Name: investigation of beige fat biology and metabolism using the crispr suntag-p65-hsf1 activation system
Uploaded: 2023-01-06T14:20:00
Description: Watch this Scientific Journal Video about Investigation of Beige Fat Biology and Metabolism Using the CRISPR SunTag-p65-HSF1 Activation System at JoVE.com

The authors thank the support received from Centro Multidisciplinar para Investiga&#231;&#227;o Biol&#243;gica na &#193;rea da Ci&#234;ncia em Animais de Laborat&#243;rio (Cemib), Unicamp, for the generation of AdipoSPH mice, the Inmmunometabolism and Cell Signaling Laboratory, and National Institute of Science and Technology on Photonics Applied to Cell Biology (INFABIC) for all experimental support. We thank the financial support from Sao Paulo Research Foundation (FAPESP): 2019/15025-5; 2020/09308-1; 2020/14725-0; 2021/11841-2.

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
<ol>
	<li>Design of single guide RNAs (sgRNAs)
	<ol>
		<li>Design sgRNAs for CRISPR activation using CHOPCHOP, available at https://chopchop.cbu.uib.no/,&#160;or any other suitable tool. Use the following parameters to design sgRNA targeting the Prdm16 gene: Target: Prdm16; In: Mus musculus; Using: Cri...

<ol>
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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...

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 stimuli1,2. 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 humans have shown that the induction of beige fat improves whole-body glucose and lipid homeostasis3. However, although our knowledge of beige fat biology has evolved rapidly in recent years, most of its metabolic benefits and related mechanisms are still not fully understood.
Clustered regularly interspaced short palindromic repeats (CRISPR) were first described in eukaryotic cells as a tool capable of generating a double-strand break (DSB) at a specific site in the genome through the nuclease activity of the Cas9 protein4,5. Cas9 is guided by a synthetic single-guide RNA (sgRNA) to target a specific genomic region, leading to a DNA DSB. In addition to using the nuclease Cas9 for editing purposes, CRISPR-Cas9 technology has evolved to be used as a sequence-specific gene regulation tool6. The development of a catalytically inactive Cas9 protein (dCas9) and the association of transcriptional modules capable of enhancing gene expression has given rise to CRISPR activation (CRISPRa) tools. Several CRISPRa systems have emerged, such as VP647,8, synergistic activation mediator (SAM)9, SunTag10,11, VPR12,13, and SunTag-p65-HSF1 (SPH)14, which combines the components of SAM and SunTag activators. It has recently been demonstrated that the induced expression of neurogenic genes in N2a neuroblasts and primary astrocytes is higher using SPH compared to other CRISPRa systems14, demonstrating SPH as a promising CRISPRa tool.
Here, we took advantage of a previously developed SPH mouse14 to generate a conditional mouse model expressing SPH specifically in adipocytes using the adiponectin Cre lineage (AdipoSPH). Using an adeno-associated virus (AAV) carrying the gRNA targeting the endogenous Prdm16 gene, browning (white to beige conversion) of inguinal WAT (iWAT) was induced to increase the expression of the thermogenic gene program. Moreover, in vitro Prdm16 overexpression enhanced oxygen consumption. Therefore, this protocol provides a versatile SPH mouse model for exploring the mechanisms of beige fat development within adipose tissue.

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			<td>3,3&#39;,5-Triiodo-L-thyronine</td>
			<td>Sigma-Aldrich</td>
			<td>T2877</td>
			<td></td>
		</tr>
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			<td>3-Isobutyl-1-methylxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>I5879</td>
			<td></td>
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		<tr>
			<td>AAVpro 293T Cell Line</td>
			<td>Takarabio</td>
			<td>632273</td>
			<td></td>
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			<td>Amicon Ultra Centrifugal Filter</td>
			<td>Merckmillipore</td>
			<td>UFC510008</td>
			<td>100 KDa</td>
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			<td>Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>D1756</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modification of Eagles Medium (DMEM)</td>
			<td>Corning</td>
			<td>10-017-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) F-12, GlutaMAX&#8482; supplement</td>
			<td>Gibco</td>
			<td>10565-018</td>
			<td>high concentrations of glucose, amino acids, and vitamins</td>
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		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (DPBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8662</td>
			<td></td>
		</tr>
		<tr>
			<td>Excelta Self-Opening Micro Scissors</td>
			<td>Fisher Scientific</td>
			<td>17-467-496</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Cell Scrapers (100 pk)</td>
			<td>Fisher Scientific</td>
			<td>08-100-241</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand High Precision Straight Tapered Ultra Fine Point Tweezers/Forceps</td>
			<td>Fisher Scientific</td>
			<td>12-000-122</td>
			<td></td>
		</tr>
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			<td>Fisherbrand Sharp-Pointed Dissecting Scissors</td>
			<td>Fisher Scientific</td>
			<td>08-940</td>
			<td></td>
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			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexadimethrine bromide (Polybrene)</td>
			<td>Sigma-Aldrich</td>
			<td>H9268</td>
			<td></td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Sigma-Aldrich</td>
			<td>I7378</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I9278</td>
			<td></td>
		</tr>
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			<td>LigaFast Rapid DNA Ligation System</td>
			<td>Promega</td>
			<td>M8225</td>
			<td></td>
		</tr>
		<tr>
			<td>Maxiprep purification kit&#160;</td>
			<td>Qiagen</td>
			<td>12162</td>
			<td></td>
		</tr>
		<tr>
			<td>Microliter syringe</td>
			<td>Hamilton</td>
			<td>80308</td>
			<td>Model 701</td>
		</tr>
		<tr>
			<td>NEB 10-beta/Stable&#160;</td>
			<td>New England Biolabs</td>
			<td>C3019H</td>
			<td>E. coli competent cells</td>
		</tr>
		<tr>
			<td>pAAV2/8&#160;</td>
			<td>Addgene&#160;</td>
			<td>112864</td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV-U6-gRNA-CBh-mCherry</td>
			<td>Addgene&#160;</td>
			<td>91947</td>
			<td></td>
		</tr>
		<tr>
			<td>pAdDeltaF6&#160;</td>
			<td>Addgene&#160;</td>
			<td>112867</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG 8000</td>
			<td>Sigma-Aldrich</td>
			<td>89510</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine</td>
			<td>Sigma-Aldrich</td>
			<td>23966</td>
			<td>Linear, MW 25000</td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td>Rioqu&#237;mica</td>
			<td>510101303</td>
			<td>Antiseptic</td>
		</tr>
		<tr>
			<td>Rosiglitazone</td>
			<td>Sigma-Aldrich</td>
			<td>R2408</td>
			<td></td>
		</tr>
		<tr>
			<td>SacI enzyme</td>
			<td>New England Biolabs</td>
			<td>R0156</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Design Premier Adson Forceps</td>
			<td>Fisher Scientific</td>
			<td>22-079-741</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Hamilton</td>
			<td>475-40417</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Promega</td>
			<td>M180B</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA ligase buffer&#160;</td>
			<td>New England Biolabs</td>
			<td>B0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 PNK enzyme kit</td>
			<td>New England Biolabs</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>Tramadol Hydrochloride</td>
			<td>SEM</td>
			<td>43930</td>
			<td></td>
		</tr>
		<tr>
			<td>Vidisic Gel&#160;</td>
			<td>Bausch + Lomb&#160;</td>
			<td>99620</td>
			<td></td>
		</tr>
	</tbody>
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investigation of beige fat biology and metabolism using the crispr suntag-p65-hsf1 activation system

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.

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...

Watch this Scientific Journal Video about Investigation of Beige Fat Biology and Metabolism Using the CRISPR SunTag-p65-HSF1 Activation System at JoVE.com

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

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.

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has prompted a revolution in biology, and recent tools have been applied far ...

This protocol presents the SPH CRISPR Activation System as an alternative strategy to conventional viral vectors to perform gain function assays in adipocytes. It allows the overexpression of single or multiple adipocyte endogenous genes within the complex cellular environment of adipose tissue by delivering a customized single guide RNA using an adeno associated virus. The challenging steps of this protocol are related to cloning the single guide RNA and the production and injection of adeno-associated virus into the adipose tissue.To begin, design single guide RNA or sgRNA for CRISPR activation using chopchop or any other suitable tool. Design the sgRNA targeting the PRDM16 gene using the following parameters:Target as PRDM16 in a Mus musculus using a CRISPR/Cas9 and for as activation. Add overhangs to the sgRNA to match the Sac1 restriction site in the vector backbone PAAVU6GRNACBHM cherry.Include 5'AGCT 3'where n corresponds to nucleotides. Obtain 3'sgRNA reverse complement sequence using the indicated tool. Next, for annealing of single-stranded complimentary oligonucleotides, add one microliter of each 5'and 3'single-stranded oligonucleotide, one microliter of T4 ligase buffer, 0.5 microliters of T4 polynucleotide kinase, and 6.5 microliters of water for a final reaction volume of 10 microliters.Anneal the complimentary single-stranded oligonucleotides using a thermocycler at 37 degrees Celsius for 30 minutes and 95 degrees Celsius for five minutes, followed by a ramp-down rate of five degrees Celsius per minute. Then for ligation of annealed sgRNA oligonucleotides add 25 nanograms of plasmid PAAVU6GRNACBHM cherry to two microliters of annealed sgRNA Oligonucleotides, one microliters of Sac1 enzyme, two microliters of 10X T4 DNA ligase buffer, one microliter of T4 DNA ligase and water to a final reaction volume of 10 microliters. Using a thermocycler, perform ligation by incubating the reaction mixture for 15 cycles at 37 degrees Celsius for five minutes and 25 degrees Celsius for five minutes followed by holding at four degrees Celsius.Next, transform the competent Escherichia coli DH10B cells with four microliters of the ligation product by heat shock at 42 degrees Celsius for 45 seconds and spread on an agar plate containing 10 micrograms per milliliter Ampicillin. Confirm transformed colonies by Colony Polymerase Chain Reaction or PCR using the PCR Master Mix, pick the colony and mix it with five microliters of Master Mix, 0.1 microliters of universal primer, 0.1 microliter of sgRNA reverse primer and five microliters of water. Run the PCR using initial denaturation at 94 degrees Celsius for two minutes, followed by 35 cycles of denaturation at 94 degrees Celsius for 20 seconds, annealing for 30 seconds at 60 degrees Celsius, extension at 72 degrees Celsius for 30 seconds, and a final elongation step at 72 degrees Celsius for five minutes.After PCR, resolve the DNA using agarose gel electrophoresis in 0.5X TAE Buffer at 90 volts for 30 minutes. Submit positive samples for Sanger Sequencing using universal primer. Next, purify the plasmid from a positive clone using a plasmid purification kit following the manufacturer's instructions.Place the anesthetized mouse in the supine position and shave a small area on the flanks proximal to the hip joints for inguinal white adipose tissue or IWAT injections with a shaver. Add depilatory cream for five minutes. Remove residual cream with water to avoid skin burns.Disinfect the skin using three alternating rounds of applying the povidone iodine solution on the skin with clean gauze and 70%alcohol. Then, make a one to two centimeter incision with sterilized scissors in the proximal area of the joints and hold the skin open using forceps to expose the fat depot. The WAT can be attached to the skin on both sides extending it from the beginning from the back and towards the testes.Using forceps, gently pull the fat depot upward through the incision to ensure the injection is at the correct location and depth. Fill the microliter syringe with 2.5 microliters of the adeno-associated virus or a AAV containing the sgRNA targeting the endogenous PRDM16 gene. Carefully insert the needle at a 30-45 degree angle into the IWAT.Repeat the injection five times into different locations of the tissue to homogeneously infect the whole IWAT fat pad. Place the mouse on the heating pad until consciousness is regained. Monitor the animal every 10 to 15 minutes until it fully recovers.After the animal regains consciousness observe the locomotive profiling, which should be linear and have no signs of distress or pain. See the stromal vascular cells or SVF's derived from adipo SPH mouse IWAT into a 6-Well plate containing complete DMEM medium for one to two hours, aspirate the medium, wash the well twice using PBS, and replace it with fresh, complete medium. Incubate the cells at 37 degrees Celsius, 5%carbon dioxide and 95%humidity until the cells reach 70-80%confluency.At Day 0, induce differentiation by treating the cells with the induction medium and after two days replace the induction medium with the maintenance medium. Again after two days, at day four, replace the maintenance medium with a fresh maintenance medium for 2-3 days. Change the maintenance medium every 48 hours until the preadipocytes are fully differentiated into adipocytes.Observe the mature adipocytes using light microscopy as the differentiated cells appear to be loaded with lipid droplets. After growing the cells on a 6-Well culture plate with the complete medium until the cells reach 70-80%confluency, mix 5.6*10^10 viral genome per microliter of AAV-carrying sgRNA PRDM16 with two milliliters of complete medium and hexadimethrine bromide. Transduce the cells by replacing the complete medium and adding the complete medium containing AAV.Incubate the transduced cells for 12 hours at 37 degrees Celsius 95%humidity and 5%carbon dioxide. Split and see the cells as described for cell proliferation and differentiation into beige adipocytes. The immunofluorescence images from IWAT of adipo SPH mice demonstrated the expression of dCas9 in both the control and PRDM16 groups.SPH-induced PRDM16 expression clearly induced a widespread accumulation of multi ocular beige adipocytes into IWAT. Moreover, quantitative PCR revealed an increased expression of PRDM16 and the thermogenic gene program in the PRDM16 group compared to the control. Similarly, in vitro gene expression analysis confirmed the increased expression of the endogenous PRDM16 gene and thermogenic genes in the PRDM16 overexpression group compared to the control.The SPH-induced PRDM16 expression resulted in higher basal and maximal oxygen consumption than that in control cells. Importantly, the data indicated enhanced uncoupled respiration in the PRDM16 group compared with that in the control group. Adipo SPH model is suitable for investigating multiple genes and regulatory elements within adipocytes.This method opens up the possibility of a deeper understanding of beige fat biology.

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