Rhesus Macaque Somatic Cell Gene Editing Resource

恒河猴体细胞基因编辑资源

基本信息

批准号：
10222805
负责人：
Jon D Hennebold
金额：
$ 77.6万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-19 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10222805
关键词：
Anatomy Animals Assisted Reproductive Technology Biological Process CRISPR/Cas technology Cell physiology Cells Cercopithecidae Clinic Cryopreservation DNA DNA Double Strand Break DNA Sequence DNA lesion Data Detection Development Developmental Biology Disease Embryo Epigenetic Process Etiology Foundations Generations Genes Genetic Genetic Diseases Genetic Materials Genome Green Fluorescent Proteins Heterogeneity Human Human Genetics Human Genome Inherited Lead Lentivirus Vector Link Macaca Macaca mulatta Mammals Mediating Methodology Methods Modeling Modification Molecular Biology Mosaicism Mus Mutation Nonhomologous DNA End Joining Nonsense Codon Nucleotides Oocytes Oregon Outcome Phenotype Physiological Point Mutation Primates Process Proteins Publishing Reporter Reporting Research Research Personnel Resources Rodent Role Safety Scientist Site Specificity Standardization System Techniques Technology Testing Therapeutic Therapeutic Uses Time Tissues Transgenic Organisms Translations Ursidae Family base clinically relevant cohort de novo mutation design disease-causing mutation ds-DNA efficacy testing experience gain of function gene correction gene function genome editing homologous recombination human disease in vivo insight nonhuman primate prevent reconstruction repaired sex somatic cell gene editing success tool

项目摘要

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.

概括直到最近，在哺乳动物中产生特定基因改变的能力主要仅限于使用过去十年，基因组编辑的新进展开启了小鼠同源重组的大门。对大多数物种（包括人类）的 DNA 进行有针对性的改变的大门。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），在创建双链后在目标基因内生成小的插入或删除链状 DNA 断裂，进而可能导致终止密码子过早出现或产生无功能的蛋白质。或者，可以通过以下方式将所需的特定 DNA 序列整合到 DNA 损伤位点：同源定向修复（HDR）也已开发出来，可以修改基因。通过 DNA 碱基编辑 (DBE) 去除单核苷酸，从而避免产生双链 DNA 虽然这些方法是纠正致病遗传突变的重大进展。胚胎或以组织特异性方式发生的从头突变，人们对功效存在担忧和安全性。基因组编辑方法有可能创造出不同突变的“马赛克”，其中一些突变是可能是纠正性的，有些可能会损害细胞功能。另一个问题包括编辑。同源的“脱靶”DNA 序列，这反过来又会对 DNA 的功能产生负面影响。因此，必须创造一种可用于报告的动物。测试现有或尚未开发的基因组编辑技术的编辑异质性和脱靶效应为了实现这一目标，我们建议创建可能用于治疗的应用。恒河猴记者动物通过两种互补的方法，将为研究人员提供评估基于 NHEJ、MMEJ、HDR 或 DBE 的效率和特异性所需的资源我们将从每个报告动物中生成个性化的基因组组装，以高度促进。准确检测随后的脱靶效应将衍生出一组动物作为重要的是，基于密切的遗传，与人类的生理和解剖关系，对这些领域的基因组编辑活动的评估恒河猴报告动物将提供对其治疗潜力的深入了解。