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Efficient CRISPR-mediated base editing in Agrobacterium spp.

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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 inter-spaced short palindromic repeats (CRISPR)-mediated base editing to enable the efficient introduction of targeted point mutations into the genomes of both Agrobacterium tumefaciens and Agro-bacterium 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.
Keywords
Multidisciplinary, Agrobacterium, plant transformation, CRISPR, hairy root disease, base editing, REGULATORS BABY-BOOM, T-DNA GENE, BINARY VECTORS, GENOMIC DNA, TUMEFACIENS, TRANSFORMATION, RHIZOGENES, PLASMID, REGION, ACTIVATION

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Citation

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MLA
Rodrigues, Savio D., et al. “Efficient CRISPR-Mediated Base Editing in Agrobacterium Spp.” PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 118, no. 2, 2021, doi:10.1073/pnas.2013338118.
APA
Rodrigues, S. D., Karimi, M., Impens, L., Van Lerberge, E., Coussens, G., Aesaert, S., … Pauwels, L. (2021). Efficient CRISPR-mediated base editing in Agrobacterium spp. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 118(2). https://doi.org/10.1073/pnas.2013338118
Chicago author-date
Rodrigues, Savio D., Mansour Karimi, Lennert Impens, Els Van Lerberge, Griet Coussens, Stijn Aesaert, Debbie Rombaut, et al. 2021. “Efficient CRISPR-Mediated Base Editing in Agrobacterium Spp.” PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 118 (2). https://doi.org/10.1073/pnas.2013338118.
Chicago author-date (all authors)
Rodrigues, Savio D., 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 Jacobs, Barbara De Coninck, and Laurens Pauwels. 2021. “Efficient CRISPR-Mediated Base Editing in Agrobacterium Spp.” PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 118 (2). doi:10.1073/pnas.2013338118.
Vancouver
1.
Rodrigues SD, Karimi M, Impens L, Van Lerberge E, Coussens G, Aesaert S, et al. Efficient CRISPR-mediated base editing in Agrobacterium spp. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. 2021;118(2).
IEEE
[1]
S. D. Rodrigues et al., “Efficient CRISPR-mediated base editing in Agrobacterium spp.,” PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 118, no. 2, 2021.
@article{8686391,
  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 inter-spaced short palindromic repeats (CRISPR)-mediated base editing to enable the efficient introduction of targeted point mutations into the genomes of both Agrobacterium tumefaciens and Agro-bacterium 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.}},
  articleno    = {{e2013338118}},
  author       = {{Rodrigues, Savio D. and Karimi, Mansour and Impens, Lennert and Van Lerberge, Els and Coussens, Griet and Aesaert, Stijn and Rombaut, Debbie and Holtappels, Dominique and Ibrahim, Heba M. M. and Van Montagu, Marc and Wagemans, Jeroen and Jacobs, Thomas and De Coninck, Barbara and Pauwels, Laurens}},
  issn         = {{0027-8424}},
  journal      = {{PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}},
  keywords     = {{Multidisciplinary,Agrobacterium,plant transformation,CRISPR,hairy root disease,base editing,REGULATORS BABY-BOOM,T-DNA GENE,BINARY VECTORS,GENOMIC DNA,TUMEFACIENS,TRANSFORMATION,RHIZOGENES,PLASMID,REGION,ACTIVATION}},
  language     = {{eng}},
  number       = {{2}},
  pages        = {{8}},
  title        = {{Efficient CRISPR-mediated base editing in Agrobacterium spp.}},
  url          = {{http://dx.doi.org/10.1073/pnas.2013338118}},
  volume       = {{118}},
  year         = {{2021}},
}

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