CRISPR-mediated activation of endogenous gene expression in the postnatal heart
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ABSTRACT:
PROVIDER: PRJNA564638 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10089761.fastq.gz | Fastqsanger.gz | |||
| SRR10089762.fastq.gz | Fastqsanger.gz | |||
| SRR10089763.fastq.gz | Fastqsanger.gz | |||
| SRR10089764.fastq.gz | Fastqsanger.gz | |||
| SRR10089765.fastq.gz | Fastqsanger.gz |
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Project description:Endogenous activation of Myocyte enhancer factor 2 D (Mef2d) in the postnatal mouse heart by cardiomyocyte-specific CRISPRactivation (Myh6-dCas9VPR) The aim of this study was to compare endogenous gene activation of the pro-hypertrophic factor Myocyte enhancer factor 2 D (Mef2d) in the postnatal mammalian heart and experimental control groups including wild-type litter mates injected with saline, wild-type litter mates injected with AAV9 TRISPR Mef2d and transgenic litter mates injected with saline. Mice were injected with saline/AAV9 TRISPR Mef2d at P4 and sacrificed 8 weeks later. RNA was isolated by TRIzol/Chloroform and isopropanol precipitation. Ventricular tissue mRNA profiles were obtained using deep sequencing, in triplicates, using Illumina HiSeq4000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by DESeq2. qPCR validation was performed using SYBR Green assays. We present in this study a mouse model for cardiomyocyte-specific endogenous gene activation in the postnatal mammalian heart by CRISPR/dCas9VPR. As a proof of concept, we activated the pro-hypertrophic factor Myocyte enhancer factor 2 D (Mef2d) leading to functional decline, increased marker expression of heart failure as well as cardiomyocyte hypertrophy over a time course of 8 weeks. We furthermore observed no phenotypic consequence of constitutive dCas9VPR transgene expression in the postnatal heart. We therefore conclude, that the Myh6-dCas9VPR mouse model is a suitable tool for endogenous gene activation studies in the postnatal heart.
2021-08-02 | GSE132992 | GEO
Project description:Endogenous activation of Myocyte enhancer factor 2 D (Mef2d) and Krüppel-like factor 15 (Klf15) in the postnatal mouse heart by cardiomyocyte-specific CRISPRactivation (Myh6-dCas9VPR) The aim of this experiment was to identify dCas9VPR on- and off-target chromatin association after gRNA delivery targeted to Myocyte enhancer factor 2 D (Mef2d) and Krüppel-like factor 15 (Klf15) in the postnatal mammalian heart. Saline injected Myh6-dCas9VPR transgenic mice were used to identify gRNA dependent and gRNA independent off-target sites. Mice were injected with saline/AAV9 TRISPR Mef2d/AAV9 TRISPR Klf15 at P4 and sacrificed at P14. Hearts were minced and nuclei were isolated, sonicated and dCas9VPR bound regions were immunoprecipitated. dCas9VPR occupancy profiles were obtained using deep sequencing, in triplicates, using Illumina HiSeq4000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by DESeq2. qPCR validation was performed using SYBR Green assays. We present in this study a mouse model for cardiomyocyte-specific endogenous gene activation in the postnatal mammalian heart by CRISPR/dCas9VPR. As a proof of concept, we activated the pro-hypertrophic factor Myocyte enhancer factor 2 D (Mef2d) leading to functional decline, increased marker expression of heart failure as well as cardiomyocyte hypertrophy over a time course of 8 weeks. We furthermore observed no phenotypic consequence of constitutive dCas9VPR transgene expression in the postnatal heart. We therefore conclude, that the Myh6-dCas9VPR mouse model is a suitable tool for endogenous gene activation studies in the postnatal heart. We additionally activated Krüppel-like factor 15 (Klf15) in neonatal heart and confirmed increased Klf15 transcript levels. dCas9VPR-On-target association was confirmed by ChIP qPCRs and sequencing for both targets.
2021-08-02 | GSE137146 | GEO
Project description:CRISPR-mediated activation of endogenous gene expression in the postnatal heart [ChIP-seq]
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Project description:CRISPR-mediated activation of endogenous gene expression in the postnatal heart [RNA-seq]
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Project description:Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 (dCas9) protein fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4 genes. The most efficient gene activation was achieved by clusters of 3 to 4 sgRNAs binding to the proximal promoters suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system. We used microarray to assay the gene expression changes after transfection of dCas9VP160 with the different sgRNAs
2013-08-10 | E-GEOD-49701 | biostudies-arrayexpress
Project description:Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 (dCas9) protein fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4 genes. The most efficient gene activation was achieved by clusters of 3 to 4 sgRNAs binding to the proximal promoters suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system.
2013-08-10 | GSE49701 | GEO
Project description:Single cell CRISPR activitaion analysis with 96 genes with the aim to build a quantative CRISPR activation model. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
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Project description:In this study, we determine whether direct activation of endogenous loci by targeting the genomic DNA (using a CRISPRa system) can fulfill reprogramming of fibroblasts into cardiac progenitors. Core promoter regions of Gata4, Nkx2-5, and Tbx5 were targeted by the CRISPRa (Synergistic Activation Mediator) in mouse tail tip fibroblasts (TTFs). RNA sequencing was performed to characterize the transcriptome of the transfected fibroblasts and reprogrammed cells. These data indicate that our CRISPRa approaches can induce cardiac reprogramming and establish a gene network for heart development.
2021-02-01 | GSE158084 | GEO
Project description:The present study aimed at determining the differences in global mRNA expression between proliferating cardiomyocytes in the 2-day-old heart and growth arrested cardiomyocytes in 13-day-old heart of ICR mice. The transcriptomic profiles of the samples at the two time points(day 2 and day 13 of postnatal stage) were analyzed by direct comparison of the transcriptional level between the two groups of heart on the same custom 12×135K microarray (Roche NimbleGen)
2015-04-04 | E-GEOD-67575 | biostudies-arrayexpress