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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

基本信息

批准号：
10666490
负责人：
Channabasavaiah Gurumurthy
金额：
$ 43.38万
依托单位：
UNIVERSITY OF NEBRASKA MEDICAL CENTER
依托单位国家：
美国
项目类别：
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

项目摘要

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.

老鼠模型对于生物医学研究的几乎每一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 R01奖项可能会使用鼠标模型。淘汰赛老鼠项目(KOMP)和国际基因敲除小鼠联盟(IKMC)成功地产生了条件基因敲除 (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，非常基于CRISPR/Cas9的绕过ES细胞的快速技术被广泛采用来产生简单的KO 小鼠，但事实证明对CKO模型来说很困难。为了解决这个问题，我们开发了EASI-CRISPR，其中单一的-CRISPR。同源臂短的滞留DNA供体替代长臂的双链DNA供体同源双臂。因为EASI-CRISPR简单、快速和高效，并且已被证明可多次重现在多个实验室，包括KOMP实验室在内的许多组织都采用了我们的方法。要延长像EASI-CRISPR这样的突破性技术的成功，我们建议解决更大的问题，对于使用鼠标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可获得性有限的问题车手系列；(3)开发基于CRISPR的技术，用于淘汰第一款车型，最优雅和在胚胎干细胞时代的KOMP中心使用的多功能设计；以及(4)创造CKO等位基因，以应对“难以达到的目标” 基因，包括那些具有单个外显子或异常长的外显子的基因，以及具有重复序列的基因，加在一起约占所有蛋白质编码基因的15%。在老鼠遗传学的许多方面做过工作；作为一名研究人员为我自己的工作设计和培育模型，作为一名转基因核心董事建议数百名研究人员为他们的研究选择最佳的鼠标模型，并作为推动该领域向前发展的突破性技术-我的经验帮助确定了以下关键问题田野。利用基因组创新者奖，我将通过开发创新的与EASI-CRISPR类似的技术。这个项目的成功完成将使个人受益通过其核心设施设计鼠标模型，并将允许KOMP/IKMC中心生产通常最有用的模型。鉴于鼠标模型的使用率非常高，开发技术以解决这样的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。