p53 exon 11 mutants generated by CRISPR/Cas9 result in aberrant alternative splicing.

The 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.Tp53基因（Tp53 gene）编码经典的肿瘤抑制蛋白p53。该基因由11个外显子（exon）组成，目前已检测到其转录本存在多种可变剪接（alternative splicing）变体。本研究借助CRISPR/Cas9基因组编辑工具，对人类结肠癌细胞系HCT116实施靶向诱变，诱变位点精准定位于Tp53基因第11外显子的5'端。将转染CRISPR/Cas9的细胞通过非同源末端连接（non-homologous end joining）途径修复双链断裂后，对突变细胞池进行单细胞克隆，并通过Sanger DNA测序（Sanger DNA sequencing）开展分析。结果显示，mut4、mut5与mut9这3个突变克隆均携带双等位纯合突变（biallelic homozygous mutations）。从突变细胞池中分离得到聚腺苷酸化RNA（poly adenylated RNA），并进行三次生物学重复的Illumina RNASeq测序。分析发现，第11外显子的突变会引发上游外显子发生可变剪接，并诱导p53β可变剪接产物的表达。这种C端截短蛋白与细胞衰老（senescence）的诱导过程密切相关。本研究进而在Tp53基因第11外显子中鉴定出一处外显子剪接沉默子（exonic splicing silencer），该元件在正常生理条件下会抑制编码p53β变体的可变剪接事件。该序列及其结合蛋白因子可作为极具潜力的靶向干预靶点，有望通过诱导p53β剪接进而触发肿瘤细胞的衰老进程。