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Learn MoreThe Tp53 gene encodes the famous tumor suppressor protein p53. This gene is encoded in 11 exons and various alternative spliced versions of the transcripts have been detected. Here we generated targeted mutations in human colon cancer cell line HCT116 using CRISPR/Cas9 genome editing tools. The mutations were targeted to the 5' end of Tp53 gene exon 11. CRISPR/Cas9 transfected cells were allowed to repair double stranded breaks by non-homologous end joining. Mutant cell pools were single cell cloned and analyzed by Sanger DNA sequencing. Three mutant clones, mut4, mut5 and mut9 were found to contain biallelic homozygous mutations. Poly adenylated RNA was isolated from mutant cell pools was isolated and sequenced by Illumina RNASeq in triplicate. We find that exon 11 mutations result in the alternative splicing of earlier exons and cause the expression of the p53beta alternative splice product. This C-terminally deleted protein has been associated with the induction of senescence. We thus identify an exonic splicing silencer in exon 11 of the Tp53 gene which normally suppressed alternative splicing events that encode the p53beta variant. This sequence and the factors that associate with it are prime candidates for targeting, which may induce p53beta splicing and the induction of senescence in tumors. SOURCE: Batu Erman (batu@sabanciuniv.edu) - Faculty of Engineering and Natural Sciences Sabanci University
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