GSE137146
CRISPR-mediated activation of endogenous gene expression in the postnatal heart [ChIP-seq]
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. Overall design: dCas9 genomic occupancy profiling from Mef2d and Klf15 endogenous gene activation in the postnatal hearts versus controls.