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Summary Agrobacterium tumefaciens, the causal agent of plant crown gall disease, has been widely used to genetically transform many plant species. The inter‐kingdom gene transfer capability madeAgrobacteriuman essential tool and model system to study the mechanism of exporting and integrating a segment of bacterial DNA into the plant genome. However, many biological processes such asAgrobacterium‐host recognition and interaction are still elusive. To accelerate the understanding of this important plant pathogen and further improve its capacity in plant genetic engineering, we adopted a CRISPR RNA‐guided integrase system forAgrobacteriumgenome engineering. In this work, we demonstrate thatINsertion ofTransposableElements byGuideRNA–AssistedTargEting (INTEGRATE) can efficiently generate DNA insertions to enable targeted gene knockouts. In addition, in conjunction with Cre‐loxPrecombination system, we achieved precise deletions of large DNA fragments. This work provides new genetic engineering strategies forAgrobacteriumspecies and their gene functional analyses.
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This content will become publicly available on November 6, 2026
IMAGE: INTEGRATE-Mediated Agrobacterium Genome Engineering
The ability to precisely engineerAgrobacteriumstrains 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 inAgrobacterium. Despite its promise, the practical application of INTEGRATE inAgrobacteriumstrain 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 forAgrobacterium, facilitating both functional genomics and strain development for plant transformation. As a demonstration, we domesticatedAgrobacterium rhizogenesK599 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,Agrobacteriumtransformation, 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 inAgrobacteriumusing the INTEGRATE system, paving the way for broader adoption and innovation in plant biotechnology.
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- Award ID(s):
- 1917138
- PAR ID:
- 10655333
- Publisher / Repository:
- Frontiersin.org
- Date Published:
- Journal Name:
- Frontiers in Microbiology
- Volume:
- 16
- ISSN:
- 1664-302X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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