Efficient CRISPR-mediated base editing in Agrobacterium spp.

Significance Agrobacteria are plant-pathogenic bacteria that can deliver DNA to plant cells as part of their infection strategy. This property has been used for decades to generate transgenic plants and, more recently, to deliver gene-editing reagents to plant cells. Notwithstanding their importance for research and industry, laboratory strains have not been improved much over the years and several aspects of Agrobacterium biology and pathogenesis remain poorly understood. Here, we developed a CRISPR-mediated base-editing approach to efficiently modify the genome of Agrobacterium. We show that single-nucleotide changes can be introduced at targeted positions in both the Agrobacterium tumefaciens and Agrobacterium rhizogenes genomes. Whole-genome analysis of edited strains revealed only a limited number of unintentional mutations. Agrobacterium spp. are important plant pathogens that are the causative agents of crown gall or hairy root disease. Their unique infection strategy depends on the delivery of part of their DNA to plant cells. Thanks to this capacity, these phytopathogens became a powerful and indispensable tool for plant genetic engineering and agricultural biotechnology. Although Agrobacterium spp. are standard tools for plant molecular biologists, current laboratory strains have remained unchanged for decades and functional gene analysis of Agrobacterium has been hampered by time-consuming mutation strategies. Here, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-mediated base editing to enable the efficient introduction of targeted point mutations into the genomes of both Agrobacterium tumefaciens and Agrobacterium rhizogenes. As an example, we generated EHA105 strains with loss-of-function mutations in recA, which were fully functional for maize (Zea mays) transformation and confirmed the importance of RolB and RolC for hairy root development by A. rhizogenes K599. Our method is highly effective in 9 of 10 colonies after transformation, with edits in at least 80% of the cells. The genomes of EHA105 and K599 were resequenced, and genome-wide off-target analysis was applied to investigate the edited strains after curing of the base editor plasmid. The off-targets present were characteristic of Cas9-independent off-targeting and point to TC motifs as activity hotspots of the cytidine deaminase used. We anticipate that CRISPR-mediated base editing is the start of “engineering the engineer,” leading to improved Agrobacterium strains for more efficient plant transformation and gene editing.