CRISPR induced exon skipping of beta-catenin reveals unexpected tumorigenic mutants driving distinct subtypes of liver cancer. CRISPR induced exon skipping of beta-catenin reveals unexpected tumorigenic mutants driving distinct subtypes of liver cancer

we induce exon skipping and generate a gain-of-function of an oncogene, β-catenin, using CRISPR/Cas9 in mouse liver cells. Specifically, a single guide RNA (sgRNA) targeting exon 3 of β-catenin induces exon skipping and gain-of-function of β-catenin in mouse hepatocytes. In synergy with YAPS127A, exon skipped hepatocytes gain tumorigenic ability and are thus enriched via tumor formation. Surprisingly, characterization of the exon-skipped tumors reveals two distinct subtypes with different histological features. Remarkably, ectopic expression of two representative exon-skipped β-catenin transcripts together with YAPS127A phenocopies the two histologically distinct subtypes of liver cancer. Finally, the transcriptome sequencing analysis reveal two subtypes of liver cancer and most importantly, one subtype of the exon-skipped tumor shows features of hepatoblastoma, while the other does not. This exon skipping model reveals CRISPR/Cas9 can lead to exon-skipped transcripts with in frame coding and gain-of-functions. Overall design: Transcriptome and histology to dinstinguish two subtypes of liver cancer driven by exon-skipped isoform of beta-catenin

我们在小鼠肝细胞中利用CRISPR/Cas9系统诱导外显子跳跃，并使致癌基因β-连环蛋白（β-catenin）获得功能增益。具体而言，靶向β-连环蛋白（β-catenin）第3号外显子的单向导RNA（single guide RNA, sgRNA）可在小鼠肝细胞中诱导外显子跳跃，同时使β-连环蛋白产生功能获得性变化。与YAPS127A协同作用时，发生外显子跳跃的肝细胞可获得致瘤能力，并通过肿瘤形成过程得到富集。令人意外的是，对该类外显子跳跃肿瘤的表征分析显示，其存在两种具有迥异组织学特征的亚型。值得注意的是，将两种代表性的外显子跳跃型β-连环蛋白转录本与YAPS127A进行异位表达，可重现两种组织学特征迥异的肝癌亚型表型。最终，转录组测序分析证实了肝癌的两种亚型，而尤为关键的是，其中一种外显子跳跃型肿瘤呈现肝母细胞瘤（hepatoblastoma）的特征，另一种则无此类特征。该外显子跳跃模型证实，CRISPR/Cas9可诱导产生带有框内编码序列且具备功能获得性的外显子跳跃型转录本。实验整体设计：通过转录组分析与组织学鉴定，区分由β-连环蛋白外显子跳跃异构体驱动的两种肝癌亚型。