Gene therapy approaches using viral vectors for the overexpression of target genes have been for several years the focus of gene therapy research against neurological disorders. These approaches deliver robust expression of therapeutic genes, but are typically limited to the delivery of single genes and often do not manipulate the expression of the endogenous locus. In the last years, the advent of CRISPR-Cas9 technologies have revolutionized many areas of scientific research by providing novel tools that allow simple and efficient manipulation of endogenous genes. One of the applications of CRISPR-Cas9, termed CRISPRa, based on the use of a nuclease-null Cas9 protein (dCas9) fused to transcriptional activators, enables quick and efficient increase in target endogenous gene expression. CRISPRa approaches are varied, and different alternatives exist with regards to the type of Cas9 protein and transcriptional activator used. Several of these approaches have been successfully used in neurons in vitro and in vivo, but have not been so far extensively applied for the overexpression of genes involved in synaptic transmission. Here we describe the development and application of two different CRISPRa systems, based on single or dual Lentiviral and Adeno-Associated viral vectors and VP64 or VPR transcriptional activators, and demonstrate their efficiency in increasing mRNA and protein expression of the Cnr1 gene, coding for neuronal CB1 receptors. Both approaches were similarly efficient in primary neuronal cultures, and achieved a 2–5-fold increase in Cnr1 expression, but the AAV-based approach was more efficient in vivo. Our dual AAV-based VPR system in particular, based on Staphylococcus aureus dCas9, when injected in the hippocampus, displayed almost complete simultaneous expression of both vectors, high levels of dCas9 expression, and good efficiency in increasing Cnr1 mRNA as measured by in situ hybridization. In addition, we also show significant upregulation of CB1 receptor protein in vivo, which is reflected by an increased ability in reducing neurotransmitter release, as measured by electrophysiology. Our results show that CRISPRa techniques could be successfully used in neurons to target overexpression of genes involved in synaptic transmission, and can potentially represent a next-generation gene therapy approach against neurological disorders.

多年来，使用病毒载体进行靶基因过表达的基因治疗方法一直是针对神经系统疾病的基因治疗研究的重点。这些方法提供了治疗基因的稳定表达，但通常仅限于单个基因的传递，并且通常不操纵内源基因座的表达。在过去的几年中，CRISPR-Cas9技术的出现彻底改变了科学研究的许多领域，它提供了新颖的工具，可以简单而有效地操纵内源基因。CRISPR-Cas9的一种应用称为CRISPRa，是基于与转录激活因子融合的无核酸酶的Cas9蛋白（dCas9）的使用，可快速有效地增加靶标内源基因的表达。CRISPRa的方法多种多样，关于Cas9蛋白的类型和使用的转录激活因子，存在不同的选择。这些方法中的几种已成功地用于神经元体外 和 体内，但到目前为止尚未广泛应用于涉及突触传递的基因的过表达。在这里，我们描述了基于单或双慢病毒和腺相关病毒载体以及VP64或VPR转录激活因子的两种不同CRISPRa系统的开发和应用，并展示了它们在增加mRNA和蛋白质表达中的效率。Cnr1基因，编码神经元CB1受体。两种方法在原代神经元培养中的效率相似，并实现了2-5倍的增加。Cnr1 表达，但基于AAV的方法更有效 体内。 我们基于双AAV的VPR系统，特别是基于 金黄色葡萄球菌 将dCas9注射入海马体后，显示两种载体几乎完全同时表达，高水平的dCas9表达以及良好的增加效率 Cnr1 通过测量mRNA 原位杂交。此外，我们还显示CB1受体蛋白显着上调体内如通过电生理学测量的那样，这可以通过减少神经递质释放的能力增强来体现。我们的结果表明，CRISPRa技术可以成功地用于神经元中，以靶向突触传递中涉及的基因的过表达，并且有可能代表下一代针对神经系统疾病的基因治疗方法。