使用CRISPR SunTag-p65-HSF1激活系统研究米色脂肪生物学和代谢
Summary
该协议提出了在脂肪细胞（AdipoSPH）中使用CRISPR SunTag-p65-HSF1（SPH）作为腺相关病毒（AAV）的替代策略来研究米色脂肪生物学。 体内 注射靶向内源性Prdm16基因的携带AAV的sgRNA足以诱导米色脂肪发育并增强产热基因程序。
Abstract
成簇规则间隔短回文重复（CRISPR）技术引发了生物学的革命，最近的工具已经远远超出了最初描述的基因编辑。CRISPR活化（CRISPRa）系统将催化无活性的Cas9（dCas9）蛋白与不同的转录模块相结合，以诱导内源性基因表达。SunTag-p65-HSF1（SPH）是最近开发的一种CRISPRa技术，它将协同激活介质（SAM）的成分与SunTag激活剂相结合。该系统通过设计定制的单向导RNA（sgRNA）允许单个或多个基因的过表达。在这项研究中，先前开发的SPH小鼠用于在脂肪细胞（脂联素Cre谱系）中产生表达SPH的条件小鼠，称为AdipoSPH。为了诱导白色至米色脂肪（褐变）表型，将携带靶向内源性Prdm16基因（一种与棕色和米色脂肪发育相关的成熟转录因子）的sgRNA的腺相关病毒（AAV）注射到腹股沟白色脂肪组织（iWAT）中。该小鼠模型诱导内源性Prdm16的表达并激活产热基因程序。此外， 体外 SPH诱导的Prdm16过表达增强了米色脂肪细胞的耗氧量，表型复制了先前Prdm16转基因小鼠模型的结果。因此，该协议描述了一种用于研究脂肪组织生物学的多功能，经济高效且具有时间效率的小鼠模型。
Introduction
米色（或卤状）脂肪细胞是解偶联表达蛋白 1 （UCP1） 和富含线粒体的脂肪细胞，位于白色脂肪组织 （WAT） 库中。米色脂肪来自脂肪细胞祖细胞或成熟白色脂肪细胞的亚群，以响应寒冷暴露和其他刺激1，2。米色脂肪细胞可以以UCP1依赖性或独立的方式将能量转化为热量3。除了产热功能如何，米色脂肪还可以通过其他方式改善代谢健康，例如脂肪因子的分泌和抗炎和抗纤维化活性。对小鼠和人类的研究表明，米色脂肪的诱导改善全身葡萄糖和脂质稳态3。然而，尽管近年来我们对米色脂肪生物学的了解发展迅速，但其大部分代谢益处和相关机制仍未完全了解。
簇状规则间隔短回文重复序列（CRISPR）首先在真核细胞中被描述为能够通过Cas9蛋白4，5的核酸酶活性在基因组的特定位点产生双链断裂（DSB）的工具。Cas9由合成的单向导RNA（sgRNA）引导，靶向特定的基因组区域，从而产生DNA DSB。除了使用核酸酶Cas9进行编辑外，CRISPR-Cas9技术还发展成为序列特异性基因调控工具6。催化无活性Cas9蛋白（dCas9）的发展和能够增强基因表达的转录模块的结合产生了CRISPR激活（CRISPRa）工具。已经出现了几种CRISPRa系统，例如VP647，8，协同激活介质（SAM）9，SunTag10，11，VPR 12，13和SunTag-p65-HSF1（SPH）14，它们结合了SAM和SunTag激活剂的组件。最近已经证明，与其他CRISPRa系统相比，使用SPH诱导的N2a神经母细胞和原代星形胶质细胞中神经源性基因的表达更高14，这表明SPH是一种有前途的CRISPRa工具。
在这里，我们利用先前开发的SPH小鼠14 ，使用脂联素Cre谱系（AdipoSPH）生成在脂肪细胞中特异性表达SPH的条件小鼠模型。使用携带靶向内源性Prdm16基因的gRNA的腺相关病毒（AAV），诱导腹股沟WAT（iWAT）的褐变（白色到米色转换）以增加产热基因程序的表达。此外， 体外 Prdm16过表达增加了耗氧量。因此，该协议提供了一种多功能的SPH小鼠模型，用于探索脂肪组织内米色脂肪发育的机制。
Protocol
Representative Results
Discussion
CRISPR技术最有用的非编辑应用之一是通过使用CRISPRa系统激活内源性基因来询问基因功能6。SPH是一种强大的CRISPRa，最初被描述为通过靶向几个神经源性基因来诱导星形胶质细胞转化为活跃的神经元14。在这项研究中，AdipoSPH被证明是通过激活脂肪细胞中内源性Prdm16的表达来研究米色脂肪生物学的合适工具。SPH诱导的Prdm16在脂肪细胞中的过表达导致产热基因程序的…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢Unicamp的Centro Multidisciplinar para Investigação Biológica na Área da Animais de Laboratório（Cemib）对AdipoSPH小鼠的产生，免疫代谢和细胞信号实验室以及国家应用于细胞生物学的光子学科学技术研究所（INFABIC）的所有实验支持。我们感谢圣保罗研究基金会（FAPESP）的财政支持：2019/15025-5;2020/09308-1;2020/14725-0;2021/11841-2.
Materials
| 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 |
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