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Homozygous CRISPR/Cas9 Knockout Generated a Novel Functionally Active Exon 1 Skipping XPA Variant in Melanoma Cells
Veronika Banicka1, Marie Christine Martens1, Rüdiger Panzer1
1Clinic and Policlinic for Dermatology and Venereology, University Medical Center Rostock, 18057 Rostock, Germany.
Abstract:
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.
Insights
Defects in Xeroderma pigmentosum (XP) DNA repair improve response to immune checkpoint inhibition (ICI). This study created XPA-disrupted melanoma cells, revealing a novel XPA protein variant linked to DNA repair and survival.
Area of Science:
- Oncology
- Genetics
- Immunology
Background:
- Defects in DNA repair pathways, particularly nucleotide excision repair (NER) in Xeroderma pigmentosum (XP), are linked to enhanced responses to immune checkpoint inhibition (ICI).
- Pretherapeutic XP gene expression in melanoma patients predicts response to anti-programmed cell death-1 (PD-1) ICI, but mechanisms remain unclear.
Purpose of the Study:
- To investigate the underlying mechanisms of improved ICI response in XP by creating and analyzing XPA-disrupted melanoma cell lines.
- To characterize novel XPA protein variants resulting from CRISPR/Cas9 gene editing and assess their functional impact on DNA repair and cell survival.
Main Methods:
- CRISPR/Cas9 gene editing was used to disrupt the XPA gene in A375 melanoma cells, generating homozygous and compound heterozygous cell lines.
- Sanger sequencing confirmed mutations, while immunoblotting, immunofluorescence, and High-Content Screening (HCR) assays evaluated protein expression and DNA repair.
- MTT assays assessed post-UV-C cell survival to determine functional consequences of XPA disruption.
Main Results:
- Established distinct XPA-disrupted melanoma cell lines, including homozygous and compound heterozygous mutations in exon 1 and compound heterozygous mutations in exon 2.
- Homozygous exon 1 mutated cells showed superior DNA damage repair (HCR) and post-UV-C survival (MTT) compared to heterozygous cells.
- These functional differences correlated with the expression of a novel XPA protein variant.
Conclusions:
- XPA disruption in melanoma cells can lead to novel protein variants impacting DNA repair and cell survival.
- The findings provide a foundation for exploring the immunological consequences of XPA disruption in melanoma and its potential role in ICI response.
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