Development of approaches to apply CRISPR/Cas9-mediated gene conversion to model complex genetic traits in mice

开发应用 CRISPR/Cas9 介导的基因转换来模拟小鼠复杂遗传性状的方法

基本信息

批准号：
10565297
负责人：
Kimberly Lynn Cooper
金额：
$ 52.14万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-02-01 至 2027-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10565297
关键词：
Alleles Alzheimer&apos s Disease Animal Model Animals Bacterial Artificial Chromosomes Biological Biological Process Biomedical Research CRISPR/Cas technology Cells Chromosomes Clinical Data Clinical Trials Complex Complex Genetic Trait Data Development Diabetes Mellitus Disease Embryonic Development Engineering Ensure Event Female Frequencies Future Gene Conversion Genes Genetic Genetic Diseases Genetic Processes Genotype Germ Lines Goals Guide RNA Health Heart Diseases Heterozygote Human Human Development Immunity Individual Inherited Knowledge Laboratories Laboratory Rat Laboratory mice Learning Link Litter Size Mediating Meiosis Meiotic Prophase I Metabolism Modeling Monophenol Monooxygenase Mus Oocytes Outcomes Research Pharmaceutical Preparations Pharmacologic Substance Phenotype Probability Rattus Research Rodent Spermatocytes Technology Testing Therapeutic Time Transgenes Transgenic Organisms Translating Work cost design egg future implementation genetic information genomic locus human disease improved insight loss of function male mathematical model next generation offspring permissiveness predictive modeling repaired sex sperm cell technology development tool transmission process

项目摘要

PROJECT SUMMARY Decades of research using laboratory mice and rats have revealed mechanisms of human development and disease. It is highly likely that rodents have provided critical supporting data for clinical trials of every pharmaceutical or therapeutic approach currently used to improve human health. There are, however, limitations to the utility of mice and rats to understand and model complex genetic problems (e.g. Alzheimer's, heart disease, and diabetes) due to the rare probability of inheriting desired alleles at four or more loci and the small litter size compared to offspring of other traditional model species. The cost, time, and number of animals needed to model complex genetic traits would be reduced by an approach to increase the probability one of the two alleles at multiple loci will be transmitted to the next generation. CRISPR/Cas9-mediated gene conversion, which is feasible in mice, can perhaps accomplish exactly this goal by copying genetic information from a donor to a recipient allele in the germline. Briefly, this occurs by germline-restricted expression of genetically encoded Cas9 and a guide RNA (gRNA) that targets only the recipient and not the donor allele. If the double strand break in the recipient allele is repaired by interchromosomal homology directed repair, the recipient allele is replaced by the donor allele. CRISPR/Cas9- mediated gene conversion therefore changes the genotype of the cell from heterozygous to homozygous and ensures any resulting sperm or egg will transmit only the donor allele. The proposed objectives will build on proof-of-feasibility to improve the efficiency of CRISPR/Cas9 mediated gene conversion in the female and male mouse germline and to test the efficiency of gene conversion at two loci in the same cell. Previous work suggests a high level of Cas9 expression timed to initiate during early meiosis I is necessary for efficient gene conversion in both sexes. Aim1 of this proposal seeks to apply this knowledge to develop and test three BAC transgenic drivers of Cas9 expression using regulatory sequences of the meiotic genes Tex12, Prdm9, and Rad51ap2. To date, CRISPR/Cas9-mediated gene conversion has been assessed at one locus – Tyrosinase. Aim 2 seeks to quantify the efficiency of gene conversion at two additional loci individually and of two loci together in the same cell. Mathematical models predict the efficiency of multi-locus gene conversion, but empirical evidence is required to determine whether the actual efficiency follows a `multiplicative' or `coordinated' probability. The outcomes of this research are critical to maximize the utility and future biological impact of CRISPR/Cas9-mediated gene conversion approaches to model and solve a variety of genetically complex challenges to human health.

项目概要使用实验室小鼠和大鼠进行的数十年研究揭示了人类发育的机制啮齿动物很可能为每种疾病的临床试验提供了关键的支持数据。然而，目前用于改善人类健康的药物或治疗方法。小鼠和大鼠在理解和模拟复杂遗传问题（例如阿尔茨海默病、心脏病和糖尿病），因为在四个或更多基因座遗传所需等位基因的可能性很小，并且与其他传统模式物种的后代相比，每窝产仔数较小。模拟复杂遗传特征所需的成本、时间和动物数量将减少增加多个基因座的两个等位基因之一被传递到下一个基因座的概率的方法 CRISPR/Cas9 介导的基因转换在小鼠中是可行的，也许可以实现这一目标。正是通过将遗传信息从供体复制到种系中的受体等位基因来实现这一目标。由基因编码的 Cas9 和靶向的向导 RNA (gRNA) 的种系限制表达而发生如果受体等位基因中的双链断裂被修复，则仅修复受体等位基因，而不是供体等位基因。染色体间同源性定向修复，受体等位基因被供体等位基因替换。因此，介导的基因转换将细胞的基因型从杂合子改变为纯合子，并且确保产生的任何精子或卵子仅传播供体等位基因。拟议的目标将建立在可行性证明的基础上，以提高 CRISPR/Cas9 的效率介导雌性和雄性小鼠种系中的基因转换并测试基因的效率先前的研究表明，在同一细胞中的两个位点发生高水平的 Cas9 表达是有时间启动的。在减数分裂早期，I 对于两性的有效基因转换都是必要的。应用这些知识利用监管来开发和测试 Cas9 表达的三个 BAC 转基因驱动程序减数分裂基因 Tex12、Prdm9 和 Rad51ap2 的序列迄今为止，CRISPR/Cas9 介导的基因。目标 2 旨在量化基因的效率。数学模型中两个额外基因座的单独转换和两个基因座的转换。预测多位点基因转换的效率，但需要经验证据来确定是否实际效率遵循“乘法”或“协调”概率。这项研究的结果是。对于最大化 CRISPR/Cas9 介导的基因转换的效用和未来生物学影响至关重要模拟和解决人类健康面临的各种遗传复杂挑战的方法。