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

项目总结 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，不能在啮齿类动物身上充分建模。因此，有必要 大脑结构和功能更接近人类的新遗传模型。出于这些原因，非人类 灵长类动物(NHP)为研究高级脑功能和脑疾病提供了一个有吸引力的模型。一个有希望的人 新兴的NHP模式是常见的绒猴，一种小型的新大陆灵长类动物，作为一种 遗传模型。尽管细菌CRISPR/Cas系统对靶向基因组工程的适应性 而模型的创建已经给现代生物学带来了革命性的变化，对绒猴基因组的编辑仍处于起步阶段。 考虑到绒猴基因组编辑所需的大量时间和金钱，增加编辑的新方法 效率，减少嵌合体，并在移植给受体雌性之前识别正确编辑的胚胎是 危急时刻。此外，控制创始动物合子的新方法是必要的，以使 对纯合的F0动物进行分析，并在针对以下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，我们提出了一个研究计划，该计划将 显著增强我们在绒猴基因组中引入多种类型编辑的能力，减少 嵌合体，控制编辑的合并度，通过产前遗传鉴定成功编辑的胚胎 测试。我们将通过面对面的直接资源共享来传播这些技术和模式 培训，向NIH支持的Marmoset协调中心交纳。加在一起，拟议的进展将 显著减少构建基因模型和意愿所需的时间、精力、成本和动物数量 释放绒猴在基础和翻译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.