International Session（Symposium）1(JSH・JSGE・JSGCS)
Thu. November 5th   9:00 - 11:20   Room 5: Portopia Hotel South Wing Ohwada A
IS-S1-2_H
 Genome-wide pooled CRISPR library screen identifies genetic mediators of hepatocyte lipotoxicity.
Takahiro Kodama1, Yuta Myojin1, Tetsuo Takehara1
1Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine
Backgrounds and aims: The prevalence of non-alcoholic steatohepatitis (NASH) is increasing worldwide. However, its pharmacotherapy has not yet been developed. To discover its new therapeutic targets, we performed a loss-of-function genetic screen and aimed to identify mediators of hepatocyte lipotoxicity caused by saturated fatty acid (SFA).
Method: Genome-wide pooled CRISPR library containing 90,709 gRNAs targeting 18,010 genes was lentivirally infected into Cas9-expressing human hepatoma cell line HLF-Cas9. Transduced cells were treated with either vehicle or palmitic acid (PA) in vitro for 3 weeks. Integrated gRNA sequences were PCR amplified and sequenced by next-generation sequencing. Alteration of gRNA abundance in transduced cells between pre- and post-treatment was compared between vehicle or PA treatment. Significantly enriched gRNAs and corresponding genes in PA-treated cells were identified by MAGeCK algorithm.
Result: Hundred forty-two genes were enriched in PA-treated cells and genes involved in the apoptosis pathway were highly enriched among them. Most significantly enriched genes were ACSL3, NF2, and INSIG1. CRISPR-mediated knockout of ACSL3 suppressed lipid droplet formation and significantly inhibited apoptosis induced by PA treatment in HLF cells. Consequently, ACSL3 deletion significantly reduced PA-mediated cytotoxicity. Mechanistically, ACSL3 deletion significantly downregulated genes involved in the lipogenesis process.
Conclusion: CRISPR library screen successfully identified multiple genes involved in SFA-mediated hepatocyte lipotoxicity. They might be a potential therapeutic target for NASH.
Index Term 1: hepatocyte lipoapotosis
Index Term 2: CRISPR library screen
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