Project Summary Despite extraordinary advances in genome engineering, tools for precise and efficient gene correction across all cell types and desired edits remain lacking. Current programmable DNA cleavage tools, such as CRISPR-Cas9, rely on cellular DNA repair mechanisms, which are inefficient and do not function in post-mitotic cells. Thus, genome editing still needs efficient, robust tools that can make a variety of specific DNA sequence alterations. These tools could have broad applications across both basic biological discovery, allowing for new modalities of screening, and therapeutics, including engineered cell therapies. The proposed work will address these needs by combining computational discovery, biochemical characterization, and enzyme engineering to develop integrase-based tools for programmable, multiplexed insertion of large genes in diverse cell types independent of DNA repair. The discovery, characterization, and engineering of these new integrase proteins will both build upon our deep history of CRISPR enzyme discovery, as well as draw from new, high-throughput approaches to mine biological diversity. Complementary to the discovery of these new enzymes, we will combine Cas9-based genome editing with integrase engineering to develop programmable, multiplexed genome integration systems that do not depend on DNA repair mechanisms, allowing integration of large sequences in any cell type. We will explore delivery mechanisms, including viruses, electroporation, and novel lipid nanoparticle formulations to edit T cells and neurons. We will engineer aspects of the integrases, including protein engineering and site mutagenesis, to boost activity of the system and screen many insertion sites to develop design rules for the technology. Moreover, through studying orthogonal integrases sites we can develop multiplexed versions of the insertion tool to edit up to three sites in a given cell with superior efficiency over other tools. We will apply these multiplexed integrases to develop a new screening system, where tagging of multiple genes can be used for determining protein interaction partners in high throughput. Our new integrase systems will also be applied to the development of multiple-edited T-cells for improved immuno-oncology therapies. The multiple technologies resulting from these discoveries and engineering efforts will overcome the limitations of existing genome and epigenome engineering approaches and serve as a valuable resource for broader biomedical research. Programmable gene integration with CRISPR-recruited integrases will allow for more advanced genome engineering applications to be pursued in cells and in vivo, accelerating the pace of biomedical research, enabling greater exploration of basic biological processes and disease mechanisms, and promoting novel therapeutic developments.

Project Narrative There are more than 5,000 human diseases caused by known genetic variation, including mutations, insertions, and deletions, but programmable tools for gene insertion to reliably study and model these diseases are lacking. We propose a two-pronged approach to developing novel platforms for programmable insertion of large sequences (up to 10 kb) by (1) mining bacterial and archaeal systems to identify novel integrases that can be used in mammalian cells and (2) combining CRISPR gene editors and integrases to insert large payloads into genomes in a programmable and multiplexed manner that is independent of cellular repair pathways and can function in both dividing and non-dividing cells. We will apply these new technologies for both basic biological discovery, such as high-throughput protein interaction assays, as well as therapeutic uses, such as modification of T-cells for immune oncology, and will accelerate the study and modeling of disease biology, providing a chassis for further engineering of programmable gene insertion tools.