Proof-of-concept for CRISPR/Cas9 gene editing in human preadipocytes: Deletion of FKBP5 and PPARG and effects on adipocyte differentiation and metabolism

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. Adipose tissue is widely regarded as an endocrine organ that plays a central role in both obesity and insulin resistance 1. Dysregulation of adipose tissue transcriptional pathways may contribute to significant changes in energy balance, glucose and lipid metabolism, and adipokine secretion, which in turn can influence the whole-body metabolic homeostasis. Thus, 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. However, the specificity and stability of such approaches are critical factors and may sometimes limit their use. The recent advancements in clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) technology have given rise to a precise and highly efficient method for gene editing in cells, tissues and whole organisms 3. However, its application to human adipose tissue is scarce. To the best of our knowledge, only one study reported using this technique in primary human adipose cells, but the phenotypic results following a single nucleotide substitution in the fat mass and obesity-associated gene were equivocal 4. In another study, immortalized human brown preadipocytes were used to knockout genes using CRISPR/Cas9 5. Both of these studies have used an expression vector to deliver the CRISPR components into the cells. However, there are some potential complications associated with the use of plasmid DNA, as mentioned in the discussion.

CRISPR-enhanced human adipocyte “browning” as cell therapy for metabolic disease

“…The electroporated cells were placed in complete media immediately following transfection, expanded, grown to confluence and differentiated into mature adipocytes for downstream applications. We found these methods improved the viability of preadipocytes and adipocytes and their ability to differentiate over methods reported while our manuscript was in preparation 44 .…”

Human adipose tissue gene expression of solute carrier family 19 member 3 (SLC19A3); relation to obesity and weight‐loss

“…The isolation of preadipocytes was performed as previously reported. 22 In brief, adipose tissue was digested with collagenase (1.2 mg/ml, from Clostridium histolyticum, Roche, Manheim, Germany) in Hank's medium (5.6 mM glucose, 4% BSA, 150 nM adenosine) for 60 min in a shaking water-bath. Isolated adipocytes were filtered through a 250 μm nylon mesh, and the media under the adipocytes was collected and centrifuged for 3 min at 1200 rpm for isolation of the and 4°C until complete phase separation.…”

“…SDS-PAGE and immunoblotting were performed with standard procedures. 22 Membranes were incubated overnight with anti-SLC19A3 (1:1000, 13407-1-AP, Proteintech, Manchester, UK) and thereafter with horseradish peroxidase-conjugated anti-rabbit (Cell Signalling Technologies) secondary antibody. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:3000, 5174, Cell Signalling) was used as a loading control protein.…”

Viral and Nonviral Transfer of Genetic Materials to Adipose Tissues: Toward a Gold Standard Approach

“…Importantly, no off-target mutations were detected and CRISPR/Cas9 did not affect the capacity to differentiate. In a second study, Kamble et al (62) electroporated human preadipocytes with a ribonucleoprotein complex of Cas9 and an sgRNA targeted to Pparg or Fkbp5. Impressively, treated cells achieved 90% efficiency and did not require selection or clonal isolation of edited cells.…”