Department of Neurobiology and Evelyn F. McKnight Brain Institute, University of Alabama at Birmingham, Birmingham, AL, 35294.
Civitan International Research Center, University of Alabama at Birmingham, Birmingham, AL, 35294.
eNeuro. 2019 Mar 7;6(1). doi: 10.1523/ENEURO.0495-18.2019. eCollection 2019 Jan-Feb.
CRISPR-based technology has provided new avenues to interrogate gene function, but difficulties in transgene expression in post-mitotic neurons has delayed incorporation of these tools in the central nervous system (CNS). Here, we demonstrate a highly efficient, neuron-optimized dual lentiviral CRISPR-based transcriptional activation (CRISPRa) system capable of robust, modular, and tunable gene induction and multiplexed gene regulation across several primary rodent neuron culture systems. CRISPRa targeting unique promoters in the complex multi-transcript gene brain-derived neurotrophic factor () revealed both transcript- and genome-level selectivity of this approach, in addition to highlighting downstream transcriptional and physiological consequences of regulation. Finally, we illustrate that CRISPRa is highly efficient , resulting in increased protein levels of a target gene in diverse brain structures. Taken together, these results demonstrate that CRISPRa is an efficient and selective method to study gene expression programs in brain health and disease.
基于 CRISPR 的技术为研究基因功能提供了新途径，但由于在有丝分裂后神经元中转基因表达的困难，这些工具在中枢神经系统（CNS）中的应用受到了阻碍。在这里，我们展示了一种高效、神经元优化的双慢病毒 CRISPR 基转录激活（CRISPRa）系统，能够在几种主要的啮齿动物神经元培养系统中实现强大、模块化和可调的基因诱导和多基因调控。CRISPRa 靶向复杂的多转录基因脑源性神经营养因子（）中的独特启动子，揭示了这种方法在转录和基因组水平上的选择性，此外还突出了 调节的下游转录和生理后果。最后，我们表明 CRISPRa 非常高效，导致目标基因在不同脑结构中的蛋白水平增加。总之，这些结果表明，CRISPRa 是一种研究大脑健康和疾病中基因表达程序的有效和选择性方法。
