A Mobile Genome Editing System Using Dicistronic CRISPR/Cas9-tRNA (CC9-tR) For Grapevine And Other Perennial Crops (Non-Transgenic Genome Editing System For Grapevine)

It is expected that 50-81% of the grapevine growing acreage in the USA will fall out of production due to weather changes by 2040. This urgently demands the breeding of varieties more resilient to periods of environmental stress, without losing fruit quality. Genome editing has revolutionized our ability to modify the genome in a targeted manner. The reliance of GE technologies on genetic transformation, in-vitro culture and sexual crosses, hampers the implementation of GE in transformation-recalcitrant and long living horticultural crops. DNA-free editing methods are used to avoid transgene integration. However, these present lower efficiency rates, the need for laborious mutant screening, and high levels of somaclonal variation. Moreover, their reliance on in-vitro culture make them unsuitable for many crops. We hypothesize that a system that capitalizes on the systemic movement of polycistronic CRISPR/Cas9-tRNA (CC9-tR) from transgenic to non-transgenic tissues could address the practical issues surrounding GE-based strategies for perennial crops.The overall goal of this innovative proposal is to advance the breeding of perennials by developing, validating and applying a novel GE system that will generate non-transgenic lines, and circumvent the need for in-vitro culture and sexual crosses. The project objectives fit the priorities of the crosscutting program “Agricultural Innovation through Gene Editing” by delivering a genome editing system that does not require plant cell transformation or the use of plant pest sequences in an economically important crop with a publicly available reference genome. The findings of the proposed research will provide knowledge transferable to other agriculturally-important perennial crops.