Oral Presentation ESA-SRB-ANZOS 2025 in conjunction with ENSA

Genome-wide CRISPR screen identifies C1orf35 as a novel regulator of hepatic lipid metabolism (127875)

Li Dong 1 , Julie Nigro 2 , Zhili Cheng 1 , Xueyi Dong 3 , Marco Herold 3 , Kumar Visvanathan 2 , Magdalene Montgomery 1
  1. Department of Anatomy & Physiology, University of Melbourne, Melbourne, VIC, Australia
  2. Department of Medicine, University of Melbourne, Melbourne, VIC, Australia
  3. Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia

Metabolic dysfunction-associated steatotic liver disease (MASLD) has served as a major driver of Hepatocellular carcinoma (HCC) due to its great potential for increasing cirrhosis. MASLD is characterised by defective lipid metabolism, which causes hepatic steatosis and inflammation, key events that precede and contribute to fibrosis and are essential for disease progression. Hence, tackling early metabolic defects (i.e., inhibiting hepatic steatosis) is likely to prevent the development of MASH and metabolic co-morbidities.

To systematically uncover novel genes required for hepatic lipid accumulation, we performed five independent, pooled genome-wide screens in HepG2 cells using the human GeCKO CRISPR library. Cells were treated with or without excess fatty acid (FA) to mimic chronic lipid overload, followed by Bodipy lipid staining and fluorescence-activated cell sorting. In Control cells, we selected the highest 10% of cells to identify mutants that retained high levels of lipid due to defects in lipid processing. In contrast, for the FA-treated cells, we selected the lowest 10% of cells, thereby screening for cells bearing mutations in genes necessary for FA-induced lipid accumulation.

Using single gRNA knockout combined with functional assessment of lipid accumulation, we identified C1orf35 (Chromosome 1 open reading frame 35) as a novel regulator of hepatic lipid metabolism.

Deletion of C1orf35 in HepG2 cells reduces lipid accumulation by >80% by suppressing lipogenesis and fatty acid uptake and enhancing triglyceride secretion. The restoration of C1orf35 expression in C1orf35-/- cells brings the triglyceride content back to baseline, indicating a dynamic response to changes in C1orf35 expression. Mechanistically, C1orf35 acts as a master transcriptional regulator of key lipid metabolism enzymes, including SREBP1, FASN and CD36. The role of C1orf35 in metabolic reprogramming was further validated in 3D liver organoids derived from patients. Future studies aim to investigate whether the deletion of C1orf35 in the liver may confer protective benefits in MASLD.