Endocrine Abstracts

FK506-binding protein-like (FKBPL) is a member of the immunophilin protein family, implicated in key biological processes including inflammation, angiogenesis, and metabolic regulation. Recent findings have unveiled a novel role for FKBPL in adipose tissue function and the pathophysiology of obesity. To explore its transcriptional upregulation, five single guide RNAs (sgRNAs) were designed to target the FKBPL promoter using a CRISPR activation (CRISPRa) system based on a nuclease-inactive Cas 9 fused to the VPR transcriptional activator (dCas9-VPR). This system was delivered to 3T3-L1 pre-adipocytes and mature adipocytes via either plasmid DNA (pDNA) or in vitro–transcribed mRNA using Lipofectamine-mediated transfection. Quantitative PCR (qPCR) was employed to assess FKBPL mRNA levels, while protein expression was evaluated through Western blotting, flow cytometry, and ELISA. In pre-adipocytes, mRNA-based CRISPRa induced robust transcriptional activation, with the most effective sgRNA yielding a greater than 200% increase in FKBPL mRNA expression, accompanied by a corresponding elevation in protein levels. Intracellular FKBPL was markedly upregulated, as confirmed by flow cytometry, and secreted protein levels peaked at 24 hours post-transfection before declining. Although pDNA-based delivery also enhanced FKBPL expression, its efficacy was comparatively limited. In differentiated adipocytes, transfection efficiency was substantially reduced, likely due to lipid droplet accumulation restricting cytoplasmic uptake. This study constitutes the first successful demonstration of CRISPRa-mediated upregulation of FKBPL in vitro, providing a foundational platform for future in vivo studies aimed at evaluating FKBPL as a potential therapeutic target in metabolic diseases such as obesity.