Effect of CRISPR activation of Zbtb7b, Vps72, Gba, or Mrpl9 in liver tumorigenesis in mice

Hepatocellular carcinomas (HCCs) undergo chromosomal amplifications with resultant increases in gene expression levels, yet the impact of these changes remains largely unknown. We sought to identify cancer driver genes at these genetic loci using CRISPR activation (CRISPRa) screening in mouse livers. We examined The Cancer Genome Atlas data for copy number-amplified and upregulated genes in HCCs. We then conducted in vivo CRISPRa tumorigenesis screens targeting the top 51 genes using dCas9 knock-in mice. Genes Zbtb7b, Vps72, Gba, and Mrpl9 emerged as putative drivers of liver tumorigenesis. Furthermore, high levels of expression of VPS72, GBA, or MRPL9, but not ZBTB7B, correlated with worse survival of HCC patients from the The Cancer Genome Atlas. For validation of the role of these genes in driving tumorigenesis, mice were injected with single CRISPRa plasmids containing Vps72-, Gba-, or Mrpl9-targeting gRNAs. This led to decreased survival and extensive liver tumorigenesis compared to non-targeting controls. We performed RNA sequencing of these tumors to understand the transcriptomic changes caused by activation of these genes. To understand how activation of Vps72, Gba, Mrpl9, and Zbtb7b could drive tumorigenesis, we injected dCas9-expressing mice with a single plasmid containing (1) Myc to drive baseline hyperproliferation, (2) transcriptional activator sequences for target gene activation, and (3) a single gRNA targeting Zbtb7b (sg-Zbtb7b), Vps72 (sg-Vps72), Gba (sg-Gba), Mrpl9 (sg-Mrpl9), or non-targeting control (sg-NT). We then performed RNA sequencing of liver tumors from dCas9-expressing Albino (Alb) mice injected with sg-NT (N=5) or sg-Vps72 (N=5) or dCas9-expressing Black (Bl) mice injected with sg-NT (N=6), sg-Zbtb7b (N=5), sg-Gba (N=5), or sg-Mrpl9 (N=5).