In Vivo Epigenome Editing with CRISPR-Based Histone Acetyltransferase Transgenic Mice

 产品说明：慢性可卡因滥用是由于持续可卡因诱导的神经元功能适应，包括mesolimbocortical脑奖励电路。可卡因诱导的基因转录变化导致了许多神经元功能的改变。此外，可卡因暴露已显示通过调节组蛋白和DNA修饰酶的表达和/或功能来动态改变表观基因组。总之，这些数据导致了一个假设，即表观基因组的长期变化可能是可卡因诱导的成瘾样行为持续存在的基础。然而，染色质调控的特定变化是否是药物诱导的行为可塑性的真正原因，仍然是一个具有挑战性的假设，以测试由于缺乏高通量的体内方法的位点特异性实验操作的表观基因组。为了克服这一限制，我们将产生两种新的基于Cre/loxP条件CRISPR/Cas9的转基因小鼠品系，其中将与p300的核心组蛋白乙酰转移酶结构域（dCas-p300）或KRAB阻遏物结构域（dCas 9-KRAB）融合的酶促死亡Cas9蛋白敲入Rosa 26基因座。我们已经表明，gRNA介导的dCas 9融合物与染色质调节剂的募集足以诱导靶向组蛋白修饰和基因转录中的高度特异性相应变化。现在，通过在转基因小鼠中表达这些融合蛋白，我们将通过使dCas 9融合蛋白表达的Cre依赖性诱导与靶向可卡因调节的增强子的经验证的gRNA的立体定位病毒递送交叉来实现体内脑中位点特异性表观基因组编辑的区域和时间控制。在该提议的R21阶段，我们将首先产生和表征条件性dCas 9-p300和dCas 9-KRAB小鼠品系，然后进行原理验证实验以验证在延髓核的神经元中dCas 9介导的Fosb调节是否足以改变可卡因诱导的运动敏化和条件性位置偏爱。在R33阶段，我们将使用dCas 9- p300和dCas 9-KRAB小鼠品系来测试以下假设：在腹侧被盖区的多巴胺能神经元中Bdnf转录的表观遗传敏化促进可卡因渴望的孵化，这是一种重要的复发啮齿动物模型。最后，为了加速未来的表观基因组编辑研究，我们将首先使用一种新的方法来捕获可卡因激活的神经元系综的细胞核，用于染色质分析，然后开发和功能验证靶向可卡因调节增强子的gRNA，供更广泛的科学界使用。我们的研究将为物质使用障碍的分子机制的功能表观基因组研究提供一个新的工具箱。

 DESCRIPTION: Chronic cocaine abuse arises as a result of persistent cocaine-induced adaptations in the function of the neurons that comprise mesolimbocortical brain reward circuits. Cocaine-induced changes in gene transcription contribute to many of these alterations in neuronal function. Furthermore cocaine exposure has been shown to dynamically alter the epigenome by regulating the expression and/or function of histone and DNA modifying enzymes. Taken together, these data have led to the hypothesis that long-lasting changes in the epigenome may underlie the persistence of cocaine-induced addictive-like behaviors. However whether specific changes in chromatin regulation are truly causative for drug-induced behavioral plasticity has remained a challenging hypothesis to test due to the lack of high-throughput in vivo methods for site-specific experimental manipulation of the epigenome. To overcome this limitation we will generate two novel Cre/loxP-conditional CRISPR/Cas9- based transgenic mouse strains in which an enzymatically dead Cas9 protein fused either to the core histone acetyltransferase domain of p300 (dCas-p300) or the KRAB repressor domain (dCas9-KRAB) is knocked into the Rosa26 locus. We have shown that gRNA-mediated recruitment of dCas9 fusions with chromatin regulators is sufficient to induce targeted histone modifications and highly specific corresponding changes in gene transcription. Now by expressing these fusion proteins in transgenic mice, we will achieve regional and temporal control of site-specific epigenome editing in the brain in vivo by intersecting Cre-dependent induction of dCas9-fusion protein expression with stereotaxic viral delivery of validated gRNAs targeting cocaine- regulated enhancers. In the R21 phase of this proposal we will first generate and characterize the conditional dCas9-p300 and dCas9-KRAB mouse strains and then conduct a proof-of-principle experiment to validate whether dCas9-mediated regulation of Fosb in neurons of the nucleus accumbens is sufficient to alter cocaine- induced locomotor sensitization and conditioned place preference. In the R33 phase we will use the dCas9- p300 and dCas9-KRAB mouse strains to test the hypothesis that epigenetic sensitization of Bdnf transcription in dopaminergic neurons of the ventral tegmental area promotes incubation of cocaine craving, an important rodent model for relapse. Finally to accelerate future epigenome editing studies we will first use a novel method to capture nuclei of cocaine-activated neuronal ensembles in the nucleus accumbens for chromatin profiling and then develop and functionally validate gRNAs targeting cocaine-regulated enhancers for use by the broader scientific community. Our studies will provide a novel toolbox for functional epigenomic studies of the molecular mechanisms underlying substance use disorders.