Engineering Highly Specific and Orthogonal CRISPR-Cas Systems

工程高度特异性和正交的 CRISPR-Cas 系统

• 批准号: 1403135
• 负责人: Chase Beisel
• 金额: $ 40万
• 依托单位: North Carolina State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-15 至 2018-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1403135&HistoricalAwards=false
• 关键词: Engineering; Highly; Specific; Orthogonal; CRISPR

1403135Beisel, Chase L.An organism's genome provides the blueprints for all cellular functions and behaviors. Accordingly, editing the genome is an essential step toward understanding and controlling any of these activities. A powerful method of genome editing recently became available with the discovery of prokaryotic defense systems. While these tools have been used to genetically engineer diverse organisms, these natural editing systems are constrained in the types of DNA sequences that can be targeted and their ability to function independently if more than one system is present. To overcome these limitations, this project seeks to engineer editing systems that are more flexible and that can act independently. The resulting systems have the potential to transform how genome editing is performed in all forms of life, thereby driving efforts to understand the basis of genetic diseases and to engineer microbes that can produce chemicals sustainably. Insights from these efforts will also help reveal how the components of these systems interact with each other and how the assembled systems identify DNA targets. Beyond these scientific advances, this project will train the next generation of scientists and engineers to harness natural biological processes as tools for studying and engineering biology. This project will also integrate topics on gene editing systems into existing coursework for undergraduate and graduate students and expand a campus-wide biotechnology seminar series to include local industry representatives and students from surrounding schools.The long-term goal of this research is to generate tools that permit facile, affordable, and efficient genome manipulation of any organism. CRISPR-Cas systems offer one of the most promising tools, yet each system can only target DNA sequences flanked by a DNA motif called a PAM. Furthermore, many of these systems utilize the same CRISPR RNAs responsible for target recognition, preventing the use of multiple systems at one time. To address these particular challenges, this project will generate orthogonal CRISPR-Cas systems that recognize different PAMs. These variants will be generated based on a well-characterized CRISPR-Cas system and a unique selection scheme that couples bacteriophage resistance and genome targeting. The resulting collection of evolved variants can specifically target diverse DNA sequences and can be implemented together without any crosstalk. As a proof-of-principle demonstration of their functionality, multiple variants will be simultaneously introduced into the dairy-culturing bacterium S. thermophilus for the combinatorial regulation of exopolysaccharide genes associated with yogurt texture.This project is co-funded by the Biotechnology and Biochemical Engineering Program of the CBET Division and by the Synthetic and Systems Biology Program of the Division of Molecular and Cell Biology.

1403135 Beisel，Chase L.生物体的基因组提供了所有细胞功能和行为的蓝图。因此，编辑基因组是理解和控制任何这些活动的重要一步。随着原核生物防御系统的发现，最近出现了一种强大的基因组编辑方法。虽然这些工具已被用于基因工程多样化的生物体，但这些天然编辑系统在可以靶向的DNA序列类型以及如果存在多个系统则独立发挥功能的能力方面受到限制。为了克服这些限制，该项目寻求设计更灵活且可以独立操作的编辑系统。由此产生的系统有可能改变基因组编辑在所有生命形式中的执行方式，从而推动了解遗传疾病的基础和设计可以可持续生产化学物质的微生物的努力。从这些努力中获得的见解也将有助于揭示这些系统的组件如何相互作用，以及组装的系统如何识别DNA靶标。除了这些科学进步之外，该项目还将培训下一代科学家和工程师利用自然生物过程作为研究和工程生物学的工具。该项目还将把基因编辑系统的主题整合到本科生和研究生的现有课程中，并扩大校园范围的生物技术研讨会系列，包括当地行业代表和周边学校的学生。该研究的长期目标是生成允许对任何生物进行简单，负担得起和有效的基因组操作的工具。CRISPR-Cas系统提供了最有前途的工具之一，但每个系统只能靶向侧翼有称为PAM的DNA基序的DNA序列。此外，这些系统中的许多系统利用相同的CRISPR RNA负责靶标识别，从而防止同时使用多个系统。为了解决这些特殊的挑战，该项目将生成识别不同PAM的正交CRISPR-Cas系统。这些变体将基于充分表征的CRISPR-Cas系统和独特的选择方案产生，该方案将噬菌体抗性和基因组靶向结合起来。所得到的进化变体的集合可以特异性地靶向不同的DNA序列，并且可以在没有任何串扰的情况下一起实施。作为其功能性的原理证明，将多个变体同时引入乳品培养细菌S中。该项目由CBET分部的生物技术和生物化学工程项目以及分子和细胞生物学分部的合成和系统生物学项目共同资助。