18-BTT Clean genome editing through the use of nonintegrating T-DNA technology

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Genetic modification of crop species is the key to both food security and sustainable agriculture. The advent of CRISPR/Cas technology has provided a great advance in our ability to engineer genomes, but barriers remain to the routine employment of these methods in the most important agricultural species. This proposal addresses the most significant problem in engineering crop plants, that genome modification is associated with untargeted and potentially mutagenic integration of the machinery used to edit the genome. This is problematic due to the increased screening required to identify targeted transformants against the high background of random integrations. In addition, for commercial use the synthetic constructs must be eliminated from the genome in a process that can be lengthy and expensive for many crops. This project will develop a clean genetic engineering methodology based on the suppression of random transgene integration. This technology builds on the identification of a DNA Polymerase Theta (PolQ)-mediated pathway that is responsible for the majority of transgene integration events. We will suppress this pathway and investigate the effect on gene targeting frequencies. Proof of principle will be provided in Arabidopsis through targeted mutation of the ABI1 gene, resulting in the production of a dominant mutation that allows germination in the presence of abscisic acid. This work will be extended to Brassica to demonstrate the application of this technology to crop species. This project will significantly advance our ability to engineer crop genomes using a knowledge-based approach and informed by the applicants' considerable experience in plant transformation and DNA recombination mechanisms.