Engineering Highly Specific and Orthogonal CRISPR-Cas Systems

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这些活动的必要步骤。随着原核生物防御系统的发现，一种强大的基因组编辑方法最近变得可行。虽然这些工具已被用于对多种生物进行基因工程改造，但如果存在多个系统，这些自然编辑系统在可靶向的DNA序列类型和它们独立工作的能力方面受到限制。为了克服这些限制，这个项目寻求设计更灵活的编辑系统，可以独立行动。由此产生的系统有可能改变在所有生命形式中进行基因组编辑的方式，从而推动人们努力了解遗传疾病的基础，并设计出能够可持续地产生化学物质的微生物。从这些努力中获得的见解也将有助于揭示这些系统的组成部分如何相互作用，以及组装系统如何识别DNA目标。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，并扩大全校范围的生物技术系列研讨会，让当地行业代表和周边学校的学生参加。这项研究的长期目标是产生一种工具，可以方便、负担得起、有效地操纵任何生物体的基因组。CRISPR-Cas系统提供了最有前途的工具之一，然而每个系统只能靶向被称为PAM的DNA基序两侧的DNA序列。此外，许多这些系统使用相同的CRISPR rna负责目标识别，防止一次使用多个系统。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。这些变异将基于一个特性良好的CRISPR-Cas系统和一种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。由此产生的进化变体集合可以特异性地针对不同的DNA序列，并且可以在没有任何串扰的情况下一起实现。作为其功能的原理证明，将同时将多个变体引入嗜热链球菌中，以组合调节与酸奶质地相关的外多糖基因。本项目由CBET部门的生物技术与生化工程项目和分子与细胞生物学部门的合成与系统生物学项目共同资助。

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.