RATIONALE Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is evolving rapidly. Recently, second-generation CRISPR/Cas9 activation systems based on nuclease inactive dead (d)Cas9 fused to transcriptional transactivation domains were developed for directing specific guide (g)RNAs to regulatory regions of any gene of interest, to enhance transcription. The application of dCas9 to activate cardiomyocyte transcription in targeted genomic loci in vivo has not been demonstrated so far. OBJECTIVE We aimed to develop a mouse model for cardiomyocyte-specific, CRISPR-mediated transcriptional modulation, and to demonstrate its versatility by targeting Mef2d and Klf15 loci (2 well-characterized genes implicated in cardiac hypertrophy and homeostasis) for enhanced transcription. METHODS AND RESULTS A mouse model expressing dCas9 with the VPR transcriptional transactivation domains under the control of the Myh (myosin heavy chain) 6 promoter was generated. These mice innocuously expressed dCas9 exclusively in cardiomyocytes. For initial proof-of-concept, we selected Mef2d, which when overexpressed, led to hypertrophy and heart failure, and Klf15, which is lowly expressed in the neonatal heart. The most effective gRNAs were first identified in fibroblast (C3H/10T1/2) and myoblast (C2C12) cell lines. Using an improved triple gRNA expression system (TRISPR [triple gRNA expression construct]), up to 3 different gRNAs were transduced simultaneously to identify optimal conditions for transcriptional activation. For in vivo delivery of the validated gRNA combinations, we employed systemic administration via adeno-associated virus serotype 9. On gRNA delivery targeting Mef2d expression, we recapitulated the anticipated cardiac hypertrophy phenotype. Using gRNA targeting Klf15, we could enhance its transcription significantly, although Klf15 is physiologically silenced at that time point. We further confirmed specific and robust dCas9VPR on-target effects. CONCLUSIONS The developed mouse model permits enhancement of gene expression by using endogenous regulatory genomic elements. Proof-of-concept in 2 independent genomic loci suggests versatile applications in controlling transcription in cardiomyocytes of the postnatal heart.

中文翻译：

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产后心脏中CRISPR介导的内源基因表达的激活。

CRISPR的RATIONALE基因组编辑（聚簇的规则间隔的短回文重复序列）/ Cas9正在迅速发展。最近，开发了基于与转录反式激活结构域融合的核酸酶无活性死（d）Cas9的第二代CRISPR / Cas9激活系统，用于将特定的引导（g）RNA引导至任何目的基因的调控区，以增强转录。迄今为止，尚未证明dCas9在体内靶向基因组基因座中激活心肌细胞转录的应用。目的我们旨在开发一种针对心肌细胞的，CRISPR介导的转录调控的小鼠模型，并通过靶向Mef2d和Klf15基因位点（两个与心肌肥大和体内平衡有关的特性良好的基因）来增强转录，从而证明其多功能性。方法和结果产生了在Myh（肌球蛋白重链）6启动子的控制下表达带有VPR转录反式激活域的dCas9的小鼠模型。这些小鼠仅在心肌细胞中无毒表达dCas9。对于最初的概念验证，我们选择了Mef2d和Klf15，Mef2d过表达会导致肥大和心力衰竭，而Klf15在新生儿心脏中低表达。最有效的gRNA首先在成纤维细胞（C3H / 10T1 / 2）和成肌细胞（C2C12）细胞系中鉴定。使用改进的三重gRNA表达系统（TRISPR [三重gRNA表达构建体]），可同时转导多达3种不同的gRNA，以鉴定转录激活的最佳条件。为了在体内递送经过验证的gRNA组合，我们通过腺相关病毒血清型9进行全身性给药。在靶向Mef2d表达的gRNA递送中，我们概括了预期的心脏肥大表型。使用靶向Klf15的gRNA，我们可以显着增强其转录，尽管在那个时间点Klf15在生理上是沉默的。我们进一步证实了dCas9VPR对靶的特异性和强效作用。结论所开发的小鼠模型可通过使用内源性调控基因组元件来增强基因表达。在2个独立的基因组基因座中的概念验证表明，在控制产后心脏的心肌细胞转录方面具有广泛的应用。尽管在那个时间点Klf15在生理上是沉默的，但我们可以显着增强其转录。我们进一步证实了dCas9VPR对靶的特异性和强效作用。结论所开发的小鼠模型可通过使用内源性调控基因组元件来增强基因表达。在2个独立的基因组基因座中的概念验证表明，在控制产后心脏的心肌细胞转录方面具有广泛的应用。尽管在那个时间点Klf15在生理上是沉默的，但我们可以显着增强其转录。我们进一步证实了dCas9VPR对靶的特异性和强效作用。结论所开发的小鼠模型可通过使用内源性调控基因组元件来增强基因表达。在2个独立的基因组基因座中的概念验证表明，在控制产后心脏的心肌细胞转录方面具有广泛的应用。