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: 10202690
• 负责人: Channabasavaiah Gurumurthy
• 金额: $ 44.5万
• 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/10202690
• 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 driver; Cre-LoxP; Development; Exons; Future; Genes; 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; conditional knockout; design; ds-DNA; embryonic stem cell; experience; genome editing; improved; innovation; innovative technologies; mouse genetics; 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.

小鼠模型实际上对于生物医学研究的各个方面都是必不可少的。 NIH最近的分析表明 多达71％的NIH R01奖项可以使用鼠标模型。淘汰鼠标项目（KOMP）和 国际淘汰小鼠财团（IKMC）成功地产生了有条件的淘汰赛 （CKO）ES细胞克隆近90％的基因；但是，其中只有大约25％通过 2013年，CRISPR技术影响KOMP/IKMC中心的工作流程的一年。随后，非常 快速CRISPR/CAS9基于绕过ES细胞的技术广泛用于生成简单的KO 小鼠，但事实证明对CKO模型很难。为了解决这个问题，我们开发了Easi-Crispr，其中 滞留的DNA供体，具有短同源性臂替代双链DNA供体的供体长 同源性臂。因为Easi-Crispr是简单，快速和高效的，并且在多个 多个实验室的基因座，包括科普实验室在内的许多团体都采用了我们的方法。扩展 Easi-Crispr等突破性技术的成功，我们建议解决更大的问题 通用使用鼠标模型的项目。这些挑战的一些例子是： （1）减少 在繁殖Cre-loxp模型上花费的时间过多； （2）解决CRE的有限可用性 驾驶员线； （3）开发基于CRISPR的淘汰赛技术，是最优雅和最优雅的技术 在ES细胞擦除的Komp中心使用的多功能设计，并且； （4）创建“难以定位”的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，这些基因 共同完成所有蛋白质编码基因的约15％。研究了小鼠遗传学的许多方面； 作为我自己工作的研究人员设计和育种模型，作为转基因核心顾问 数百名研究人员研究了他们的研究最佳鼠标模型选项，并作为开发人员 突破性技术以推动领域向前发展 - 我的经验有助于确定这些关键问题 使用基因组创新奖，我将通过开发创新来解决这些问题 沿Easi-Crispr的技术。成功完成该项目将使个人受益 通过其核心设施设计鼠标模型，还将允许KOMP/IKMC中心生产 最有用的模型。鉴于鼠标模型的使用非常高，开发技术来解决 预计将来，这种普遍问题将对生物医学研究的所有领域产生重大影响。