CRISPR‐induced exon skipping of β‐catenin reveals tumorigenic mutants driving distinct subtypes of liver cancer

Abstract

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 YAP S127A 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 YAP S127A 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.

A review of the literature on the use of CRISPR/Cas9 gene therapy to treat hepatocellular carcinoma

“…Mouse liver tumors were made by Ctnnb1 exon skipping using CRISPR/Cas9 in conjunction with YAPS127A. Oncogene gain-of-function mutations in vivo under β-catenin-dependent tumorigenesis are helpful using CRISPR/Cas9 [ 70 ].…”

Escaping from CRISPR–Cas-mediated knockout: the facts, mechanisms, and applications

“…Owing to the lack of proper antibodies, this was not confirmed [ 10 ]. Since then, more than 20 studies have identified in-frame transcripts or truncated proteins from knockout organisms generated by the CRISPR–Cas system (Table 1 ) [ 10 – 37 ]. One interesting study from the Farber group provided direct evidence explaining the low frequency of mutant phenotypes by reverse genetics in zebrafish.…”

“…Functional analysis has also been conducted in mice with genes knocked out, giving similar results as in cells or zebrafish [ 27 , 29 , 37 ]; For example, different gene knockout strategies were compared for the mutant phenotypes in mice, namely a definitive-null strategy using a bacterial artificial chromosome (BAC) to remove the entire genomic sequence of the target gene, a KO-first strategy that excised exons 4–11 flanked by the loxP sites with the help of Cre recombinase, and CRISPR–Cas9-mediated knockout targeting exon 2 and 3 of the RHBDF1 gene. The phenotypes displayed by the knockout mice from the three strategies were strikingly different.…”

“…Following their previous finding that targeting exon 3 of Ctnnb1 resulted in exon skipping and in-frame transcript [ 15 ], Mou and colleagues studied the possibility of exon skipping in mice via tail vein injection of plasmid expressing Cas9 and sgRNA. RT-PCR detected a PCR band corresponding to the transcript with exon skipped, and immunohistochemistry also found a small number of hepatocytes with nuclear β-catenin, indicating the occurrence of exon skipping and residual protein expression in vivo [ 37 ].…”

Diet-induced rewiring of the Wnt gene regulatory network connects aberrant splicing to fatty liver and liver cancer in DIAMOND mice

“…In the nucleus, β-catenin interacts with LEF/TCF transcription factors to regulate gene expression. Recent reports have demonstrated that similar activation of β-catenin, either via engineered deletions or CRISPR targeting of Ctnnb1 exon 3 in the liver, is sufficient to generate tumours in mice 7 , 8 .…”