METHODS article
Front. Microbiol.
Sec. Microbiotechnology
Volume 16 - 2025 | doi: 10.3389/fmicb.2025.1676008
Agrobacterium Genome Engineering using INTEGRATE
Provisionally accepted
- 1Department of Agronomy, Iowa State University, Ames, United States
- 2Iowa State University of Science and Technology Crop Bioengineering Center, Ames, United States
- 3Iowa State University, Interdepartmental Plant Biology Major, Ames, United States
- 4Iowa State University of Science and Technology Department of Agronomy, Ames, United States
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The ability to precisely engineer Agrobacterium strains is crucial for advancing their utility in plant biotechnology. We recently implemented the CRISPR RNA-guided transposase system, INTEGRATE, as an efficient tool for genetic modification in Agrobacterium. Despite its promise, the practical application of INTEGRATE in Agrobacterium strain engineering remains underexplored. Here, we present a standardized and optimized workflow that enables researchers to harness INTEGRATE for targeted genome modifications. By addressing common challenges, such as crRNA design, transformation efficiency, and vector eviction, this protocol expands the genetic toolkit available for Agrobacterium, facilitating both functional genomics and strain development for plant transformation. As a demonstration, we domesticated Agrobacterium rhizogenes K599 strain by deleting the 15-kb T-DNA region from its root-inducing plasmid pRi2659 and inactivating a thymidylate synthase gene to render the strain auxotrophic for thymidine. The protocol provides detailed guidance for each step, including target site selection, crRNA spacer cloning, Agrobacterium transformation, screening for targeted insertion and Cre/loxP-mediated deletion, and vector removal. This resource will empower new users to perform efficient and reproducible genome engineering in Agrobacterium using the INTEGRATE system, paving the way for broader adoption and innovation in plant biotechnology.
Keywords: Agrobacterium rhizogenes, Agrobacterium tumefaciens, Cre-Lox, CRISPR Integrase, disarming, large DNA fragment deletion, Targeted DNA insertion
Received: 29 Jul 2025; Accepted: 08 Sep 2025.
Copyright: © 2025 Aliu, Chen, Lee and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Keunsub Lee, klee@iastate.edu
Kan Wang, kanwang@iastate.edu
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