Development of Modular CRISPR Genome Editing Technologies and Tools

模块化CRISPR基因组编辑技术和工具的开发

• 批准号: 9815153
• 负责人: Channabasavaiah Gurumurthy
• 金额: $ 45.57万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9815153
• 关键词: Address; Adopted; Affect; Alleles; Award; Biomedical Research; Breeding; Bypass; CRISPR/Cas technology; Clone Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Communities; Complex; Core Facility; Cre-LoxP; Development; Exons; Future; Genes; Genetic; Genetic Research; Genomics; Individual; Institutes; International; Knock-out; Knockout Mice; Laboratories; Methods; Modeling; Molecular Genetics; Mus; Problem Solving; Proteins; Repetitive Sequence; Reproducibility; Research; Research Personnel; Single-Stranded DNA; Technology; Time; Transgenic Organisms; United States National Institutes of Health; Work; arm; base; design; ds-DNA; embryonic stem cell; experience; genome editing; improved; innovation; innovative technologies; mouse model; success; tool; virtual

Mouse models are essential for virtually every aspect of biomedical research. A recent NIH analysis indicates that as many as 71% of NIH R01 awards may use mouse models. The Knockout Mouse Project (KOMP) and the International Knockout Mouse Consortium (IKMC) were successful in generating conditional knockout (cKO) ES cell clones for nearly 90% of genes; however, only about 25% of these were converted into mice by 2013, the year when CRISPR technologies affected the workflow at KOMP/IKMC centers. Subsequently, very rapid CRISPR/Cas9-based technologies that bypass ES cells were widely adopted for generating simple KO mice, but proved difficult for cKO models. To solve this problem, we developed Easi-CRISPR, in which single- stranded DNA donors with short homology arms substitute for double-stranded DNA donors with long homology arms. Because Easi-CRISPR is simple, rapid, and efficient, and has proved reproducible at multiple loci in multiple laboratories, many groups, including KOMP laboratories, have adopted our method. To extend the success of breakthrough technologies like Easi-CRISPR, we propose to solve much bigger problems that are universal to projects using mouse models. Some examples of these challenges are; (1) reducing the excessive amount of time spent in breeding Cre-LoxP models; (2) addressing the limited availability of Cre driver lines; (3) developing CRISPR-based technologies for knock-out first models, the most elegant and versatile design used at KOMP centers in the ES cell era, and; (4) creating cKO alleles for “difficult-to-target” genes, including those with single exons or unusually long exons, and genes with repetitive sequences, which together comprise about 15% of all protein coding genes. Having worked on many aspects of mouse genetics; as a researcher designing and breeding models for my own work, as a transgenic core director advising hundreds of investigators on the best mouse model options for their research, and as a developer of breakthrough technologies to move the field forward—my experience helped to identify these key problems in the field. Using the Genomic Innovator award, I will solve each of these problems by developing innovative technologies along the lines of Easi-CRISPR. Successful completion of this project will benefit individuals designing mouse models through their core facilities and will also allow KOMP/IKMC centers to produce the most generally useful models. Given the very high use of mouse models, developing technologies to address such universal problems is expected to have a major impact on all fields of biomedical research in the future.

小鼠模型对于生物医学研究的几乎每个方面都是必不可少的。美国国立卫生研究院最近的一项分析表明， 多达71%的NIH R 01奖项可能使用小鼠模型。敲除小鼠项目（KOMP） 国际敲除小鼠联盟（IKMC）成功地产生了条件敲除 (cKO)ES细胞克隆了近90%的基因;然而，只有大约25%的基因被转化为小鼠。 2013年，CRISPR技术影响了KOMP/IKMC中心的工作流程。随后，非常 绕过ES细胞的基于CRISPR/Cas9的快速技术被广泛用于产生简单的KO 小鼠，但事实证明对于cKO模型来说很困难。为了解决这个问题，我们开发了一种CRISPR， 具有短同源臂的双链DNA供体取代具有长同源臂的双链DNA供体 同源臂由于CRISPR简单、快速、高效，并且已被证明在多个环境中具有可重复性， 基因座在多个实验室，许多团体，包括KOMP实验室采用了我们的方法。延长 像CRISPR这样的突破性技术的成功，我们建议解决更大的问题， 对于使用小鼠模型的项目是通用的。这些挑战的一些例子是：（1）减少 在培育Cre-LoxP模型上花费的时间过多;（2）解决Cre有限的可用性问题 （3）开发基于CRISPR的技术，用于淘汰第一个模型，最优雅， 在ES细胞时代KOMP中心使用的通用设计，以及（4）为“难以靶向”的cKO等位基因创建cKO等位基因 基因，包括具有单个外显子或异常长外显子的基因，以及具有重复序列的基因， 总共包含约15%的蛋白质编码基因。研究过小鼠遗传学的许多方面; 作为一名研究人员，为我自己的工作设计和繁殖模型，作为一名转基因核心主任， 数百名研究人员为他们的研究提供了最佳的小鼠模型选择， 突破性技术推动该领域向前发展-我的经验有助于确定这些关键问题， 外地利用基因组创新者奖，我将通过开发创新的 沿着CRISPR的路线。这个项目的成功完成将使个人受益 通过他们的核心设施设计小鼠模型，也将允许KOMP/IKMC中心生产 最常用的模型。鉴于小鼠模型的使用率非常高，开发技术来解决 这种普遍性问题预计将对未来生物医学研究的所有领域产生重大影响。