A single-cell CRISPRi platform for characterizing candidate genes relevant to metabolic disorders in human adipocytes.
A single-cell CRISPRi platform for characterizing candidate genes relevant to metabolic disorders in human adipocytes.
复制标题
用于表征与人类脂肪细胞代谢紊乱相关的候选基因的单细胞 CRISPRi 平台。
DOI：
10.1152/ajpcell.00148.2023
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Li,Jiehan
中科院分区：
文献类型：
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作者：
Bielczyk-Maczynska,Ewa;Sharma,Disha;Blencowe,Montgomery;SalibaGustafsson,Peter;Gloudemans,MichaelJ;Yang,Xia;Carcamo-Orive,Ivan;Wabitsch,Martin;Svensson,KatrinJ;Park,ChongY;Quertermous,Thomas;Knowles,JoshuaW;Li,Jiehan
CROP-Seq将使用CRISPR干扰的基因沉默与单细胞RNA测序相结合。在这里，我们应用CROP-Seq研究脂肪形成和脂肪细胞生物学。用sgRNA文库转导表达KRAB-dCas 9的人前脂肪细胞SGBS细胞系。选择后，在脂肪形成期间的不同时间点使用微流体捕获单个细胞。使用转录组数据的生物信息学分析来确定敲除效应、失调的途径并预测细胞表型。单细胞转录组再现脂肪形成状态。对于所有靶标，至少在一个时间点鉴定出超过400个差异表达基因。作为我们方法的验证，PPARGandCEBPB（编码关键的前脂肪形成转录因子）的敲低导致脂肪形成的抑制。基因集富集分析表明，MAFF基因敲除可下调前脂肪细胞对促炎细胞因子TNF-α的转录反应，减少CXCL-16和IL-6的分泌，TIPARP基因敲除可增加脂肪形成标志物的表达。总之，这个强大的，无假设的工具可以识别新的调节脂肪形成，前脂肪细胞，脂肪细胞功能与代谢diseases.NEW相关的努力导致许多基因组位点的识别与代谢性状，其中许多是与脂肪组织功能相关。然而，确定致病基因及其在代谢中的作用机制是一个耗时的过程。在这里，我们使用一种方法来确定候选基因敲低多个基因在同一时间在人类细胞模型的脂肪形成的转录结果。
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 knockdown 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 knockdown ofPPARGandCEBPB(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.MAFFknockdown led to downregulation of transcriptional response to proinflammatory cytokine TNF-α in preadipocytes and to decreased CXCL-16 and IL-6 secretion.TIPARPknockdown 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.NEW & NOTEWORTHYGenomics efforts led to the identification of many genomic loci that are associated with metabolic traits, many of which are tied to adipose tissue function. However, determination of the causal genes, and their mechanism of action in metabolism, is a time-consuming process. Here, we use an approach to determine the transcriptional outcome of candidate gene knockdown for multiple genes at the same time in a human cell model of adipogenesis.