Establishment of a PEG-mediated protoplast transformation system based on DNA and CRISPR/Cas9 ribonucleoprotein complexes for banana

文献类型： 外文期刊

第一作者： Wu, Shaoping

作者： Wu, Shaoping;Wu, Shaoping;Yang, Qiaosong;Shao, Xiuhong;Bi, Fangcheng;Hu, Chunhua;Yi, Ganjun;Zhu, Haocheng;Liu, Jinxing;Chen, Kunling;Zhu, Haocheng;Huo, Heqiang

作者机构：

关键词： PEG-mediated; Protoplast transformation; Deep amplicon sequencing; Genome editing; DNA-free

期刊名称：BMC PLANT BIOLOGY （ 影响因子：4.215； 五年影响因子：4.96 ）

ISSN： 1471-2229

年卷期： 2020 年 20 卷 1 期

页码：

收录情况： SCI

摘要： Background To date, CRISPR/Cas9 RNP editing tools have not been applied to the genetic modification of banana. Here, the establishment of a PEG-mediated banana protoplast transformation system makes it possible to build an efficient DNA-free method for a site-directed mutagenesis system. Results Protoplasts constitute a versatile platform for transient expression in plant science. In this study, we established a PEG-mediated banana protoplast transformation system. This system was further optimized for successfully delivering CRISPR/Cas9 and CRISPR/Cas12a plasmids and CRISPR/Cas9 ribonucleoproteins (RNPs) for targeted delivery of thePDSgene into banana protoplasts. Specific bands were observed in PCR-Restriction Enzyme Digestion (PCR-RE) assays, and Sanger sequencing of single clones further confirmed the occurrence of indels at target sites. Deep amplicon sequencing results showed that the editing efficiency of the CRISPR/Cas9 system was higher than that of the other two systems. Conclusions The PEG-mediated banana protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in banana. The application of the CRISPR/Cas9 RNP system enables the generation of banana plants engineered by DNA-free gene editing.

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