CRISPR induced exon skipping of beta-catenin reveals unexpected tumorigenic mutants driving distinct subtypes of liver cancer
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA688910 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13337600_1.fastq.gz | Fastqsanger.gz | |||
| SRR13337600_2.fastq.gz | Fastqsanger.gz | |||
| SRR13337601_1.fastq.gz | Fastqsanger.gz | |||
| SRR13337601_2.fastq.gz | Fastqsanger.gz | |||
| SRR13337602_1.fastq.gz | Fastqsanger.gz |
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Project description: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.
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Project description:CRISPR/Cas9-driven cancer modeling studies are based on the disruption of tumor suppressor genes by small insertions or deletions (indels) that lead to frame-shift mutations. In addition, CRISPR/Cas9 is widely used to define the significance of cancer oncogenes and genetic dependencies in loss-of-function studies. However, how CRISPR/Cas9 influences gain-of-function oncogenic mutations is elusive. Here, we demonstrate that single guide RNA targeting exon 3 of Ctnnb1 (encoding β-catenin) results in exon skipping and generates gain-of-function isoforms in vivo. CRISPR/Cas9-mediated exon skipping of Ctnnb1 induces liver tumor formation in synergy with YAPS127A in mice. We define two distinct exon skipping-induced tumor subtypes with different histological and transcriptional features. Notably, ectopic expression of two exon-skipped β-catenin transcript isoforms together with YAPS127A phenocopies the two distinct subtypes of liver cancer. Moreover, we identify similar CTNNB1 exon-skipping events in patients with hepatocellular carcinoma. Collectively, our findings advance our understanding of β-catenin-related tumorigenesis and reveal that CRISPR/Cas9 can be repurposed, in vivo, to study gain-of-function mutations of oncogenes in cancer. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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Project description:Pre-mRNA splicing is functionally coupled to transcription, and genotoxic stresses can enhance alternative exon inclusion by affecting elongating RNA polymerase II. We report here that various genotoxic stress inducers, including camptothecin, inhibit the interaction between EWS, an RNA polymerase II-associated factor, and YB-1, a spliceosome-associated factor. This results in the cotranscriptional skipping of several exons of the MDM2 gene encoding the main p53 ubiquitin-ligase. This reversible exon skipping participates in the timely regulation of MDM2 expression, and may contribute to the accumulation of p53 during stress exposure and its rapid shut off when stress is removed. Finally, a splicing-sensitive microarray identified numerous exons that are skipped in response to camptothecin and EWS/YB-1 depletion. These data demonstrate genotoxic stress-induced alteration of the communication between the transcriptional and splicing machineries, resulting in widespread exon skipping and playing a central role in the genotoxic stress response. 6 samples of MCF7 cells exposed to different treatments were analyzed: 3 x control_6 hours; 3 x camptothecin_6 hours.
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