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

项目概要 虽然小鼠是神经科学许多领域的重要模型，但也有许多方面 更高的大脑功能和功能障碍无法在啮齿类动物中充分建模。因此，需要 具有更接近人类大脑结构和功能的新遗传模型。由于这些原因，非人类 灵长类动物（NHP）为研究高级大脑功能和大脑疾病提供了一个有吸引力的模型。一个有前途的 新兴的 NHP 模型是普通狨猴，这是一种小型新大陆灵长类动物，作为灵长类动物具有许多优势 遗传模型。尽管细菌 CRISPR/Cas 系统适用于靶向基因组工程 模型的创建彻底改变了现代生物学，狨猴基因组的编辑仍处于起步阶段。 鉴于狨猴基因组编辑需要大量时间和金钱，增加编辑的新方法 效率，减少嵌合，并在移植到受体雌性之前识别正确编辑的胚胎 至关重要的。此外，需要新的方法来控制创始人动物的接合性，以实现 分析纯合 F0 动物，并在针对重要基因时避免纯合编辑 双等位基因破坏时导致胚胎致死。为此，我们提出了一项研究计划 显着增强我们将多种类型的编辑引入狨猴基因组的能力，减少 嵌合体，控制编辑的接合性，并通过产前遗传识别成功编辑的胚胎 测试。我们将通过直接资源共享、面对面的方式传播这些技术和模型 培训、向 NIH 支持的狨猴协调中心提交材料。总之，拟议的进展将 显着减少狨猴遗传模型所需的时间、精力、成本和动物数量 释放狨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.