Rationale: Genome-wide association studies have identified genetic loci associated with insulin resistance (IR) but pinpointing the causal genes of a risk locus has been challenging. Objective: To identify candidate causal genes for IR, we screened regional and biologically plausible genes (16 in total) near the top 10 IR-loci in risk-relevant cell types, namely preadipocytes and adipocytes. Methods and Results: We generated 16 human Simpson-Golabi-Behmel syndrome preadipocyte knockout lines each with a single IR-gene knocked out by lentivirus-mediated CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system. We evaluated each gene knockout by screening IR-relevant phenotypes in the 3 insulin-sensitizing mechanisms, including adipogenesis, lipid metabolism, and insulin signaling. We performed genetic analyses using data on the genotype-tissue expression portal expression quantitative trait loci database and accelerating medicines partnership type 2 diabetes mellitus Knowledge Portal to evaluate whether candidate genes prioritized by our in vitro studies were expression quantitative trait loci genes in human subcutaneous adipose tissue, and whether expression of these genes is associated with risk of IR, type 2 diabetes mellitus, and cardiovascular diseases. We further validated the functions of 3 new adipose IR genes by overexpression-based phenotypic rescue in the Simpson-Golabi-Behmel syndrome preadipocyte knockout lines. Twelve genes, PPARG, IRS-1, FST, PEPD, PDGFC, MAP3K1, GRB14, ARL15, ANKRD55, RSPO3, COBLL1, and LYPLAL1, showed diverse phenotypes in the 3 insulin-sensitizing mechanisms, and the first 7 of these genes could affect all the 3 mechanisms. Five out of 6 expression quantitative trait loci genes are among the top candidate causal genes and the abnormal expression levels of these genes (IRS-1, GRB14, FST, PEPD, and PDGFC) in human subcutaneous adipose tissue could be associated with increased risk of IR, type 2 diabetes mellitus, and cardiovascular disease. Phenotypic rescue by overexpression of the candidate causal genes (FST, PEPD, and PDGFC) in the Simpson-Golabi-Behmel syndrome preadipocyte knockout lines confirmed their function in adipose IR. Conclusions: Twelve genes showed diverse phenotypes indicating differential roles in insulin sensitization, suggesting mechanisms bridging the association of their genomic loci with IR. We prioritized PPARG, IRS-1, GRB14, MAP3K1, FST, PEPD, and PDGFC as top candidate genes. Our work points to novel roles for FST, PEPD, and PDGFC in adipose tissue, with consequences for cardiometabolic diseases.

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