Towards Robust Multiplex Genome Engineering Beyond CRISPR-Cas9

迈向 CRISPR-Cas9 之外的稳健多重基因组工程

• 批准号: 10251146
• 负责人: Le Cong
• 金额: $ 47.31万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10251146
• 关键词: Autoimmune Diseases; Bar Codes; Biological Models; CRISPR/Cas technology; Cancer Model; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Complex; Computational Technique; Computer Models; DNA; Disease; Drug resistance; Engineering; Epigenetic Process; Genes; Genetic; Genetic Recombination; Genome; Genome engineering; Genomics; Goals; Human; Human Genome; In Vitro; Knowledge; Lead; Malignant Neoplasms; Medicine; Methods; Modality; Modification; Nerve Degeneration; Proteins; RNA; Resolution; Single Nucleotide Polymorphism; Structure; System; Technology; Testing; Therapeutic; Toxic effect; Variant; Work; cell type; design; genetic variant; genome editing; genomic locus; human disease; improved; in vivo; mammalian genome; microbial; success; tool; tumor

Project Abstract Towards Robust Multiplex Genome Engineering Beyond CRISPR-Cas9 Exemplified by the CRISPR-Cas9 system, gene-editing technology is a powerful collection of tools for probing the hidden mechanisms of human diseases by understanding and controlling the functions of human genome variants. However, existing CRISPR genome technologies have three major limitations: (1) low efficiency and lack of accuracy when making large genome modifications such as structural variants in complex diseases; (2) uncontrollable off-target effects that lead to unwanted editing and cellular toxicity; (3) variable activity and precision when performing CRISPR editing in mammalian genome across different contexts, e.g. genomic loci, cell types, and model systems. To overcome these limitations, many groups including our own have sought to develop improved CRISPR tools using experimental methods and computational techniques. Building on my previous expertise, I will work towards multiplex, robust and error- free genome engineering. My group will seek to design new microbial proteins with sequence- independent recombination and RNA-to-DNA editing capabilities (Focus 1). Then, to provide robust gene-editing tools for studying single-cell genomics, I propose to leverage versatile CRISPR designs to enable high-capacity cell barcoding to define genome dynamics at single- cell resolution (Focus 2). To validate our new tools and as initial demonstration, we will use in human cancer models, with a focus on studying the cellular dynamics that lead to tumor drug resistance through genetic perturbation (Focus 3). The ultimate goal of my lab is to enable error- free engineering of genomic variants at any sizes, with robust activities across in vitro and in vivo applications. I will use this precise toolkit to uncover the functions of long genome alterations in human diseases, a major “black box” in our genome. The success of the proposal has the promise to generate safe, reliable genome correction tools for therapeutics.

项目摘要 超越CRISPR-Cas9的强大多重基因组工程 以CRISPR-Cas9系统为例，基因编辑技术是一个强大的集合， 通过理解和控制人类疾病的隐藏机制来探索人类疾病的工具 人类基因组变异的功能。然而，现有的CRISPR基因组技术 三大局限性：（1）制作大型基因组时效率低、准确性差 复杂疾病中的结构变异等修饰;（2）无法控制的脱靶 导致不需要的编辑和细胞毒性的影响;（3）当 在不同背景下，例如基因组基因座，在哺乳动物基因组中进行CRISPR编辑， 细胞类型和模型系统。为了克服这些限制，包括我们在内的许多团体 已经寻求使用实验方法和计算方法开发改进的CRISPR工具， 技术.在我以前的专业知识的基础上，我将努力实现多元化，强大和错误- 免费基因工程我的团队将设计新的微生物蛋白质， 独立重组和RNA到DNA编辑能力（焦点1）。为了提供 强大的基因编辑工具研究单细胞基因组学，我建议利用多功能 CRISPR设计使高容量细胞条形码能够在单个细胞中定义基因组动态 细胞分辨率（焦点2）。为了验证我们的新工具并作为初始演示，我们将使用 人类癌症模型，重点是研究导致肿瘤药物的细胞动力学 通过遗传干扰的抗性（焦点3）。我实验室的最终目标是让错误- 任何大小的基因组变体的自由工程，在体外和体内都具有强大的活性， 体内应用。我将用这个精确的工具来揭示长基因组的功能 人类疾病的改变，我们基因组中的一个主要“黑匣子”。提案的成功 有望为治疗学提供安全可靠的基因组校正工具。