CROP-Seq combines gene silencing using CRISPR interference with single-cell RNA sequencing. Here, we applied CROP-Seq to study adipogenesis and adipocyte biology. Human preadipocyte SGBS cell line expressing KRAB-dCas9 was transduced with a sgRNA library. Following selection, individual cells were captured using microfluidics at different timepoints during adipogenesis. Bioinformatic analysis of transcriptomic data was used to determine the knock-down effects, the dysregulated pathways, and to predict cellular phenotypes. Single-cell transcriptomes recapitulated adipogenesis states. For all targets, over 400 differentially expressed genes were identified at least at one timepoint. As a validation of our approach, the knock-down of PPARG and CEBPB (which encode key proadipogenic transcription factors) resulted in the inhibition of adipogenesis. Gene set enrichment analysis generated hypotheses regarding the molecular function of novel genes. MAFF knock-down led to downregulation of transcriptional response to proinflammatory cytokine TNF-a in preadipocytes and to decreased CXCL-16 and IL-6 secretion. TIPARP knock-down resulted in increased expression of adipogenesis markers. In summary, this powerful, hypothesis-free tool can identify novel regulators of adipogenesis, preadipocyte and adipocyte function associated with metabolic disease.
View details for DOI 10.1152/ajpcell.00148.2023
View details for PubMedID 37486064