Engineering Highly Specific and Orthogonal CRISPR-Cas Systems

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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.