EAGER: Development of a Novel Method for Rapid and Efficient Genome Editing in Tomatoes

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Recent technological developments have made it possible to alter the genetic makeup of plants in deliberate and precise ways that were unthinkable just a few years ago. The new technology, called genome editing, can have a transformative impact on plant breeding and agriculture because it will be possible to introduce favorable genetic variants to increase yields and food quality. Genome editing techniques are based on a bacterial mechanism that is used for defense against infectious agents by changing their DNA. The great advantage of adapting this bacterial technique is that once fully developed, the technology will not leave foreign genes in the edited plant like previous technologies did. There are remaining challenges to the genome editing technique. However, this project will develop new methods to facilitate genome editing in plants. These methods make use of a group of cells in young plant parts that retain the potential to develop new organs, similar to the same property of animal stem cells. The research will test new ways of introducing the molecules that carry out genome editing in tomato stem cells. The high risk experiments will have a major impact on advancing the new genome editing technology.This project will create a tomato transgenic line carrying both the Cas9 endonuclease and a conditional (lethal in the presence of a substrate) counter-selectable marker (CSM) driven by a shoot meristem-specific promoter. The latter represents a conditional and tissue-specific meristem suicide gene. A sgRNA targeting both the CSM and a gene of interest will be delivered to young stems by Agrobacterium-mediated infection of CSM-transgenic tomato seedlings. Expression of the sgRNA in the meristem will lead to inactivation of both CSM and target genes. The injected plants will then be decapitated at the inoculation site to induce new shoot regeneration. The CSM will be expressed exclusively in shoot apical meristems. Thus, application of the selective substrate will lead to meristem death in non-mutated regenerating meristems, but will allow the stem of the plant to continue support of newly formed, target-edited, and substrate-resistant shoots. This approach can significantly expedite genome editing at an unprecedented rate, and in addition can make genome editing a reality for crop species that are recalcitrant to transformation and regeneration with traditional methodologies.