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

Summary

This protocol presents the use of CRISPR SunTag-p65-HSF1 (SPH) in adipocytes (AdipoSPH) as an alternative strategy to adeno-associated virus (AAV) for investigating beige fat biology. In vivo injection of AAV-carrying sgRNA targeting the endogenous Prdm16 gene is sufficient to induce beige fat development and enhance the thermogenic gene program.

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 adeno-associated 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.

Introduction

Beige (or brite) adipocytes are uncoupling protein 1 (UCP1)-expressing and mitochondrial-rich adipocytes that reside within white adipose tissue (WAT) depots. Beige fat emerges from a subset of adipocyte progenitors or mature white adipocytes in response to cold exposure and other stimuli^1,2. Beige adipocytes can convert energy into heat in a UCP1-dependent or independent manner³. Regardless of its thermogenic function, beige fat can also improve metabolic health by other means, such as the secretion of adipokines and anti-inflammatory and anti-fibrotic activities. Studies in mice and h....

Protocol

Animal studies were performed in accordance with the University of Campinas Guide for the Care and Use of Laboratory Animals (protocol CEUA #5810-1/2021).

1. Molecular cloning

1. Design of single guide RNAs (sgRNAs)
   1. Design sgRNAs for CRISPR activation using CHOPCHOP, available at https://chopchop.cbu.uib.no/, or any other suitable tool. Use the following parameters to design sgRNA targeting the Prdm16 gene: Target: Prdm16; In: Mus musculus; Using: Crispr/Cas9; For: Activation.
      NOTE: Design sgRNAs for each region of interest spread over a 200 bp upstream transcription start site (TSS) region....

Results

AdipoSPH mice were developed by breeding SPH and Adipoq-Cre mouse strains. Both mouse strains were in a hybrid C57BL6J-DBA/2J background (according to the commercial supplier; see Table of Materials). The SPH mouse lineage was originally described by Zhou et al.¹⁴.

In vivo beige adipocyte development through AdipoSPH-mediated Prdm16 overexpression
To evaluate the capacity of the model described in this st.......

Discussion

One of the most useful non-editing applications of CRISPR technology is the interrogation of gene function through the activation of endogenous genes using CRISPRa systems⁶. SPH is a powerful CRISPRa that was originally described to induce the conversion of astrocytes into active neurons by targeting several neurogenic genes¹⁴. In this study, AdipoSPH was demonstrated to be a suitable tool for investigating beige fat biology by activating the expression of endogenous Prdm16.......

Materials

Name	Company	Catalog Number	Comments
3,3',5-Triiodo-L-thyronine	Sigma-Aldrich	T2877
3-Isobutyl-1-methylxanthine	Sigma-Aldrich	I5879
AAVpro 293T Cell Line	Takarabio	632273
Amicon Ultra Centrifugal Filter	Merckmillipore	UFC510008	100 KDa
Dexamethasone	Sigma-Aldrich	D1756
Dulbecco's Modification of Eagles Medium (DMEM)	Corning	10-017-CV
Dulbecco's Modified Eagle Medium (DMEM) F-12, GlutaMAX™ supplement	Gibco	10565-018	high concentrations of glucose, amino acids, and vitamins
Dulbecco's phosphate buffered saline (DPBS)	Sigma-Aldrich	D8662
Excelta Self-Opening Micro Scissors	Fisher Scientific	17-467-496
Fetal bovine serum	Sigma-Aldrich	F2442
Fisherbrand Cell Scrapers (100 pk)	Fisher Scientific	08-100-241
Fisherbrand High Precision Straight Tapered Ultra Fine Point Tweezers/Forceps	Fisher Scientific	12-000-122
Fisherbrand Sharp-Pointed Dissecting Scissors	Fisher Scientific	08-940
Glycerol	Sigma-Aldrich	G5516
HEPES	Sigma-Aldrich	H3375-25G
Hexadimethrine bromide (Polybrene)	Sigma-Aldrich	H9268
Indomethacin	Sigma-Aldrich	I7378
Insulin	Sigma-Aldrich	I9278
LigaFast Rapid DNA Ligation System	Promega	M8225
Maxiprep purification kit	Qiagen	12162
Microliter syringe	Hamilton	80308	Model 701
NEB 10-beta/Stable	New England Biolabs	C3019H	E. coli competent cells
pAAV2/8	Addgene	112864
pAAV-U6-gRNA-CBh-mCherry	Addgene	91947
pAdDeltaF6	Addgene	112867
PEG 8000	Sigma-Aldrich	89510
Penicillin/streptomycin	Gibco	15140-122
Polyethylenimine	Sigma-Aldrich	23966	Linear, MW 25000
Povidone-iodine	Rioquímica	510101303	Antiseptic
Rosiglitazone	Sigma-Aldrich	R2408
SacI enzyme	New England Biolabs	R0156
Surgical Design Premier Adson Forceps	Fisher Scientific	22-079-741
Syringe	Hamilton	475-40417
T4 DNA Ligase	Promega	M180B
T4 DNA ligase buffer	New England Biolabs	B0202S
T4 PNK enzyme kit	New England Biolabs	M0201S
Tramadol Hydrochloride	SEM	43930
Vidisic Gel	Bausch + Lomb	99620