Homozygous CRISPR/Cas9 Knockout Generated a Novel Functionally Active Exon 1 Skipping XPA Variant in Melanoma Cells.

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.

PRG3 and PRG5 C-Termini: Important Players in Early Neuronal Differentiation.

The functional importance of neuronal differentiation of the transmembrane proteins' plasticity-related genes 3 (PRG3) and 5 (PRG5) has been shown. Although their sequence is closely related, they promote different morphological changes in neurons. PRG3 was shown to promote neuritogenesis in primary neurons; PRG5 contributes to spine induction in immature neurons and the regulation of spine density and morphology in mature neurons. Both exhibit intracellularly located C-termini of less than 50 amino acids. Varying C-termini suggested that these domains shape neuronal morphology differently. We generated mutant EGFP-fusion proteins in which the C-termini were either swapped between PRG3 and PRG5, deleted, or fused to another family member, plasticity-related gene 4 (PRG4), that was recently shown to be expressed in different brain regions. We subsequently analyzed the influence of overexpression in immature neurons. Our results point to a critical role of the PRG3 and PRG5 C-termini in shaping early neuronal morphology. However, the results suggest that the C-terminus alone might not be sufficient for promoting the morphological effects induced by PRG3 and PRG5.