Abstract Backgrounds and Aims: Hepatocellular carcinoma (HCC) is a deadly cancer and its mortality rate ranks third among all cancer types worldwide. To comprehensively understand its genetic drivers, over 500 HCC genomes have been sequenced but they reveal the existence of thousands of infrequently mutated genes. This intra- and inter-tumor heterogeneity, combined with large number of passenger mutations, has made it difficult to identify the true drivers of HCC. In addition, sorafenib is the currently only FDA-approved molecular target drug in HCC but its anti-tumor effect is very limited. To overcome these problems, we performed two forward genetic screens using recently innovated whole-genome pooled CRISPR/Cas library. Methods: Two human liver cancer cell lines, Huh-7 and SNU-398, were lentivirally transduced with Geckov2 library and transduced cells were selected with puromycin. First, transduced cells were orthotopically injected into the liver of the Nude mice and monitored for tumor formation. To identify potential tumor suppressor genes (TSGs) in the liver, gRNAs enriched in the orthotopic liver tumors were determined by sequencing of gDNA in these tumors. Next, transduced cells were treated with vehicle or sorafenib in vitro for 14 days. To identify potential targets which inhibition show synergistic anti-tumor effect with sorafenib, we determined gRNAs depleted in cells with sorafenib treatment compared to cells with vehicle treatment. Results: In the first in vivo screening, we identified 62 genes targeted by 2 or more highly enriched gRNAs in Huh-7 cells-derived tumors and 47 genes in SNU-398 cells-derived tumors. Twenty-nine genes were common including 4 miRNAs. TCGA analysis revealed that HCC patients with mutation or copy number alteration in any of these genes showed worse overall survival and disease free survival compared to HCC patients without them. In addition, gene expression levels of 3 candidate TSGs, BPHL, CTSZ and PLCG2 were significantly negatively correlated with poor survival of HCC patients. In the second in vitro screening, CYP7B1, HS3ST5, NLRC4, and Mir-1237 were identified as potential drug targets in combination with sorafenib in HCC. Conclusion: Whole-genome pooled CRISPR/Cas library screens discovered potential new hepatocellular carcinoma drivers and drug targets in combination with sorafenib. Citation Format: Takahiro Kodama, Justin Y. Newberg, Nancy A. Jenkins, Tetsuo Takehara, Neal G. Copeland. Whole-genome pooled CRISPR/Cas library screens to identify new hepatocellular carcinoma drivers and drug targets in combination with sorafenib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 407. doi:10.1158/1538-7445.AM2017-407