Loth Meredith K, Mesch Kendall T, Herrera-Garcia Celine, Brusman Liza E, Donaldson Zoe R
Department of Molecular, Cellular, and Developmental Biology, University of Colorado Boulder, Boulder, CO, United States.
Department of Psychology and Neuroscience, University of Colorado Boulder, Boulder, CO, United States.
Front Genome Ed. 2025 May 30;7:1602983. doi: 10.3389/fgeed.2025.1602983. eCollection 2025.
Prairie voles () are a powerful model for studying the neurobiology of social bonding, yet tools for region- and cell type-specific gene regulation remain underdeveloped in this species. Here, we present a lentivirus-mediated CRISPR activation and interference (CRISPRa/i) platform for somatic gene modulation in the prairie vole brain. This system enables non-mutagenic, titratable regulation of gene expression in the adult brain without germline modification. Our dual-vector system includes one construct expressing dCas9-VPR (VP64-p65-Rta) referred to as CRISPRa or dCas9-KRAB-MeCP2 (Kruppel-associated box-methyl CpG binding protein 2), referred to as CRISPRi under a neuron-specific promoter, and a second construct delivering a U6-driven sgRNA (single guide RNA) alongside an elongation factor 1 alpha (EF1α)-driven mCherry reporter. We detail the design, production, and stereotaxic delivery of these tools and demonstrate their application by targeting four genes implicated in social behavior ( and ) across two mesolimbic brain regions: the nucleus accumbens and ventral pallidum. Gene expression analyses confirmed robust, bidirectional transcriptional modulation for selected targets, establishing a proof of concept for CRISPRa/i in this non-traditional model. The dual-vector design is readily adaptable to other gene targets, cell types, and brain regions, and can be multiplexed to provide a flexible and scalable framework for investigating gene function in behaviorally relevant circuits. These advances represent the first successful implementation of somatic CRISPRa/i in prairie voles and expand the genetic toolkit available for this species.
草原田鼠是研究社会联结神经生物学的有力模型，但针对该物种区域和细胞类型特异性基因调控的工具仍未充分开发。在此，我们展示了一种用于草原田鼠大脑体细胞基因调控的慢病毒介导的CRISPR激活和干扰（CRISPRa/i）平台。该系统能够在不进行种系修饰的情况下，对成年大脑中的基因表达进行非诱变、可滴定的调控。我们的双载体系统包括一个在神经元特异性启动子下表达dCas9-VPR（VP64-p65-Rta，称为CRISPRa）或dCas9-KRAB-MeCP2（Kruppel相关框-甲基CpG结合蛋白2，称为CRISPRi）的构建体，以及另一个与延伸因子1α（EF1α）驱动的mCherry报告基因一起递送U6驱动的sgRNA（单向导RNA）的构建体。我们详细介绍了这些工具的设计、生产和立体定位递送，并通过在两个中脑边缘脑区（伏隔核和腹侧苍白球）靶向四个与社会行为相关的基因（和）来证明它们的应用。基因表达分析证实了对选定靶点的强大双向转录调控，为在这个非传统模型中应用CRISPRa/i建立了概念验证。双载体设计易于适应其他基因靶点、细胞类型和脑区，并且可以进行多重操作，以提供一个灵活且可扩展的框架，用于研究行为相关回路中的基因功能。这些进展代表了体细胞CRISPRa/i在草原田鼠中的首次成功应用，并扩展了该物种可用的遗传工具包。
