Intrahepatic cholangiocarcinoma (ICC) is the second most common malignancy among primary liver cancers, with an increasing overall incidence and poor prognosis. The intertumoral and intratumoral heterogeneity of ICC makes it difficult to find efficient drug therapies. Therefore, it is essential to identify tumor suppressor genes and oncogenes that induce ICC formation and progression. Here, we performed CRISPR/Cas9-mediated genome-wide screening in a liver-specific Smad4/Pten knockout mouse model (Smad4 ;Pten ;Alb-Cre, abbreviated as SPC), which normally generates ICC after 6 months, and detected that mutations in Trp53, Fbxw7, Inppl1, Tgfbr2, or Cul3 markedly accelerated ICC formation. To illustrate the potential mechanisms, we conducted transcriptome sequencing and found that multiple receptor tyrosine kinases were activated, which mainly upregulated the PI3K pathway to induce cell proliferation. Remarkably, the Cul3 mutation stimulated cancer progression mainly by altering the immune microenvironment, whereas other mutations promoted the cell cycle. Moreover, Fbxw7, Inppl1, Tgfbr2, and Trp53 also affect inflammatory responses, apelin signaling, mitotic spindles, ribosome biogenesis, and nucleocytoplasmic transport pathways, respectively. We further examined FDA-approved drugs for the treatment of liver cancer and performed high-throughput drug screening of the gene-mutant organoids. Different drug responses and promising drug therapies, including chemotherapy and targeted drugs, have been discovered for ICC.