Efficient genome engineering in Agrobacterium tumefaciens C58 using recombineering assisted by CRISPR/Cas9

Recombineering, a technique derived from phage-encoded homologous recombination, has emerged as a vital approach for bacterial genome engineering. Agrobacterium tumefaciens is extensively utilized to transfer DNA into the host plant genomes. To facilitate the transformation of various plant species, particularly those of considerable economic value, genetic modifications of Agrobacterium strains are essential. Our previous studies established an Agrobacterium-specific phage-encoded homologous recombination system for Agrobacterium species. Yet, recent investigations have indicated that there is a substantial variability in the recombination efficiency of these recombineering systems for gene editing across different genome loci in A. tumefaciens. In this work, we present the development of an efficient genome engineering tool for A. tumefaciens by integrating recombineering with CRISPR/Cas9 technology. Initially, we found that lengthening the homology arms significantly enhanced genome editing efficiency. Nevertheless, at certain genomic sites, even when the length of the homology arms was increased, the editing efficiency remained suboptimal. Subsequently, combination of the Agrobacterium-specific recombineering system with the CRISPR/Cas9 system markedly enhanced the genome engineering efficiency. This study offers an enhanced and efficient genome engineering tool for A. tumefaciens, which could potentially be applied to other species within the Agrobacterium genus.