UC San Diego

The development of efficient tools for plant genome engineering is key to dramatically expedite basic research approaches and, at the same time, facilitate translational and biotechnological strategies in agriculture to improve crops and meet future food demand. For decades, technologies enabling precise Gene Targeting (GT) and efficient DNA knock-in or sequence replacement via Homology-Directed Repair (HDR) in plants has remained challenging, and although some recent reports suggest that progress is being made, no efficient and reproducible protocols have yet been established. Agrobacterium tumefaciens-mediated insertion entails the mobilization of the T-DNA into the host plant genome. However, this process occurs randomly and the system is very inefficient for targeted knock-ins. For this project, we capitalize on the extensive knowledge of Agrobacterium tumefaciens biology and combine it with CRISPR-Cas9 genome editing technologies to develop a novel strategy for high efficiency targeted knock-in. To do so, we decided to engineer Agrobacterium tumefaciens to help us reduce the distance between the T-DNA (harboring the cassette for knock-in) and the Cas9 cut site that marks the location where specifically the targeted insertion will occur. The set of tools we are presenting here will serve not to only accelerate basic research but also assist in engineering crop genomes in order to meet our future food demand in an efficient and sustainable manner.