Liver Cancer Gene Discovery Using Gene Targeting, Sleeping Beauty, and CRISPR/Cas9

Abstract

Hepatocellular carcinoma (HCC) is a devastating and prevalent cancer with limited treatment options. Technological advances have enabled genetic screens to be employed in HCC model systems to characterize genes regulating tumor initiation and growth. Relative to traditional methods for studying cancer biology, such as candidate gene approaches or expression analysis, genetic screens have several advantages: they are unbiased, with no a priori selection; can directly annotate gene function; and can uncover gene-gene interactions. In HCC, three main types of screens have been conducted and are reviewed here: (1) transposon-based mutagenesis screens, (2) knockdown screens using RNA interference (RNAi) or the CRISPR/Cas9 system, and (3) overexpression screens using CRISPR activation (CRISPRa) or cDNAs. These methods will be valuable in future genetic screens to delineate the mechanisms underlying drug resistance and to identify new treatments for HCC.

Figure 1:. Mouse models to examine specific genes involved in the initiation and progression of hepatocellular carcinoma.

(A) Examples of traditional genetically engineered mouse models (GEMMs). Transgenic and knock-in mice express oncogenes in the liver, often using liver-specific promoters (Top). Conditional expression systems such as the tTA/TRE system, in which administration of doxycycline turns off expression (middle). Loss-of-function models target tumor suppressor genes for mutations such as deletion of an exon (bottom). (B) The hydrodynamic tail vein injection (HTVI) technique leads to expression of oncogene cDNA (top) or knock-down of tumor suppressors using shRNA (bottom) in hepatocytes. The transposon systems enable integration and stable expression of the oncogenic sequences in a subset of the hepatocytes.

Figure 2:. Transposon-based mutagenesis systems.

The transposase enzyme mobilizes an engineered mutagenic transposon (e.g. T2/onc), which can disrupt tumor suppressor gene expression or activate oncogene expression, according to the position and the orientation of its insertion. Liver-specific expression of the transposase is regulated by albumin promoter-driven Cre recombinase.

Figure 3:. Genetic screens performed by hydrodynamic tail vein injection (HTVI) of plasmid pools.

(A) RNAi screens by HDI of a pool of plasmids, each carrying a unique shRNA sequence (top) or a cDNA expression vector (bottom). The shRNA or cDNA linked to the resultant tumors is determined by sequencing the shRNA coding sequence or the unique barcode linked to each cDNA. (B) CRISPR/Cas9 screening systems use either a wild type Cas9 enzyme to cleave target gene DNA (for loss-of-function studies, top), or a dead Cas9 (dCas9) that binds to transcriptional activators (to enhance target gene expression, bottom).