CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates

文献类型： 外文期刊

第一作者： Yan, Fang

作者： Yan, Fang;Wang, Jingwen;Zhang, Sujie;Lu, Zhenwan;Li, Shaofang;Xu, Jin;Feng, Jie;Zhou, Xueping;Zhou, Huanbin;Zhang, Sujie;Lu, Zhenwan;Zhou, Huanbin;Ji, Zhiyuan;Song, Congfeng;Chen, Gongyou;Zhou, Xueping

作者机构：

期刊名称：PLOS PATHOGENS （ 影响因子：6.7； 五年影响因子：6.7 ）

ISSN： 1553-7366

年卷期： 2023 年 19 卷 1 期

页码：

收录情况： SCI

摘要： Author summaryGenetic manipulation of bacterial genomes has greatly advanced our understanding of bacterial physiology and pathogenesis, which further helps us build up cell factories for the production of natural products and breed custom-designed crops for improved disease resistance. Compared with the donor-DNA-mediated homologous recombination methods commonly used in bacterial genome manipulation so far, the donor-DNA-free and CRISPR/Cas-based approaches widely developed in eukaryotic genome editing are more attractive due to their simplicity, versatility, high specificity, and efficiency. However, ectopic expression of Cas proteins causes cell death in many bacterial species, and CRISPR technology has been rarely studied in plant bacterial pathogens. In this work, by co-expressing mtLigD and mtKu genes, we confirm that the CRISPR/FnCas12a-induced lethal DSBs can be repaired in an error-prone manner, which results in efficient targeted genome editing in both Xoo and Pst strains. As a result, we have fulfilled various genome editing applications through simple gRNA design and construction, including sequentially knocking out all 25 xop genes in Xoo PXO99(A) strain, plasmid curing, etc. This study highlights the importance and promising application of utilizing heterologous NHEJ proteins in genome editing in microorganisms lacking NHEJ machines in the future. CRISPR-based genome editing technology is revolutionizing prokaryotic research, but it has been rarely studied in bacterial plant pathogens. Here, we have developed a targeted genome editing method with no requirement of donor templates for convenient and efficient gene knockout in Xanthomonas oryzae pv. oryzae (Xoo), one of the most important bacterial pathogens on rice, by employing the heterologous CRISPR/Cas12a from Francisella novicida and NHEJ proteins from Mycobacterium tuberculosis. FnCas12a nuclease generated both small and large DNA deletions at the target sites as well as it enabled multiplex genome editing, gene cluster deletion, and plasmid curing in the Xoo PXO99(A) strain. Accordingly, a non-TAL effector-free polymutant strain PXO99(A)D25E, which lacks all 25 xop genes involved in Xoo pathogenesis, has been engineered through iterative genome editing. Whole-genome sequencing analysis indicated that FnCas12a did not have a noticeable off-target effect. In addition, we revealed that these strategies are also suitable for targeted genome editing in another bacterial plant pathogen Pseudomonas syringae pv. tomato (Pst). We believe that our bacterial genome editing method will greatly expand the CRISPR study on microorganisms and advance our understanding of the physiology and pathogenesis of Xoo.

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