Generation and Characterization of a CRISPR/Cas9-Mediated SNAP29 Knockout in Human Fibroblasts

Martens, Marie Christine; Edelkamp, Janin; Seebode, Christina; Schäfer, Mirijam; Staehlke, Susanne; Krohn, Saskia; Jung, Ole; Escobar, Hugo Murua; Emmert, Steffen; Boeckmann, Lars

doi:10.3390/ijms22105293

Cited by 6 publications

(3 citation statements)

References 35 publications

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“…In our study, a transfection based CRISPR/Cas 9 approach was performed due to its lower probability of off-target effects [ 40 ]. Nevertheless, and as one limitation of our study, off-target effects may not be fully excluded.…”

Section: Discussionmentioning

confidence: 99%

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

Banicka

Martens

Panzer

et al. 2022

IJMS

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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.

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Section: Discussionmentioning

confidence: 99%

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

Banicka

Martens

Panzer

et al. 2022

IJMS

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“…The gene for synaptosome-associated protein 29 (SNAP29) has been shown to be involved in the formation of primary cilia, epidermal differentiation, membrane fusion, and autophagy. SNAP29 has been implicated in recessive neurocutaneous cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (Martens et al, 2021). The gene for the DGCR8 microprocessor complex subunit (DGCR8) encodes a subunit of the microprocessor complex, which mediates the biogenesis of microRNAs from the primary microRNA transcript.…”

Section: The Impact Of Reciprocal Translocation On Genomic Instabilit...mentioning

confidence: 99%

Case Report: Balanced Reciprocal Translocation t (17; 22) (p11.2; q11.2) and 10q23.31 Microduplication in an Infertile Male Patient Suffering From Teratozoospermia

Huang

Wu²,

Qi³

et al. 2022

Front. Genet.

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Two chromosomal abnormalities are described in an infertile man suffering from teratozoospermia: balanced reciprocal translocation t (17; 22) (p11.2; q11.2) and a microduplication in the region 10q23.31. Twenty genes located on the breakpoints of translocation (e.g., ALKBH5, TOP3A, SPECC1L, and CDC45) are selected due to their high expression in testicular tissues and might be influenced by chromosome translocation. Four genes located on the breakpoints of microduplication including FLJ37201, KIF20B, LINC00865, and PANK1 result in an increased dosage of genes, representing an imbalance in the genome. These genes have been reported to be associated with developmental disorders/retardation and might be risk factors affecting spermatogenesis. Bioinformatics analysis is carried out on these key genes, intending to find the pathogenic process of reproduction in the context of the translocation and microduplication encountered in the male patient. The combination of the two chromosomal abnormalities carries additional risks for gametogenesis and genomic instability and is apparently harmful to male fertility. Overall, our findings could contribute to the knowledge of male infertility caused by genetic factors.

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“…Previously, we were able to show that lentiviral transduction was more efficient than transient transfection to generate SNAP29 knockout human fibroblast cell lines. Although lentiviral transduction efficiency was higher, genome integration and therefore cumulating off-target effects and insertional mutation need to be considered [37].…”

Section: Splice Variants Of Xeroderma Pigmentosum Genesmentioning

confidence: 99%

Xeroderma Pigmentosum: Gene Variants and Splice Variants

Martens

Emmert

Boeckmann

2021

Genes

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The nucleotide excision repair (NER) is essential for the repair of ultraviolet (UV)-induced DNA damage, such as cyclobutane pyrimidine dimers (CPDs) and 6,4-pyrimidine-pyrimidone dimers (6,4-PPs). Alterations in genes of the NER can lead to DNA damage repair disorders such as Xeroderma pigmentosum (XP). XP is a rare autosomal recessive genetic disorder associated with UV-sensitivity and early onset of skin cancer. Recently, extensive research has been conducted on the functional relevance of splice variants and their relation to cancer. Here, we focus on the functional relevance of alternative splice variants of XP genes.

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Generation and Characterization of a CRISPR/Cas9-Mediated SNAP29 Knockout in Human Fibroblasts

Cited by 6 publications

References 35 publications

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

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

Case Report: Balanced Reciprocal Translocation t (17; 22) (p11.2; q11.2) and 10q23.31 Microduplication in an Infertile Male Patient Suffering From Teratozoospermia

Xeroderma Pigmentosum: Gene Variants and Splice Variants

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