BTT EAGER: 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 project, in collaboration with Dr. Christopher West, University of Leeds, UK, 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 because of 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, especially those with long generation times (such as tree species) and those propagated vegetatively (such as potato, sweet potato, banana, etc.). This project, informed by the applicants' considerable experience in plant transformation and DNA recombination mechanisms, will develop a clean genetic engineering methodology based on the suppression of random transgene integration. Most current plant genome engineering technology platforms require, or have as an unintended consequence, the integration of introduced genome engineering reagents into the host chromosomes. The technology that will be tested in this project builds on the identification of a DNA Polymerase Theta (PolQ)-mediated pathway that is responsible for the majority of Agrobacterium-mediated transgene integration events. Suppression of this pathway, using dominant negative fragments of DNA polymerase theta introduced into the plant either as expressible T-DNA-encoded genes or directly as peptides, will mitigate T-DNA integration into the host genome. Inhibition of ectopic T-DNA integration will enhance the ability to detect gene targeting events mediated by homology-dependent repair. Proof of principle will be provided in Arabidopsis through targeted mutation of the ABI1 gene through homology-directed repair, 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.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.