Knock-out of Flotillins in Human Cells Using the CRISPR-Cas9 Genome Editing System: Effects on mRNA Splicing

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KapahnkeMarcel_2020_10_28.pdf (3.28 MB)

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2020

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DOI:
http://dx.doi.org/10.22029/jlupub-14859

Abstract

The CRISPR-Cas9 system has emerged as a versatile tool for genome editing. Using this novel technique, specific DNA modification can be achieved by designing a simple gRNA sequence complementary to the target site guiding the nuclease Cas9 towards it. The introduced double strand break is expected to be repaired by the error-prone nonhomologous end joining repair, causing a frameshift leading to a premature stop codon and subsequently a highly truncated protein. In this study, the novel CRISPR-Cas9 approach was used to generate flotillin-1, flotillin-2 and double knock-out cell lines.The screening of the single cell clones was performed by Western blot and could detect several cell clones with absence of flotillin-1, flotillin-2 or both flotillin proteins. These cell clones were characterised in the following experiments. The real-time PCR showed a persistent presence of the flotillin mRNA, but a fundamental reduction in some cell clones, indicating degradation through NMD. EGF stimulation of knock-out cell lines did not show significantly different results compared to HeLa wild-type, which can be interpreted as a consequence of adaptation to flotillin depleted conditions. The double-KO cell lines could be shown to react more sensitive to apoptosis induction upon staurosporin, but more precise results might be obtained in a time series study.Sequence analysis of the genomic DNA and cDNA derived from mRNA showed the expected indel mutations on genomic DNA, but in addition to these mutations, several different mRNA variants were detected with apparently random missing exons. In silico translation of most mRNA variants resulted in a premature stop codon, but in two mRNA variants, a frameshift caused by a non-multiple of three indel mutation was corrected by excision of a subsequent exon containing the appropriate nucleotide number. The existence of the predicted protein was proven by Western blot after inhibition of protein degradation, upon which a band corresponding to the calculated molecular weight of the truncated protein was observed. Since this mutated protein could be detected only upon degradation inhibition, it is likely to be dysfunctional and unstable, but the functional consequences of such proteins cannot be predicted. In most studies using CRISPR-Cas9 for gene knock-out, the success is monitored only by sequencing of the genomic DNA. Regarding the results of this study, it is strongly recommended to verify the results also on mRNA and protein level.

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