Abstract Objective CROP-Seq combines gene silencing using CRISPR interference (CRISPRi) with single-cell RNA sequencing (scRNA-Seq) to conduct a functional reverse genetic screen of novel gene targets associated with adipocyte differentiation or function, with single-cell transcriptomes as the readout. Methods We created a human preadipocyte SGBS cell line with stable expression of KRAB-dCas9 for CRISPRi-mediated gene knock-down. This line was transduced with a lentiviral library of sgRNAs targeting 6 genes of interest (3 sgRNAs / gene, 18 sgRNAs), 6 positive control genes (3 sgRNAs / gene, 18 sgRNAs), and non-targeting control sgRNAs (4 sgRNAs). Transduced cells were selected and differentiated, and individual cells were captured using microfluidics at day 0, 4 and 8 of adipogenic differentiation. Next, expression and sgRNA libraries were created and sequenced. Bioinformatic analysis of resulting scRNA-Seq expression data was used to determine the effects of gene knock-down and the dysregulated pathways, and to predict cellular phenotypes. Results Single-cell transcriptomes obtained from SGBS cells following CRISPRi recapitulate different states of differentiation from preadipocytes to adipocytes. We confirmed successful knock-down of targeted genes. Transcriptome-wide changes were observed for all targeted genes, with over 400 differentially expressed genes identified per gene at least at one timepoint. Knock-down of known adipogenesis regulators PPARG and CEBPB inhibited adipogenesis. Gene set enrichment analyses revealed molecular processes for adipose tissue differentiation and function for novel genes. MAFF knock-down led to a downregulation of transcriptional response to proinflammatory cytokine TNF-α in preadipocytes. TIPARP knock-down resulted in an increase in the expression of a beiging marker UCP1 at D8 of adipogenesis. Conclusions The CROP-Seq system in SGBS cells can determine the consequences of target gene knock-down at the transcriptome level. This powerful, hypothesis-free tool can identify novel regulators of adipogenesis, preadipocyte and adipocyte function associated with metabolic disease. Highlights CRISPR interference screen coupled with single-cell RNA sequencing (CROP-Seq) Parallel screening of 12 genes in human SGBS adipocytes and preadipocytes Uncovered novel regulators of adipogenesis and adipocyte function Graphical abstract

CROP-Seq: a single-cell CRISPRi platform for characterizing candidate genes relevant to metabolic disorders in human adipocytes