IMAGE: INTEGRATE-Mediated Agrobacterium Genome Engineering

The ability to precisely engineer <i>Agrobacterium</i> strains is crucial for advancing their utility in plant biotechnology. We recently implemented the CRISPR RNA-guided transposase system, INTEGRATE, as an efficient tool for genetic modification in <i>Agrobacterium</i>. Despite its promise, the practical application of INTEGRATE in <i>Agrobacterium</i> strain engineering remains underexplored. Here, we present a standardized and optimized workflow that enables researchers to harness INTEGRATE for targeted genome modifications. By addressing common challenges, such as crRNA design, transformation efficiency, and vector eviction, this protocol expands the genetic toolkit available for <i>Agrobacterium</i>, facilitating both functional genomics and strain development for plant transformation. As a demonstration, we domesticated <i>Agrobacterium rhizogenes</i> K599 strain by deleting the 15-kb T-DNA region from its root-inducing plasmid pRi2659 and inactivating a thymidylate synthase gene to render the strain auxotrophic for thymidine. The protocol provides detailed guidance for each step, including target site selection, crRNA spacer cloning, <i>Agrobacterium</i> transformation, screening for targeted insertion and Cre/<i>loxP</i>-mediated deletion, and vector removal. This resource will empower new users to perform efficient and reproducible genome engineering in <i>Agrobacterium</i> using the INTEGRATE system, paving the way for broader adoption and innovation in plant biotechnology.