dCas9-BE 3 and dCas12a-BE3 Systems Mediated Base Editing in Kiwifruit Canker Causal Agent Pseudomonas syringae pv. actinidiae

pv. ( ) causes bacterial canker of kiwifruit with heavy economic losses. However, little is known about the pathogenic genes of . CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas-mediated genome editing technology has dramatically facilitated the characterization of gene functi...

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Bibliographic Details
Published in:International journal of molecular sciences 2023-02, Vol.24 (5)
Main Authors: Liu, Bo, Song, Wenpeng, Wang, Linchao, Wu, Yantao, Xu, Xiaoting, Niu, Xiangli, Huang, Shengxiong, Liu, Yongsheng, Tang, Wei
Format: Article
Language:English
Subjects:
Actinidia - genetics
Gene Editing
Gene Knockout Techniques
Plant Diseases - microbiology
Pseudomonas syringae
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Summary:pv. ( ) causes bacterial canker of kiwifruit with heavy economic losses. However, little is known about the pathogenic genes of . CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas-mediated genome editing technology has dramatically facilitated the characterization of gene function in various organisms. However, CRISPR genome editing could not be efficiently employed in due to lacking homologous recombination repair. The base editor (BE) system, which depends on CRISPR/Cas, directly induces single nucleoside C to T without homology recombination repair. Here, we used dCas9-BE3 and dCas12a-BE3 systems to create substitutions of C to T and to convert CAG/CAA/CGA codons to stop codons (TAG/TAA/TGA) in . The dCas9-BE3 system-induced single C-to-T conversion frequency of 3 to 10 base positions ranged from 0% to 100%, with a mean of 77%. The dCas12a-BE3 system-induced single C-to-T conversion frequency of 8 to 14 base positions in the spacer region ranged from 0% to 100%, with a mean of 76%. In addition, a relatively saturated gene knockout system covering more than 95% of genes was developed based on dCas9-BE3 and dCas12a-BE3, which could knock out two or three genes at the same time in the genome. We also found that and were involved in the virulence of kiwifruit. The HopF2 effector can potentially interact with proteins such as RIN, MKK5, and BAK1, while the HopAO2 effector can potentially interact with the EFR protein to reduce the host's immune response. In conclusion, for the first time, we established a PSA.AH.01 gene knockout library that may promote research on elucidating the gene function and pathogenesis of .
ISSN:1422-0067