New toolkit expands genetic engineering capabilities for plant transformation using Agrobacterium

These mutations improve biocontainment, transformation stability, and overall performance in genetic engineering of plants. Complementing the strains, researchers also developed new plasmids and a user-friendly guide RNA filtering plugin for Geneious Prime to accelerate CRISPR editing.

Agrobacterium tumefaciens and A. rhizogenes are vital tools in plant biotechnology due to their natural ability to transfer DNA into plant genomes. Historically, plant scientists have relied on a narrow set of disarmed strains from two lineages—C58 and Ach5—for transformation. However, these strains are largely wild-type beyond virulence gene deletions, lacking enhancements for efficiency or containment. In contrast, E. coli has long benefited from specialized laboratory strains optimized for cloning and expression. Bringing similar improvements to Agrobacterium, such as auxotrophy for easier elimination or recA deletions to improve plasmid stability, has remained a challenge due to limitations in editing methods. CRISPR base-editing now offers a precise and scalable approach to engineer bacterial strains safely without lethal DNA breaks.

A study (DOI:10.1016/j.bidere.2025.100001) published in BioDesign Research on 27 February 2025 by Vincent J. Pennetti’s team, University of Georgia, creats a powerful new suite of genetically modified Agrobacterium strains and tools that promise to enhance plant transformation workflows across research and agriculture.

To generate improved Agrobacterium strains for plant transformation, researchers employed two genome engineering strategies: a modified allelic replacement method and a streamlined CRISPR-mediated base-editing system. Initially, they attempted to disarm the A. rhizogenes K599 and A. tumefaciens Chry5 strains using traditional allelic replacement. While K599 was successfully disarmed and validated via PCR and sequencing, disarming Chry5 required an enhanced marker-excision system with dual chromoproteins and antibiotic resistance markers for efficient visual and chemical selection. This approach ultimately produced a fully disarmed Chry5 strain (PEN400), which demonstrated no tumorigenicity in soybean infection assays. To expand strain functionality, researchers then applied a novel, single-component CRISPR base-editor system that incorporated visible chromoproteins into the plasmid backbones, allowing visual confirmation of plasmid loss without UV or molecular tools. These editors enabled efficient insertion of thymidine auxotrophy and recA deficiency across several widely used Agrobacterium strains. The team also developed a Python-based guide RNA filtering tool, BEEF, with an integrated Geneious Prime plugin (“PrimeGradeBeef”), which simplified the design and annotation of knockout guides. Resulting strains showed expected auxotrophic growth defects and recombination deficiencies, confirmed by phenotyping, sequencing, and methyl methanesulfonate sensitivity tests. Although the recA mutation reduced bacterial fitness in some genetic backgrounds—particularly K599—no such effect was observed in Chry5, highlighting strain-dependent variability. Importantly, transformation efficiency remained unaffected in thymidine auxotrophs, validating their use in genetic engineering workflows. This work provides a versatile toolkit for generating high-performance, biosafe Agrobacterium strains to meet growing needs in plant biotechnology.

This new toolkit enhances the genetic transformation workflow by offering safer, more manageable, and customizable Agrobacterium strains. Thymidine auxotrophy helps ensure environmental containment by reducing bacterial persistence, while recA deficiency improves plasmid stability—critical for precise gene delivery. The simplified CRISPR editing workflow and pre-engineered plasmids reduce technical barriers for labs with limited molecular biology experience. Moreover, the expanded strain repertoire—including disarmed versions of high-virulence backgrounds—opens the door for species previously recalcitrant to transformation. This innovation supports global crop improvement efforts and could accelerate functional genomics studies and gene editing pipelines in diverse plant species.

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References

DOI

10.1016/j.bidere.2025.100001

Original Source URL

https://doi.org/10.1016/j.bidere.2025.100001

Funding information

This material is based upon work supported by the Center for Bioenergy Innovation (CBI), U.S. Department of Energy, Office of Science, Biological and Environmental Research Program under Award Number ERKP886.

About BioDesign Research

BioDesign Research is dedicated to information exchange in the interdisciplinary field of biosystems design. Its unique mission is to pave the way towards the predictable de novo design and assessment of engineered or reengineered living organisms using rational or automated methods to address global challenges in health, agriculture, and the environment.