CRISPR-Cas9 Screening Reveals Microproteins Regulating Adipocyte Proliferation and Lipid Metabolism [RNA-seq]
Small open reading frames (smORFs) encode microproteins that play crucial roles in various biological processes, yet their functions in adipocyte biology remain largely unexplored. In a previous study, we identified thousands of smORFs in white and brown adipocytes derived from the stromal vascular fraction (SVF) of mice using ribosome profiling (Ribo-Seq). Here, we expand on this work by identifying additional smORFs related to adipocytes using the in vitro 3T3-L1 preadipocyte model. To systematically investigate the functional relevance of these smORFs, we designed a custom CRISPR/Cas9 guide RNA (sgRNA) library and screened for smORFs influencing adipocyte proliferation and differentiation. Through a dropout screen and fluorescence-assisted cell sorting (FACS) of lipid droplets, we identified dozens of smORFs that regulate either cell proliferation or lipid accumulation. Among these, we validated a novel microprotein as a key regulator of adipocyte differentiation. These findings highlight the potential of CRISPR/Cas9-based screening to uncover functional smORFs and provide a framework for further exploration of microproteins in adipocyte biology and metabolic regulation. Overall design: Riboseq and RNA seq profiling of undifferentiated and differentiated 3T3-L1 Cells. 3T3-L1 cells are induced to differentiate into mature adipoctes through adding a cocktail containing insulin, IBMX, and dexamethasone. Differentiated adipocytes were collected at Days 3 and 7.