Rhesus Macaque Somatic Cell Gene Editing Resource

摘要 直到最近，在哺乳动物身上创造特定基因改变的能力主要局限于使用 小鼠体内的同源重组。在过去的十年里，基因组编辑的新发展开启了 为对包括人类在内的大多数物种的DNA引入有针对性的改变打开了大门。新的编辑技术 依赖于容易出错的修复过程非同源末端连接(NHEJ)和微同源介导的- 末端连接(MMEJ)：在创建双基因后，在目标基因内产生小的插入或缺失 搁浅的DNA断裂，进而可能导致过早的终止密码子或无功能的蛋白质。 或者，可以通过以下方式在DNA损伤部位整合所需的特定DNA序列 同源定向修复(HDR)。还开发了其他系统，其允许修改 通过DNA碱基编辑(DBE)获得单核苷酸，从而避免产生双链DNA 休息一下。虽然这些方法是在纠正致病的遗传突变方面的重大进步 以组织特异性方式发生的胚胎或从头突变，有关于疗效的担忧 和安全。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 可能是矫正的，也可能是对细胞功能有害的。另一个问题包括在中进行编辑 同源的脱靶DNA序列，而这反过来又会对一种 不相关的基因或一组基因。因此，当务之急是创造一种可以用来 测试现有的或尚未开发的基因组编辑技术的编辑异质性和非目标效果 这可能会被用于治疗应用。为了实现这一目标，我们建议创建 一只恒河猴记者动物通过两种免费的方法为调查人员提供 评估基于NHEJ、MMEJ、HDR或DBE的效率和特异性所需的资源 接近了。我们将从每个报告动物中生成个性化的基因组组装，以促进高度 准确检测后续偏离目标的效果。将派生出一群动物，作为 为后续的记者评估项目奠定基础。重要的是，基于近亲的基因， 与人类的生理和解剖关系，对这些生物的基因组编辑活动的评估 猕猴报告动物将提供对它们治疗潜力的洞察。

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.