PNAS|农杆菌属中的高效CRISPR介导的碱基编辑

农杆菌是一种重要的植物病原菌，是引起冠瘿或毛状根病的病原菌。它们独特的感染策略依赖于将部分DNA传递到植物细胞。

正是由于这种能力，这些植物病原菌成为植物基因工程和农业生物技术不可缺少的有力工具。

虽然农杆菌是植物分子生物学家的标准工具，但目前的实验室菌株几十年来一直没有变化，而且耗时的突变策略阻碍了农杆菌功能基因的分析。

本研究，我们开发了成簇的规则间隔的短回文重复序列（CRISPR）介导的碱基编辑技术，从而能够有效地将定点突变导入根癌农杆菌和发根农杆菌的基因组中。

作为一个例子，我们培育了具有recA功能缺失突变的EHA105菌株，这些菌株对玉米（Zea mays）的转化具有完全功能，并证实了RolB和RolC对发根发育的重要性。

我们的方法在转化后的10个菌落中有9个是高效的，至少80%的细胞都进行了编辑。对EHA105和K599的基因组进行了重测序，并对碱基编辑质粒进行了全基因组脱靶分析。

目前的脱靶点是Cas9非依赖性脱靶点的特征，并指向TC基序作为所用胞苷脱氨酶的活性热点。

我们预计CRISPR介导的碱基编辑是“工程设计工程师”（“engineering the engineer）的开始，从而改进农杆菌菌株，以实现更有效的植物转化和基因编辑。

Agrobacterium spp. are important plant pathogens that are the causative agents of crown gall or hairy root disease. Their unique infection strategy depends on the delivery of part of their DNA to plant cells. Thanks to this capacity, these phytopathogens became a powerful and indispensable tool for plant genetic engineering and agricultural biotechnology. Although Agrobacterium spp. are standard tools for plant molecular biologists, current laboratory strains have remained unchanged for decades and functional gene analysis of Agrobacterium has been hampered by time-consuming mutation strategies. Here, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-mediated base editing to enable the efficient introduction of targeted point mutations into the genomes of both Agrobacterium tumefaciens and Agrobacterium rhizogenes. As an example, we generated EHA105 strains with loss-of-function mutations in recA, which were fully functional for maize (Zea mays) transformation and confirmed the importance of RolB and RolC for hairy root development by A. rhizogenes K599. Our method is highly effective in 9 of 10 colonies after transformation, with edits in at least 80% of the cells. The genomes of EHA105 and K599 were resequenced, and genome-wide off-target analysis was applied to investigate the edited strains after curing of the base editor plasmid. The off-targets present were characteristic of Cas9-independent off-targeting and point to TC motifs as activity hotspots of the cytidine deaminase used. We anticipate that CRISPR-mediated base editing is the start of “engineering the engineer,” leading to improved Agrobacterium strains for more efficient plant transformation and gene editing.

https://www.pnas.org/content/pnas/118/2/e2013338118.full.pdf

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