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.

美国国立卫生研究院 (NIH) 最近的一项分析表明，小鼠模型对于生物医学研究的几乎各个方面都至关重要。 多达 71% 的 NIH R01 奖项可能使用淘汰小鼠项目 (KOMP) 和 国际基因敲除小鼠联盟（IKMC）成功产生条件基因敲除 (cKO) 近 90% 基因的 ES 细胞克隆，然而，其中只有约 25% 被转化为小鼠； 2013 年，CRISPR 技术影响了 KOMP/IKMC 中心的工作流程。 基于快速 CRISPR/Cas9 的绕过 ES 细胞的技术被广泛用于生成简单的 KO 小鼠，但事实证明对于 cKO 模型来说很难解决这个问题，我们开发了 Easi-CRISPR，其中单- 具有短同源臂的链DNA供体替代具有长同源臂的双链DNA供体 因为 Easi-CRISPR 简单、快速、高效，并且已被证明可在多个条件下重现。 多个实验室、许多团体，包括 KOMP 实验室，都采用了我们的方法来扩展基因座。 随着 Easi-CRISPR 等突破性技术的成功，我们建议解决更大的问题 这些挑战的一些例子是：（1）减少 (2) 解决 Cre 的可用性有限问题 驱动线；（3）开发基于 CRISPR 的技术，用于淘汰第一个模型，最优雅和 ES细胞时代KOMP中心使用的多功能设计，以及（4）创建“难以靶向”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， 总共包含约 15% 的蛋白质编码基因，对小鼠遗传学的许多方面进行了研究； 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主任提供建议 数百名研究人员为他们的研究选择最佳小鼠模型，并作为开发者 突破性技术推动该领域向前发展——我的经验有助于识别这些关键问题 利用基因组创新者奖，我将通过开发创新来解决这些问题。 Easi-CRISPR 等技术的成功完成将使个人受益。 通过其核心设施设计小鼠模型，并且还允许 KOMP/IKMC 中心生产 鉴于小鼠模型的使用率非常高，开发技术来解决这一问题。 这些普遍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。