Rhesus Macaque Somatic Cell Gene Editing Resource

总结 直到最近，在哺乳动物中创造特定遗传改变的能力主要限于使用 小鼠中的同源重组。在过去的十年里，基因组编辑的新发展开启了 这是一扇向包括人类在内的大多数物种的DNA引入靶向改变的大门。新的编辑技术 依赖于非同源末端连接（NHEJ）和微同源介导的易错修复过程， 末端连接（MMEJ），以在产生双末端连接后在靶基因内产生小的插入或缺失。 链DNA断裂，这又可能导致过早终止密码子或无功能蛋白质。 或者，所需的特异性DNA序列可以通过以下方式整合在DNA损伤位点处： 同源定向修复（HDR）。也已经开发了允许修改通信协议的其他系统。 通过DNA碱基编辑（DBE），从而避免产生双链DNA 休息.虽然这些方法是纠正致病遗传突变的主要进展， 胚胎或以组织特异性方式发生的新生突变，存在疗效方面的问题 和安全性基因组编辑方法有可能创造出不同突变的“马赛克”，其中一些突变 可能是矫正性的，有些可能对细胞功能有害。另一个问题包括编辑在 同源的“脱靶”DNA序列，这反过来又会对细胞的功能产生负面影响。 不相关的基因或一组基因。因此，当务之急是创造一种报告动物， 测试现有或有待开发的基因组编辑技术的编辑异质性和脱靶效应 有可能被用于治疗应用。为了实现这一目标，我们建议建立 恒河猴记者动物通过两个免费的方法，将提供调查人员， 评估NHEJ、MMEJ、HDR或DBE的效率和特异性所需的资源 接近。我们将从每只报告动物中生成个性化的基因组组装，以促进高度 准确检测后续脱靶效应。将获得一组动物，其将用作 为后续记者评估项目奠定基础。重要的是，基于相近的基因， 与人类的生理和解剖关系，评估这些基因组编辑活动， 恒河猴报告动物将提供对其治疗潜力的深入了解。

SUMMARY Until recently, the ability to create specific genetic alterations in mammals was primarily restricted to using homologous recombination in mice. Over the past decade, new developments in genome editing opened the door for introducing targeted changes to the DNA of most species, including humans. New editing techniques rely on the error-prone repair processes nonhomologous-end joining (NHEJ) and microhomology mediated- end joining (MMEJ) to generate small insertions or deletions within a target gene after the creation of a double stranded DNA break, which in turn may lead to a premature stop codon or a nonfunctional protein. Alternatively, a desired specific DNA sequence can be integrated at the site of the DNA lesion through homology-directed repair (HDR). Other systems also have been developed that allow for the modification of a single nucleotide through DNA base editing (DBE), thereby avoiding the creation of double stranded DNA breaks. While these approaches are major advances toward correcting disease-causing inherited mutations in embryos or de novo mutations that occur in a tissue specific manner, there are concerns regarding efficacy and safety. Genome editing methods have the potential to create a “mosaic” of different mutations, some that may be corrective and some that may be detrimental to cellular function. Another concern includes editing in homologous “off-target” DNA sequences, which in turn can have negative impacts on the function of an unrelated gene or set of genes. Therefore, it is imperative that a reporter animal is created that can be used to test editing heterogeneity and off-target effects for existing or yet to be developed genome editing techniques that would potentially be used for therapeutic applications. To accomplish this objective, we propose to create a rhesus macaque reporter animal through two complimentary approaches that will provide investigators with the resources needed for assessing the efficiency and specificity of NHEJ-, MMEJ-, HDR-, or DBE-based approaches. We will generate personalized genome assemblies from each reporter animal to facilitate highly accurate detection of subsequent off-target effects. A cohort of animals will be derived that will serve as the foundation for subsequent reporter assessment projects. Importantly, based on the close genetic, physiological, and anatomical relationship with humans, assessment of genome editing activities in these rhesus macaque reporter animals will provide insight into their therapeutic potential.