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

• 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
• 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' 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

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.

项目总结 几十年来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。啮齿动物很可能为每一项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，有一些， 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)，这是由于罕见的在四个或更多基因座上遗传所需等位基因的可能性以及 与其他传统模式物种的后代相比，它们的产仔数较小。 建立复杂遗传特征模型所需的成本、时间和动物数量将减少 增加多个座位上两个等位基因中的一个传递给下一个等位基因的概率的方法 一代。CRISPR/Cas9介导的基因转化在小鼠身上是可行的，也许可以实现 正是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，这是 通过种系限制性表达遗传编码的Cas9和靶向于 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 染色体间同源定向修复，受者等位基因被供体等位基因取代。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子，并 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 介导性基因在雌性和雄性小鼠生殖系中的转化及基因效率的检测 同一细胞内两个基因座的转换。先前的研究表明，Cas9的高水平表达在启动时间上是 在减数分裂早期，I是两性间有效的基因转换所必需的。这项提案的目的是为了 应用这些知识开发和测试三个Cas9表达的BAC转基因驱动器 减数分裂基因Tex12、Prdm9和Rad51ap2的序列。到目前为止，CRISPR/Cas9介导的基因 对酪氨酸酶的转化率进行了评估。目标2试图量化基因的效率 在同一细胞中，两个额外基因座单独或同时在两个基因座上的转换。数学模型 预测多位点基因转化的效率，但需要经验证据来确定 实际效率遵循“乘法”或“协调”的概率。这项研究的结果是 最大限度地发挥CRISPR/Cas9介导的基因转化的效用和未来的生物学影响至关重要 模拟和解决人类健康面临的各种基因复杂挑战的方法。