Savio D. Rodrigues,  Mansour Karimi,  Lennert Impens,  Els Van Lerberge,  Griet Coussens,  Stijn Aesaert, Debbie Rombaut,  Dominique Holtappels,  Heba M. M. Ibrahim,  Marc Van Montagu,  Jeroen Wagemans,  Thomas B. Jacobs,  Barbara De Coninck, and Laurens Pauwels.

PNAS January 12, 2021 118 (2) e2013338118

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.

Abstract

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.

See: https://www.pnas.org/content/118/2/e2013338118

Figure 1: Efficient base editing in A. tumefaciens. (A) Schematic overview of the Agrobacterium Target-AID CBE and sgRNA expression cassettes. dCas9, a nuclease-dead Cas9 with mutations indicated; L1, R1, L4, and L2, Gateway recombination sites; LVA, protein degradation tag; PglpT, E. coli glpT promoter; PJ23119, artificial promoter; PmCDA1, P. marinus cytidine deaminase; PvirB, promoter fragment of the A. tumefaciens pTiBo542 virB gene; Scaf, sgRNA scaffold; sfGFP, superfolder GFP; T3, terminator of the RNA polymerase III of bacteriophage T3; TrrfB, E. coli rrfB terminator; UGI, uracil DNA glycosylase inhibitor. (B) Overview of the virB promoter used. “A” and “C,” GreenGate overhangs; RBS, ribosome-binding site; vir-box, VirG recognition site. The start codon of the base editor is highlighted in orange. (C) The Atu1060 genomic locus. Locations of the protospacer used for the sgRNA are indicated together with the position of the primers used for genotyping. (D) Base-editing outcomes. (Top) Sequence obtained from EHA101 with the PAM (green highlighted), protospacer (yellow), and target codon (orange). The targeted C is indicated with a triangle, together with the position relative to the PAM. The solid circle denotes a bystander C. (Middle) Representative sequences from the colony obtained directly after transformation. (Bottom) Representative sequences obtained after streak plating without any prior AS induction (final clone). Edited bases are highlighted in red, the relevant codons are translated, and an asterisk indicates a stop codon. Gln, glutamine.