Bidirectional manipulation of gene expression in adipocytes using CRISPRa and siRNA

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Bidirectional manipulation of gene expression in adipocytes using CRISPRa and siRNA. / Lundh, Morten; Pluciñska, Kaja; Isidor, Marie S.; Petersen, Patricia S.S.; Emanuelli, Brice.

In: Molecular Metabolism, Vol. 6, No. 10, 10.2017, p. 1313-1320.

Research output: Contribution to journalJournal articleResearchpeer-review

Harvard

Lundh, M, Pluciñska, K, Isidor, MS, Petersen, PSS & Emanuelli, B 2017, 'Bidirectional manipulation of gene expression in adipocytes using CRISPRa and siRNA', Molecular Metabolism, vol. 6, no. 10, pp. 1313-1320. https://doi.org/10.1016/j.molmet.2017.07.001

APA

Lundh, M., Pluciñska, K., Isidor, M. S., Petersen, P. S. S., & Emanuelli, B. (2017). Bidirectional manipulation of gene expression in adipocytes using CRISPRa and siRNA. Molecular Metabolism, 6(10), 1313-1320. https://doi.org/10.1016/j.molmet.2017.07.001

Vancouver

Lundh M, Pluciñska K, Isidor MS, Petersen PSS, Emanuelli B. Bidirectional manipulation of gene expression in adipocytes using CRISPRa and siRNA. Molecular Metabolism. 2017 Oct;6(10):1313-1320. https://doi.org/10.1016/j.molmet.2017.07.001

Author

Lundh, Morten ; Pluciñska, Kaja ; Isidor, Marie S. ; Petersen, Patricia S.S. ; Emanuelli, Brice. / Bidirectional manipulation of gene expression in adipocytes using CRISPRa and siRNA. In: Molecular Metabolism. 2017 ; Vol. 6, No. 10. pp. 1313-1320.

Bibtex

@article{a239cc954d5844008a43a785ad83a04a,
title = "Bidirectional manipulation of gene expression in adipocytes using CRISPRa and siRNA",
abstract = "OBJECTIVE: Functional investigation of novel gene/protein targets associated with adipocyte differentiation or function heavily relies on efficient and accessible tools to manipulate gene expression in adipocytes in vitro. Recent advances in gene-editing technologies such as CRISPR-Cas9 have not only eased gene editing but also greatly facilitated modulation of gene expression without altering the genome. Here, we aimed to develop and validate a competent in vitro adipocyte model of controllable functionality as well as multiplexed gene manipulation in adipocytes, using the CRISPRa {"}SAM{"} system and siRNAs to simultaneously overexpress and silence selected genes in the same cell populations.METHODS: We introduced a stable expression of dCas9-VP64 and MS2-P65, the core components of the CRIPSRa SAM system, in mesenchymal C3H/10T1/2 cells through viral delivery and used guide RNAs targeting Pparγ2, Prdm16, Zfp423, or Ucp1 to control the expression of key genes involved in adipocyte differentiation and function. We additionally co-transfected mature adipocytes with sgRNA plasmids and siRNA to simultaneously up-regulate and silence selected genes. Quantitative gene expression, oxygen consumption, fluorescence-activated cell sorting and immunocytochemistry served as validation proxies in pre- or mature adipocytes.RESULTS: CRISPRa SAM-mediated up-regulation of a key adipogenic gene, Pparγ2, was successfully achieved using selected sgRNAs targeting the Pparγ2 promoter region (i.e. up to 104 fold); this induction was long lasting and sufficient to promote adipogenesis. Furthermore, co-activation of Pparγ2 with either Prdm16 or Zfp423 transcripts drove distinct thermogenic gene expression patterns associated with increased or decreased oxygen consumption, respectively, mimicking typical characteristics of brite/beige or white cell lineages. Lastly, we demonstrated that up-regulation of endogenous genes in mature adipocytes was also easily and efficiently achieved using CRISPRa SAM, here exemplified by targeted Ucp1 overexpression (up to 4 × 103 fold), and that it was compatible with concomitant gene silencing using siRNA, allowing for bidirectional manipulation of gene expression in the same cell populations.CONCLUSIONS: We demonstrate that the CRISPRa SAM system can be easily adopted and used to efficiently manipulate gene expression in pre- and mature adipocytes in vitro. Moreover, we describe a novel methodological approach combining the activation of endogenous genes and siRNA-mediated gene silencing, thus providing a powerful tool to functionally decipher genetic factors controlling adipogenesis and adipocyte functions.",
keywords = "Journal Article",
author = "Morten Lundh and Kaja Pluci{\~n}ska and Isidor, {Marie S.} and Petersen, {Patricia S.S.} and Brice Emanuelli",
note = "Copyright {\circledC} 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.",
year = "2017",
month = "10",
doi = "10.1016/j.molmet.2017.07.001",
language = "English",
volume = "6",
pages = "1313--1320",
journal = "Molecular Metabolism",
issn = "2212-8778",
publisher = "Elsevier",
number = "10",

}

RIS

TY - JOUR

T1 - Bidirectional manipulation of gene expression in adipocytes using CRISPRa and siRNA

AU - Lundh, Morten

AU - Pluciñska, Kaja

AU - Isidor, Marie S.

AU - Petersen, Patricia S.S.

AU - Emanuelli, Brice

N1 - Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

PY - 2017/10

Y1 - 2017/10

N2 - OBJECTIVE: Functional investigation of novel gene/protein targets associated with adipocyte differentiation or function heavily relies on efficient and accessible tools to manipulate gene expression in adipocytes in vitro. Recent advances in gene-editing technologies such as CRISPR-Cas9 have not only eased gene editing but also greatly facilitated modulation of gene expression without altering the genome. Here, we aimed to develop and validate a competent in vitro adipocyte model of controllable functionality as well as multiplexed gene manipulation in adipocytes, using the CRISPRa "SAM" system and siRNAs to simultaneously overexpress and silence selected genes in the same cell populations.METHODS: We introduced a stable expression of dCas9-VP64 and MS2-P65, the core components of the CRIPSRa SAM system, in mesenchymal C3H/10T1/2 cells through viral delivery and used guide RNAs targeting Pparγ2, Prdm16, Zfp423, or Ucp1 to control the expression of key genes involved in adipocyte differentiation and function. We additionally co-transfected mature adipocytes with sgRNA plasmids and siRNA to simultaneously up-regulate and silence selected genes. Quantitative gene expression, oxygen consumption, fluorescence-activated cell sorting and immunocytochemistry served as validation proxies in pre- or mature adipocytes.RESULTS: CRISPRa SAM-mediated up-regulation of a key adipogenic gene, Pparγ2, was successfully achieved using selected sgRNAs targeting the Pparγ2 promoter region (i.e. up to 104 fold); this induction was long lasting and sufficient to promote adipogenesis. Furthermore, co-activation of Pparγ2 with either Prdm16 or Zfp423 transcripts drove distinct thermogenic gene expression patterns associated with increased or decreased oxygen consumption, respectively, mimicking typical characteristics of brite/beige or white cell lineages. Lastly, we demonstrated that up-regulation of endogenous genes in mature adipocytes was also easily and efficiently achieved using CRISPRa SAM, here exemplified by targeted Ucp1 overexpression (up to 4 × 103 fold), and that it was compatible with concomitant gene silencing using siRNA, allowing for bidirectional manipulation of gene expression in the same cell populations.CONCLUSIONS: We demonstrate that the CRISPRa SAM system can be easily adopted and used to efficiently manipulate gene expression in pre- and mature adipocytes in vitro. Moreover, we describe a novel methodological approach combining the activation of endogenous genes and siRNA-mediated gene silencing, thus providing a powerful tool to functionally decipher genetic factors controlling adipogenesis and adipocyte functions.

AB - OBJECTIVE: Functional investigation of novel gene/protein targets associated with adipocyte differentiation or function heavily relies on efficient and accessible tools to manipulate gene expression in adipocytes in vitro. Recent advances in gene-editing technologies such as CRISPR-Cas9 have not only eased gene editing but also greatly facilitated modulation of gene expression without altering the genome. Here, we aimed to develop and validate a competent in vitro adipocyte model of controllable functionality as well as multiplexed gene manipulation in adipocytes, using the CRISPRa "SAM" system and siRNAs to simultaneously overexpress and silence selected genes in the same cell populations.METHODS: We introduced a stable expression of dCas9-VP64 and MS2-P65, the core components of the CRIPSRa SAM system, in mesenchymal C3H/10T1/2 cells through viral delivery and used guide RNAs targeting Pparγ2, Prdm16, Zfp423, or Ucp1 to control the expression of key genes involved in adipocyte differentiation and function. We additionally co-transfected mature adipocytes with sgRNA plasmids and siRNA to simultaneously up-regulate and silence selected genes. Quantitative gene expression, oxygen consumption, fluorescence-activated cell sorting and immunocytochemistry served as validation proxies in pre- or mature adipocytes.RESULTS: CRISPRa SAM-mediated up-regulation of a key adipogenic gene, Pparγ2, was successfully achieved using selected sgRNAs targeting the Pparγ2 promoter region (i.e. up to 104 fold); this induction was long lasting and sufficient to promote adipogenesis. Furthermore, co-activation of Pparγ2 with either Prdm16 or Zfp423 transcripts drove distinct thermogenic gene expression patterns associated with increased or decreased oxygen consumption, respectively, mimicking typical characteristics of brite/beige or white cell lineages. Lastly, we demonstrated that up-regulation of endogenous genes in mature adipocytes was also easily and efficiently achieved using CRISPRa SAM, here exemplified by targeted Ucp1 overexpression (up to 4 × 103 fold), and that it was compatible with concomitant gene silencing using siRNA, allowing for bidirectional manipulation of gene expression in the same cell populations.CONCLUSIONS: We demonstrate that the CRISPRa SAM system can be easily adopted and used to efficiently manipulate gene expression in pre- and mature adipocytes in vitro. Moreover, we describe a novel methodological approach combining the activation of endogenous genes and siRNA-mediated gene silencing, thus providing a powerful tool to functionally decipher genetic factors controlling adipogenesis and adipocyte functions.

KW - Journal Article

U2 - 10.1016/j.molmet.2017.07.001

DO - 10.1016/j.molmet.2017.07.001

M3 - Journal article

VL - 6

SP - 1313

EP - 1320

JO - Molecular Metabolism

JF - Molecular Metabolism

SN - 2212-8778

IS - 10

ER -

ID: 188785557