Developing a CRISPR-free mammalian recombineering system Project Summary / Abstract The ability to manipulate large segments of DNA in mammalian cells is essential for research and therapeutics. However, current programmable genome editing methods for creating kilobase-scale DNA manipulations require cytotoxic double-strand breaks (DSBs) or cannot create all required manipulations1. To address this major challenge, my laboratory at Scripps Research is developing a platform for mammalian recombineering that enables programmable large DNA insertions, deletions, and substitutions without the introduction of DSBs or DNA scars. Our preliminary data demonstrates that bacterial recombinases such as RecT from Enterococcus faecium2 are sufficient to catalyze whole gene insertion in the human genome. In this proposal, I describe the further characterization, optimization, and application of this system. First, we will fully characterize the mechanism and capabilities of efmRecT to create different size insertions, deletions, and substitutions across the genome using multiple methods of detection. Then we will engineer our recombineering system using supplemental bacterial recombineering proteins, mammalian repair protein inhibitors, and other manipulations informed by our mechanistic studies. We also aim to improve the efficiency of efmRecT through directed evolution in bacteria and/or mammalian cells. Finally, we will demonstrate the utility of our developed system by applying it to cell models of Gaucher Disease. The small size of RecT enables facile biomolecule delivery as the RecT gene, homology donor, and regulatory sequences can be encoded on a single recombinant AAV genome. As such, I am proposing an “all-in-one” AAV therapeutic where a single recombinant AAV can correct any of the hundreds of mutations associated with Gaucher Disease. If developed into a functional system for efficient genome editing, this method has the potential to impact fields spanning research to the clinic, including genomic perturbations of complex cell lines and organisms, methods for mammalian synthetic biology, and therapeutics.

Developing a CRISPR-free mammalian recombineering system Project Narrative The ability to manipulate large segments of DNA in mammalian cells is essential for research and therapeutics, but we lack genome editing techniques capable of making kilobase-scale DNA changes without cytotoxic double-strand breaks. In this proposal, I describe the development and use of a mammalian recombineering platform, a CRISPR-free genome editing system that enables large DNA insertions, deletions, and substitutions on the human genome without the introduction of double-strand breaks or DNA scars. Our compact recombineering machinery can enable “all-in-one” single-AAV correction of multi-allelic diseases, thus impacting fields from basic research to therapeutics.