A Genetic Engineering Toolbox for Marmosets (GETMarm): Development and optimization of genome editing and assisted reproduction techniques for marmoset models

项目摘要 虽然小鼠是神经科学许多领域的基本模型，但也有许多方面， 更高的大脑功能和功能障碍，不能在啮齿动物中充分建模。因此，需要 新的遗传模型，具有更接近人类的大脑结构和功能。因此，非人类 灵长类动物（NHP）提供了一个有吸引力的模型来研究高级脑功能和脑疾病。一个有前途 新兴的NHP模型是普通的绒猴，一种小型的新世界灵长类动物，作为一种 遗传模型尽管细菌CRISPR/Cas系统用于靶向基因组工程的适应性 模型的创建已经彻底改变了现代生物学，但对绒猴基因组的编辑仍处于起步阶段。 鉴于绒猴基因组编辑需要大量的时间和金钱， 效率，减少嵌合现象，并在转移到受体雌性之前识别正确编辑的胚胎， 很危险此外，控制创始动物的接合性的新方法是必要的， 分析纯合F0动物，并在靶向必需基因时避免纯合编辑， 在双等位基因破坏时导致胚胎死亡。为此，我们提出了一项研究计划， 显着增强我们将多种类型编辑引入绒猴基因组的能力，减少 嵌合体，控制编辑的接合性，并通过产前遗传学鉴定成功编辑的胚胎 试验.我们将通过直接资源共享、面对面等方式传播这些技术和模式 培训，沉积到NIH支持的绒猴协调中心。总之，拟议的进展将 显著减少了绒猴遗传模型所需的时间、精力、成本和动物数量， 揭示绒猴在基础和转化神经科学研究中的真正潜力。

PROJECT SUMMARY While mice are essential models for many areas of neuroscience, there are also many aspects of higher brain function and dysfunction that cannot be adequately modeled in rodents. Thus, there is a need for new genetic models that have brain structure and function closer to humans. For these reasons, non-human primates (NHP) provide an attractive model to study higher brain function and brain disorders. A promising emerging NHP model is the common marmoset, a small New World primate that has many advantages as a genetic model. Although the adaptation of bacterial CRISPR/Cas systems for targeted genome engineering and model creation has revolutionized modern biology, editing of the marmoset genome is still in its infancy. Given the significant time and money required for marmoset genome editing, new methods to increase editing efficiency, decrease mosaicism, and identify correctly-edited embryos prior to transfer to recipient females are critical. Additionally, new methods for controlling the zygosity of founder animals are necessary to enable analysis of homozygous F0 animals and to avoid homozygous editing when targeting essential genes that cause embryonic lethality upon biallelic disruption. To these ends, we propose a research program that will significantly enhance our ability to introduce multiple types of edits into the marmoset genome, reduce mosaicism, control the zygosity of edits, and identify successfully edited embryos through prenatal genetic testing. We will disseminate these technologies and models through direct resource sharing, in-person trainings, deposition to NIH-supported Marmoset Coordination Center. Together, the proposed advances will significantly reduce the time, effort, costs and animal numbers necessary to marmoset genetic models and will unlock the true potential of marmosets for basic and translational neuroscience research.