Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System

Summary

In this protocol, we describe the technical procedures to generate brown adipose tissue (BAT)-specific knockout mice leveraging a combined Cre-LoxP, CRISPR-Cas9, and adeno-associated virus (AAV) single-guide RNA (sgRNA) system. The described steps include the design of the sgRNAs, the preparation of the AAV-sgRNA particles, and the microinjection of AAV into the BAT lobes.

Abstract

Brown adipose tissue (BAT) is an adipose depot specialized in energy dissipation that can also serve as an endocrine organ via the secretion of bioactive molecules. The creation of BAT-specific knockout mice is one of the most popular approaches for understanding the contribution of a gene of interest to BAT-mediated energy regulation. The conventional gene targeting strategy utilizing the Cre-LoxP system has been the principal approach to generate tissue-specific knockout mice. However, this approach is time-consuming and tedious. Here, we describe a protocol for the rapid and efficient knockout of a gene of interest in BAT using a combined Cre-LoxP, CRISPR-Cas9, and adeno-associated virus (AAV) single-guide RNA (sgRNA) system. The interscapular BAT is located in the deep layer between the muscles. Thus, the BAT must be exposed in order to inject the AAV precisely and directly into the BAT within the visual field. Appropriate surgical handling is crucial to prevent damage to the sympathetic nerves and vessels, such as the Sultzer's vein that connects to the BAT. To minimize tissue damage, there is a critical need to understand the three-dimensional anatomical location of the BAT and the surgical skills required in the technical steps. This protocol highlights the key technical procedures, including the design of sgRNAs targeting the gene of interest, the preparation of AAV-sgRNA particles, and the surgery for the direct microinjection of AAV into both BAT lobes for generating BAT-specific knockout mice, which can be broadly applied to study the biological functions of genes in BAT.

Introduction

Obesity is increasing at a significant rate worldwide, leading to a broad spectrum of metabolic diseases^1,2,3. The adipose tissue is key to these pathologies. The two functionally distinct types of adipose tissue that exist are white adipose tissue (WAT), which stores excess calories, and brown adipose tissue (BAT) and its related beige/brite fat, which dissipate energy for thermogenesis. While BAT has been recognized for its energy-dissipating function, it also has endocrine functions via the production of bioactive molecules that regulate metabolism in distal organ....

Protocol

All the animal experiments and care procedures were approved by the Institutional Animal Care and Use Committee at Joslin Diabetes Center.

1. Screening effective sgRNAs in cultured cells

NOTE: To make the assay cost-effective, before packaging the sgRNAs into AAV particles, we recommend testing different sgRNAs in cultured cells via a lentivirus-based system for Cas9/sgRNA expression (Figure 1) and selecting the sgRNAs that give the highest knockdown efficiency for in vivo experiments.

1. CRISPR-Cas9 sgRNA design
   1. Design sgRNAs u....

Results

Throughout the above procedures, the precise delivery of AAV-sgRNA to the BAT is crucial to the success of the protocol. To maximize the effect of AAV-sgRNA and minimize the tissue damage during surgery, it is essential to understand the three-dimensional (3D) anatomical location of the BAT. As shown in Figure 3E, it is hard to identify the precise location of the BAT without exposing the tissue. However, excess BAT exposure may damage the tissue (Figure 3

Discussion

The existing published methods for AAV-mediated gene delivery to the interscapular BAT do not contain detailed pictures and videos describing the surgical techniques and approach for direct AAV injection into the BAT. In most of the published methods^33,34, the AAV is injected into the fat tissues surrounding the interscapular BAT instead of the BAT itself. Hence, there are several key steps in this protocol that determine the success of the study. These include t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chemicals, Peptides and Recombinant Proteins
OPTI-MEM serum-free medium	Invitrogen	31985
Polybrene Infection / Transfection Reagent	Sigma	TR-1003-G
TransIT-LT1 Transfection Reagent	Mirus	MIR 2300
Recombinant DNA
lentiCRISPR v2 vector	Addgene	52961
pAAV-U6-BbsI-gRNA-CB-EmGFP	Addgene	89060
pMD2.G envelope plasmid	Addgene	12259
psPAX2 packaging plasmid	Addgene	12260
Experimental Models: Cell Lines
HEK-293 cells	ATCC	CRL-1573
WT-1 cells	Developed in Tseng lab	PMID: 14966273
Experimental Models: Organisms/Strains
Cas9 knockin mice	Jackson Laboratories	24857
Ucp1-CRE mice	Jackson Laboratories	24670
Others
Banamine	Patterson	07-859-1323
Hamilton syringe	Hamilton	Model 710 RN Syringe
Hydrogen peroxide solution	Fisher Scientific	H325-500
Needle	Hamilton	32 gauge, small hub RN needle, needle point style 2
Suture 5-0 Unify PCL25 P-3 Undyed 18 inch Monofilament 12/Bx	Henry Schein	1273504
Suture 5-0 Unify Silk P-3 Black 18 inch 12/Bx	Henry Schein	1294522