Summary Agrobacterium tumefaciens, the causal agent of plant crown gall disease, has been widely used to genetically transform many plant species. The inter‐kingdom gene transfer capability made Agrobacterium an essential tool and model system to study the mechanism of exporting and integrating a segment of bacterial DNA into the plant genome. However, many biological processes such as Agrobacterium‐host recognition and interaction are still elusive. To accelerate the understanding of this important plant pathogen and further improve its capacity in plant genetic engineering, we adopted a CRISPR RNA‐guided integrase system for Agrobacterium genome engineering. In this work, we demonstrate that INsertion of Transposable Elements by Guide RNA–Assisted TargEting (INTEGRATE) can efficiently generate DNA insertions to enable targeted gene knockouts. In addition, in conjunction with Cre‐loxP recombination system, we achieved precise deletions of large DNA fragments. This work provides new genetic engineering strategies for Agrobacterium species and their gene functional analyses.

Agrobacterium-mediated transformation is a widely used gene delivery method for fundamental researches and crop trait improvement projects. Auxotrophic Agrobacterium tumefaciens strains are highly desirable for plant transformation because they can be easily removed from the explants after co-cultivation due to their dependence on essential nutrient supplementation. The thymidine auxotrophic A. tumefaciens strain LBA4404Thy- has been successfully used for plant transformation, however, auxotrophic version of other commonly used strains are not available yet to public laboratories. Here we report the generation of EHA101, EHA105 and EHA105D thymidine auxotrophic strains. These strains exhibited thymidine-dependent growth in the bacterial medium, and the transient GUS expression assay using Arabidopsis seedling showed that they retain the equivalent T-DNA transfer capability as the original strains thus are suitable for plant transformation.