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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 Ppar?2, 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, Ppar?2, was successfully achieved using selected sgRNAs targeting the Ppar?2 promoter region (i.e. up to 104 fold); this induction was long lasting and sufficient to promote adipogenesis. Furthermore, co-activation of Ppar?2 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.

SUBMITTER: Lundh M

PROVIDER: S-EPMC5641601 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Bidirectional manipulation of gene expression in adipocytes using CRISPRa and siRNA.

Lundh Morten M Pluciñska Kaja K Isidor Marie S MS Petersen Patricia S S PSS Emanuelli Brice B

Molecular metabolism 20170708 10

<h4>Objective</h4>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 contr ...[more]

PMID: 29031730

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Project description:SF-1 (NR5A1) was overexpressed (Over) or knocked down with shRNA (shRNA) in NCI-H295R human adrenocortical tumor cells and differential global gene expression analysed 48 hours later using Affymetrix GeneChip Human Gene 1.0ST arrays. Over: 5 million cells were transfected (Amaxa Nucleofection) with 10 ug of a pIRES2-AcGPF1-Nuc construct co-expressing SF-1 cDNA and GFP. For experimental control, a mutagenized pIRES2 construct, bearing the G35E mutation in SF-1 that impairs its transactivation function in vivo and in vitro was used. shRNA: 5 million cells were transfected (Amaxa Nucleofection) with 10 ug of the SureSilencing shRNA Plasmid for Human NR5A1 with GFP marker kit (SABioscience). For experimental control, mismatch constructs provided in the kit were used. In both experiments (Over and shRNA), cells were harvested, prepared, and submitted to fluorescence-activated cell sorting (FACS) in a MoFlo XDP sorter 48 hours after transfection. Viable GFP-expressing cells were pooled and resuspended in TRIzol reagent for RNA extraction. Total RNA was extracted, and RNA quality control performed using a 2100 Bioanalyzer. RNA samples were processed using the Affymetrix GeneChip WT Sense Target Labeling kit, starting with 200 ng total RNA. Four independent Over experiments and five independent shRNA experiments were performed and samples of labeled fragmented cDNA were hybridized to Affymetrix GeneChip Human Gene 1.0 ST Arrays. Based on quality control of array data (R/Bioconductor and Partek Genomics Suite), two Over arrays (one SF-1 wild type and one mutant control) and one shRNA array (mismatch control) were excluded and are not deposited here. Differential gene expression analysis was performed using the limma package in R/Bioconductor. A Benjamini-Hochberg-corrected P value cut-off of 0.05 was used to select significant differentially expressed genes in each setting (Over and shRNA). Finally, results from both analyses were combined to identify a subset of positively-regulated SF-1 target genes in these cells, ie transcripts up-regulated by SF-1 overexpression (Over) and down-regulated by SF-1 knockdown (shRNA). Four SF-1 overexpression experiments (Over; intervention = overexpression of wild type SF-1; control = overexpression of functionally-impaired mutant G35E SF-1) and five SF-1 knockdown experiments (shRNA; intervention = SF-1-specific small hairpin RNA; control = shRNA mismatch control) were performed in NCI-H295R cells. Differential gene expression in each setting was analysed using GeneChip Human Gene 1.0ST arrays. Data for arrays that met quality control are presented here: Over, 3 intervention and 3 control arrays; shRNA, 5 intervention and 4 control arrays. Total of 15 arrays.

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Bidirectional manipulation of gene expression in adipocytes using CRISPRa and siRNA.

Publications

Bidirectional manipulation of gene expression in adipocytes using CRISPRa and siRNA.

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