Abstract
CRISPR/Cas9 has revolutionized the genome-editing field. So far, successful application in human adipose tissue has not been convincingly shown. We present a method for gene knockout using electroporation in preadipocytes from human adipose tissue that achieved at least 90% efficiency without any need for selection of edited cells or clonal isolation. We knocked out the FKBP5 and PPARG genes in preadipocytes and studied the resulting phenotypes. PPARG knockout prevented differentiation into adipocytes. Conversely, deletion of FKBP51, the protein coded by the FKBP5 gene, did not affect adipogenesis. Instead, it markedly modulated glucocorticoid effects on adipocyte glucose metabolism and, furthermore, we show some evidence of altered transcriptional activity of glucocorticoid receptors. This has potential implications for the development of insulin resistance and type 2 diabetes. The reported method is simple, easy to adapt, and enables the use of human primary preadipocytes instead of animal adipose cell models to assess the role of key genes and their products in adipose tissue development, metabolism and pathobiology.
Highlights
Adipose tissue is widely regarded as an endocrine organ that plays a central role in both obesity and insulin resistance[1]
To check whether our method is suitable for editing other genes, we knocked out a well-established adipocyte-specific gene, peroxisome proliferator-activated receptor gamma (PPARG), a master regulator of adipogenesis
Western blot data showed that FK506 binding protein 51 (FKBP51) protein levels were undetectable in FK-G57 knockout cultures compared to wild type on days 0, 7, and 14 of differentiation (n = 3, Fig. 2c)
Summary
Adipose tissue is widely regarded as an endocrine organ that plays a central role in both obesity and insulin resistance[1]. Identification of genes and functional assessment of their corresponding proteins involved in such pathways could help discover novel disease mechanisms that could be used for drug development. Different approaches such as pharmacological inhibition using receptor antagonists or neutralizing antibodies, as well as genetic manipulation using small interfering RNA-mediated knockdown[2] have been widely opted for studying the function of different gene products in human adipose cells. In another study, immortalized human brown preadipocytes were used to knockout genes using CRISPR/Cas[95] Both of these studies have used an expression vector to deliver the CRISPR components into the cells. To validate our method with other genes we knocked out peroxisome proliferator-activated receptor gamma (PPARG), a master regulator of adipogenesis
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