Journal
PLANT BIOTECHNOLOGY JOURNAL
Volume 20, Issue 10, Pages 1916-1927Publisher
WILEY
DOI: 10.1111/pbi.13872
Keywords
Cre-loxP recombination; Large DNA fragment deletion; Transposon; Targeted DNA insertion; T-DNA
Funding
- National Science Foundation Plant Genome Research Program [IOS-1725122, IOS-1917138]
- Iowa State University Interdepartmental Plant Biology Major fellowship
- seed grant fund from Crop Bioengineering Center of Iowa State University
- USDA NIFA Hatch project [IOW04714]
- State of Iowa funds
- National Science Foundation
- Iowa State University Library
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In this study, a CRISPR RNA-guided integrase system was used for Agrobacterium genome engineering, resulting in targeted gene knockouts and precise deletions of large DNA fragments. This research provides new strategies for genetic engineering of Agrobacterium species and gene functional analysis.
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 made Agrobacterium an 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 as Agrobacterium-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 for Agrobacterium genome engineering. In this work, we demonstrate that INsertion of Transposable Elements by Guide RNA-Assisted TargEting (INTEGRATE) can efficiently generate DNA insertions to enable targeted gene knockouts. In addition, in conjunction with Cre-loxP recombination system, we achieved precise deletions of large DNA fragments. This work provides new genetic engineering strategies for Agrobacterium species and their gene functional analyses.
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