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

项目摘要 几十年来，使用实验室小鼠和大鼠进行的研究揭示了人类发育的机制 和疾病啮齿类动物很可能为每种药物的临床试验提供了关键的支持数据。 目前用于改善人类健康的药物或治疗方法。然而， 小鼠和大鼠理解和模拟复杂遗传问题（例如阿尔茨海默氏病， 心脏病和糖尿病），这是由于在四个或更多个基因座上遗传所需等位基因的概率很低， 与其他传统模式物种的后代相比，窝仔数较小。 建立复杂遗传性状模型所需的成本、时间和动物数量将减少， 一种增加多个基因座上两个等位基因之一传递到下一个的概率的方法 一代CRISPR/Cas9介导的基因转换在小鼠中是可行的， 通过将遗传信息从供体复制到生殖系中的受体等位基因来实现这一目标。简而言之，这 通过遗传编码的Cas9和靶向Cas9的向导RNA（gRNA）的种系限制性表达发生。 只有受体而不是供体等位基因。如果受体等位基因中的双链断裂被修复， 在染色体间同源定向修复中，受体等位基因被供体等位基因替换。CRISPR/Cas9- 因此，介导的基因转换将细胞的基因型从杂合改变为纯合， 确保任何产生的精子或卵子将只传递供体等位基因。 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。 在雌性和雄性小鼠生殖系中介导的基因转化，并测试基因转化的效率。 在同一个细胞中的两个位点上的转换。以前的工作表明，高水平的Cas9表达时间启动 在减数分裂早期，I是两性有效基因转换所必需的。本提案的目标1旨在 应用这些知识开发和测试三种BAC转基因驱动的Cas9表达，使用调节 减数分裂基因Tex 12、Prdm 9和Rad 51 ap 2的序列。迄今为止，CRISPR/Cas9介导的基因 在一个位点-酪氨酸酶处评估转化。目标2旨在量化基因的效率 在同一个细胞中，两个额外的基因座单独转换和两个基因座一起转换。数学模型 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“倍增”或“协调”概率。这项研究的结果是 对于最大化CRISPR/Cas9介导的基因转化的效用和未来生物学影响至关重要 方法来模拟和解决人类健康面临的各种遗传复杂挑战。

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.