Towards a toolbox for mammalian gene editing: Removing off-target activity and protein interactions of Cas9

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In the past 30 years, molecular genetics research has resulted in unprecedented breakthroughs in sequencing and annotating the mammalian genome. One of the main challenges now facing researchers is how to efficiently assign function to each gene. While researchers typically analyze phenotypic changes associated with gene loss-of-function, classical techniques for performing gene knockout in mammalian organisms are slow, laborious, and costly. Recently, DNA sequence-specific nucleases (SSNs) such as TALENs and CRISPR/Cas9 have emerged as tools that are poised to revolutionize this process. An over-arching goal of my lab is to develop robust technology for genetically altering mammalian organisms with high precision. Through a combination of molecular evolution and proteomics approaches, this program will develop a gene editing toolbox for mammals that will realize this possibility. Initially, we will focus on improving the current state-of-the-art gene editing tool, CRISPR/Cas9, which has been used successfully in diverse organisms. Cas9 employs a user-defined RNA target sequence to guide cleavage of a complementary DNA sequence within a gene. After DNA cleavage, cellular repair processes can result in gene inactivation, or can be exploited to add in new genetic material by using a repair template. However, while Cas9 is quite specific for its target sequence, it does cleave off-target sites in the genome, which could confound phenotypic analysis of mutants created using this technology. In addition, the possibility that the Cas9 protein binds to and influences proteins in mammalian cells, needs to be examined. To address these issues, we will set up the first platform for the continuous directed evolution of DNA-binding proteins in Canada, and use it to improve the DNA-binding specificity of Cas9. In addition, we will identify mammalian proteins that bind to Cas9 in cells and study the effects of these interactions on cell biology. We will then engineer Cas9 variants that are incapable of binding to these cellular factors. In the longer term we will extend this program to include other members of the CRISPR-Cas family with different DNA-binding specificities and mechanisms of DNA-cleavage, and explore the possibility of engineering highly specific RNA-guided RNA cleaving enzymes as a non-genetic alternative for regulating gene expression. We anticipate that his research program will benefit researchers in numerous fields by leading to the development of next-generation gene editing and gene regulating tools that will cut down on time and research costs associated with functional genomics studies in mammals and other organisms. In addition, commercialization of any new tools that we develop could lead to economic benefits for Canada. Finally, this proposal will bring cutting-edge protein evolution technology to Canada, and provide training for highly qualified personnel in its use.