TY  - JOUR
A1  - Banicka, Veronika
A1  - Martens, Marie Christine
A1  - Panzer, Rüdiger
A1  - Schrama, David
A1  - Emmert, Steffen
A1  - Boeckmann, Lars
A1  - Thiem, Alexander
T1  - Homozygous CRISPR/Cas9 knockout generated a novel functionally active exon 1 skipping XPA variant in melanoma cells
JF  - International Journal of Molecular Sciences
N2  - 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.
KW  - DNA repair
KW  - nucleotide excision repair
KW  - XPA
KW  - CRISPR
KW  - knockout
KW  - protein variant
KW  - melanoma
KW  - A375
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-290427
SN  - 1422-0067
VL  - 23
IS  - 19
ER  -