EAGER: Lambda-Red-enhanced homologous recombination in plants

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The ability to edit the information in an organism's genome is transforming not only the way scientists address basic biological questions, but also how solutions to deal with critical societal problems such as human diseases, food security, biofuel production, etc. are sought. The predominant genome editing technologies currently in use in plants and animal systems require the introduction of breaks in the genome at the precise locations where the changes or edits are wanted. Although highly efficient, current genome editing procedures are still laborious and not free of risks due, among other things, to undesired breaks in the genome. Although high-efficiency genome editing approaches that do not rely on the introduction of chromosomal breaks in the genome already exist, they have only been proven to work in bacterial systems. Using the current understanding of how DNA-break-free genome editing approaches work and what their requirements are, this project aims to modify components of an existing bacterial system and adapt them to function in plant cells. In addition to the benefits of developing a new DNA-break-free genome editing approach for plants, the generated knowledge would inform similar developments in animal systems. This project will provide an ideal training platform for researchers and undergraduate students to experience first-hand how basic biological knowledge can be translated into potentially transformative new technologies. To further inform the public and motivate young scientists, the project will lead workshops at a local museum on principles of plant biology and genetics. The main goal of this project is to develop a Lambda-Red-based homologous recombination (HR) system for eukaryotic cells. The implementation of such system would not only provide an alternative to the current genome editing approaches, but may also offer important advantages, such as the elimination of the need to induce potentially dangerous dsDNA breaks in the genome, very short sequence homology requirements, high efficiency, and the potential to generate large arrays of modifications, from single nucleotide changes to large insertions and deletions. Importantly, the molecular mechanisms behind the Lambda-Red system have been thoroughly investigated, thus providing critical clues on the requirements that need to be fulfilled in order to test the feasibility of Lambda-Red-mediated HR in a eukaryotic system. These requirements include: 1) an actively replicating genome, 2) a linear DNA fragment that can serve as the repair template, and 3) active recombineering enzymes targeted to the nucleus of the cell. To fulfill these requirements, codon-optimized, nuclear-localized recombineering enzymes will be expressed in plant cells together with a virus-derived replicon that can serve as the source for repair template DNA. A codon-optimized, nuclear-localized meganuclease will be also expressed to linearize the viral replicons to generate the required linear repair template. The work, if successful, will open doors to new means of precise genome manipulation for the benefits of agriculture and beyond.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.