Efficient CRISPR-mediated base editing in Agrobacterium spp.

Abstract

Agrobacteriumspp. 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. AlthoughAgrobacteriumspp. are standard tools for plant molecular biologists, current laboratory strains have remained unchanged for decades and functional gene analysis ofAgrobacteriumhas 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 bothAgrobacterium tumefaciensandAgrobacterium rhizogenes. As an example, we generated EHA105 strains with loss-of-function mutations inrecA, which were fully functional for maize (Zea mays) transformation and confirmed the importance of RolB and RolC for hairy root development byA. rhizogenesK599. 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 improvedAgrobacteriumstrains for more efficient plant transformation and gene editing.

Highly Efficient CRISPR-Mediated Base Editing in Sinorhizobium meliloti

“…Notably, the CBE systems did not prefer a particular substrate combination. Interestingly, the editing window of both the ABE and CBE systems was seven bases (positions 4-10, with PAM at positions 21-23) and 10 bases (positions 1-8, 12, and 15, with PAM at positions 21-23), respectively, in S. meliloti and was, therefore, broader than the five-base window that was reported for other prokaryotes (Chen et al, 2018;Gu et al, 2018;Zheng et al, 2018;Tong et al, 2019;Wang et al, 2020;Rodrigues et al, 2021). Considerable effort has been devoted to expanding the editing window of base editors.…”

“…For instance, multiplexed modifications of gene families could be very time-and laborconsuming using allelic replacement approaches. CRISPRmediated base editing systems have been widely applied in a variety of microbes because of their simplicity and efficiency (Chen et al, 2018;Gu et al, 2018;Zheng et al, 2018;Tong et al, 2019;Rodrigues et al, 2021). S. meliloti is an ideal organism for base editing because the genome has a high GC content (62.7%) and thus, "NGG" PAM sites appear frequently in target loci.…”

“…(E,F) Editing efficiency assay for multigene mutations using the CBE system. was successfully mutated (Chen et al, 2017(Chen et al, , 2018Rodrigues et al, 2021). The sucrose-inducible sacB gene product from Bacillus subtilis is lethal for gram-negative bacteria and has been used as an efficient counter-selectable marker for the construction of suicide vectors in rhizobia (Quandt and Hynes, 1993).…”

“…The base editing systems are highly efficient, lower the rate of off-target effects, and do not require either DNA double-strand cleavage or the repair of a donor template. These dramatic improvements have made base editing a widely applicable tool for gene disruption in a variety of bacteria (Banno et al, 2018;Gu et al, 2018;Zheng et al, 2018;Tong et al, 2019;Rodrigues et al, 2021).…”

IRI-CCE: A Platform for Evaluating CRISPR-based Base Editing Tools and Its Components

“…Although the p35S is less active, this aspect is of great significance for rapid validation of cloned plasmids in E. coli or design a heterologous expression platform. Moreover, the toxic effects of BE components fused with nickase (nCas9) or dead Cas9 (dCas9) under the control of strong promoters are earlier reported in bacteria [16, 17]. In this regard, combination of differential-strength promoters for the optimal expression of base editing (BE) components including sgRNAs preferably might avoid the toxicity issue in E. coli .…”