Effect of CRISPR activation of Zbtb7b, Vps72, Gba, or Mrpl9 in liver tumorigenesis in mice. 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. Overall design: 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).

肝细胞癌（Hepatocellular carcinomas, HCCs）可发生染色体扩增，进而导致基因表达水平升高，但此类改变的生物学影响迄今尚未明确。本研究旨在通过在小鼠肝脏中开展CRISPR激活（CRISPR activation, CRISPRa）筛选，鉴定上述染色体扩增位点上的癌症驱动基因。我们分析了癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据库中肝细胞癌的拷贝数扩增及表达上调基因，随后利用dCas9敲入小鼠，针对筛选出的前51个候选基因开展了体内CRISPRa致瘤筛选实验。最终鉴定出Zbtb7b、Vps72、Gba及Mrpl9作为潜在的肝脏肿瘤驱动基因。进一步分析显示，VPS72、GBA或MRPL9的高表达（而非ZBTB7B）与癌症基因组图谱队列中肝细胞癌患者的不良生存预后显著相关。

为验证上述基因在驱动肿瘤发生中的功能，我们向小鼠注射了携带靶向Vps72、Gba或Mrpl9的向导RNA（guide RNA, gRNA）的单组分CRISPRa质粒。与非靶向对照相比，该处理显著缩短了小鼠的生存期，并诱发了广泛的肝脏肿瘤病变。我们对这些肿瘤组织进行了RNA测序（RNA sequencing, RNA-seq），以解析这些靶基因激活所引发的转录组学变化。

总体实验设计：为阐明Vps72、Gba、Mrpl9及Zbtb7b的激活如何驱动肿瘤发生，我们向表达dCas9的小鼠注射了单质粒载体，该载体包含三部分元件：(1) 用于驱动基础增殖亢进的Myc序列；(2) 用于介导靶基因激活的转录激活子序列；(3) 单条靶向Zbtb7b（sg-Zbtb7b）、Vps72（sg-Vps72）、Gba（sg-Gba）、Mrpl9（sg-Mrpl9）的向导RNA，或非靶向对照向导RNA（sg-NT）。随后，我们对如下两组小鼠的肝脏肿瘤组织进行了RNA测序：第一组为注射了sg-NT（N=5）或sg-Vps72（N=5）的表达dCas9的白化（Albino, Alb）小鼠；第二组为注射了sg-NT（N=6）、sg-Zbtb7b（N=5）、sg-Gba（N=5）或sg-Mrpl9（N=5）的表达dCas9的黑色（Black, Bl）小鼠。