Directed Evolution and Engineering of CRISPR-Associated Nucleases

Abstract

CRISPR-Cas systems provide prokaryotes with a remarkable mechanism for adaptive immunity, and recent efforts to understand these systems have provided tremendous insight into the complex nature of these systems. Despite being highly diverse, all CRISPR-Cas systems are able to mediate genomic integration of DNA fragments, sequence-specific RNA processing, and programmable DNA cleavage. Excitingly, each of these processes has numerous uses in biotechnology, medicine, and biological research, and significant effort in the last five years has focused on repurposing CRISPR-Cas systems for applications in these areas. While many Cas-proteins have been successfully applied outside of their native context, protein engineering and directed evolution can be used to enhance their function or optimize their activity for a specific application. This thesis presents our work on engineering two CRISPR-associated nucleases: Csy4, a sequence-specific endoribonuclease, and Cas9, an RNA-guided endonuclease.

We report the development of a phage-assisted continuous evolution (PACE) system for Csy4 that enables the directed evolution of Csy4 variants with optimized activity or altered specificity. We demonstrate that this system can be used to evolve Csy4 variants with improved cleavage rates and to evolve Csy4 variants with orthogonal RNA-binding specificity that are more specific than wild-type Csy4. We also report the development of intein-Cas9 nucleases that are activated by the presence of a cell-permeable small molecule. We demonstrate that these intein-Cas9s enable small-molecule control of Cas9 activity and have improved genome-editing specificity compared to wild type Cas9.