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.

中文翻译：

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CRISPR RNA引导整合酶可在根癌农杆菌中实现高效靶向基因组工程


根癌农杆菌是植物冠瘿病的病原体，已被广泛用于对许多植物物种进行基因改造。跨界基因转移能力使农杆菌成为研究将细菌 DNA 片段输出并整合到植物基因组中的机制的重要工具和模型系统。然而，许多生物过程，例如农杆菌宿主识别和相互作用仍然难以捉摸。为了加速对这一重要植物病原体的了解并进一步提高其在植物基因工程方面的能力，我们采用了CRISPR RNA引导的整合酶系统进行农杆菌基因组工程。在这项工作中，我们证明通过引导RNA辅助靶向（INTEGRATE）插入转座元件可以有效地生成DNA插入，​​从而实现靶向基因敲除。此外，结合CreloxP重组系统，我们实现了大DNA片段的精确删除。这项工作为农杆菌物种及其基因功能分析提供了新的基因工程策略。