Investigation of Beige Fat Biology and Metabolism Using the CRISPR SunTag-p65-HSF1 Activation System

Author(s)

Bladimir, Fernando ; de Oliveira, Vanessa ; Henrique, Carlos

• Document type: Journal article
• Source: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS; v. N/A, n. 191, p. 1-pg., 2023-01-01.

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has prompted a revolution in biology, and recent tools have been applied far beyond the originally described gene editing. The CRISPR activation (CRISPRa) system combines the catalytically inactive Cas9 (dCas9) protein with distinct transcription modules to induce endogenous gene expression. SunTag-p65-HSF1 (SPH) is a recently developed CRISPRa technology that combines components of synergistic activation mediators (SAMs) with the SunTag activators. This system allows the overexpression of single or multiple genes by designing a customized single-guide RNA (sgRNA). In this study, a previously developed SPH mouse was used to generate a conditional mouse expressing SPH in adipocytes (adiponectin Cre lineage), named AdipoSPH. To induce a white-to-beige fat (browning) phenotype, an adenoassociated virus (AAV) carrying sgRNA targeting the endogenous Prdm16 gene (a well-established transcription factor related to brown and beige fat development) was injected into the inguinal white adipose tissue (iWAT). This mouse model induced the expression of endogenous Prdm16 and activated the thermogenic gene program. Moreover, in vitro SPH-induced Prdm16 overexpression enhanced the oxygen consumption of beige adipocytes, phenocopying the results of a previous Prdm16 transgenic mouse model. Thus, this protocol describes a versatile, cost-effective, and time-effective mouse model for investigating adipose tissue biology. (AU)

• FAPESP's process: 19/15025-5 - Unveiling the PRDM16 transcriptional complex related to the inhibition of ceramides synthesis genes in adipocytes
• Grantee: Carlos Henrique Grossi Sponton
• Support Opportunities: Research Grants - Young Investigators Grants
• FAPESP's process: 20/14725-0 - Unveiling the PRDM16 transcriptional complex related to the inhibition of ceramides synthesis genes in adipocytes
• Grantee: Fernando Bladimir Valdivieso Rivera
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 21/11841-2 - Unveiling the PRDM16 transcriptional complex related to the inhibition of ceramides synthesis genes in adipocytes
• Grantee: Vanessa de Oliveira Furino
• Support Opportunities: Scholarships in Brazil - Technical Training Program - Technical Training
• FAPESP's process: 20/09308-1 - Unveiling the PRDM16 transcriptional complex related to the inhibition of ceramides synthesis genes in adipocytes
• Grantee: Carlos Henrique Grossi Sponton
• Support Opportunities: Scholarships in Brazil - Young Researchers