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

 描述：慢性可卡因滥用是持续的可卡因诱导的神经元功能适应的结果，这些神经元组成了中脑皮质奖赏回路。可卡因诱导的基因转录变化导致了神经元功能的许多变化。此外，可卡因暴露已被证明通过调节组蛋白和DNA修饰酶的表达和/或功能来动态改变表观基因组。综上所述，这些数据导致了一种假设，即表观基因组的长期变化可能是可卡因诱导的成瘾行为持续存在的基础。然而，染色质调节的特定变化是否真的导致药物诱导的行为可塑性仍然是一个具有挑战性的假说，因为缺乏高通量的体内方法来对表观基因组进行特定部位的实验操作。为了克服这一限制，我们将产生两个新的基于Cre/loxP条件的CRISPR/Cas9转基因小鼠品系，其中酶死的Cas9蛋白融合到p300的核心组蛋白乙酰转移酶结构域(DCAS-p300)或KRAB抑制器域(dCas9-KRAb)中，被敲入rosa26基因座。我们已经证明，gRNA介导的dCas9与染色质调节因子的融合募集足以诱导有针对性的组蛋白修饰和基因转录中高度特异的相应变化。现在，通过在转基因小鼠中表达这些融合蛋白，我们将通过交叉Cre依赖的dCas9融合蛋白表达的诱导和针对可卡因调节增强子的验证gRNA的立体定位病毒传递，在活体中实现对大脑中特定部位表观基因组编辑的区域和时间控制。在这个建议的R21阶段，我们将首先产生并表征条件dCas9-p300和dCas9-KRAb小鼠品系，然后进行原则证明实验，以验证dCas9介导的伏核神经元FosB的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dCas9-p300和dCas9-KRAb小鼠品系来测试这一假设，即腹侧被盖区多巴胺能神经元中BDNF转录的表观遗传敏化促进了可卡因渴求的孵化，可卡因渴求是一种重要的复吸啮齿动物模型。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伏隔核中可卡因激活的神经元集合的核，用于染色质分析，然后开发并功能验证针对可卡因调节的增强子的gRNAs，以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学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.