sequence":"additional","affiliation":[{"name":"Clinic and Policlinic for Dermatology and Venereology, University Medical Center Rostock, 18057 Rostock, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0098-6546","authenticated-orcid":false,"given":"Lars","family":"Boeckmann","sequence":"additional","affiliation":[{"name":"Clinic and Policlinic for Dermatology and Venereology, University Medical Center Rostock, 18057 Rostock, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9504-4281","authenticated-orcid":false,"given":"Alexander","family":"Thiem","sequence":"additional","affiliation":[{"name":"Clinic and Policlinic for Dermatology and Venereology, University Medical Center Rostock, 18057 Rostock, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,10,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"6717","DOI":"10.1158\/0008-5472.CAN-18-2652","article-title":"Development of PARP and Immune-Checkpoint Inhibitor Combinations","volume":"78","author":"Stewart","year":"2018","journal-title":"Cancer Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"675","DOI":"10.1158\/2159-8290.CD-17-0226","article-title":"DNA Damage and Repair Biomarkers of Immunotherapy Response","volume":"7","author":"Mouw","year":"2017","journal-title":"Cancer Discov."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"838","DOI":"10.1158\/2159-8290.CD-20-1680","article-title":"The Next Decade of Immune Checkpoint Therapy","volume":"11","author":"Sharma","year":"2021","journal-title":"Cancer Discov."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"3753","DOI":"10.1158\/1078-0432.CCR-18-4070","article-title":"FDA Approval Summary: Pembrolizumab for the Treatment of Microsatellite Instability-High Solid Tumors","volume":"25","author":"Marcus","year":"2019","journal-title":"Clin. 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