Establishment of a PEG-mediated protoplast transformation system based on DNA and CRISPR/Cas9 ribonucleoprotein complexes for banana
Establishment of a PEG-mediated protoplast transformation system based on DNA and CRISPR/Cas9 ribonucleoprotein complexes for banana
复制标题
基于DNA和CRISPR/Cas9核糖核蛋白复合物的PEG介导的香蕉原生质体转化系统的建立
DOI：
10.1186/s12870-020-02609-8
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发表时间：
2020-09-15
影响因子：
5.3
通讯作者：
Yi, Ganjun
中科院分区：
文献类型：
--
作者：
Wu, Shaoping;Zhu, Haocheng;Yi, Ganjun
背景迄今为止，CRISPR/Cas9 RNP编辑工具尚未应用于香蕉的遗传修饰。在这里，PEG介导的香蕉原生质体转化系统的建立使得有可能建立一个有效的DNA-免费的方法，定点诱变system.ResultsProtoplasts构成一个通用的平台，在植物科学中的瞬时表达。本研究建立了PEG介导的香蕉原生质体转化体系。该系统被进一步优化以成功地递送CRISPR/Cas9和CRISPR/Cas 12 a质粒以及CRISPR/Cas9核糖核蛋白（RNP），用于将PDS基因靶向递送到香蕉原生质体中。在PCR-限制酶消化（PCR-RE）测定中观察到特异性条带，并且单克隆的桑格测序进一步证实了在靶位点处indel的存在。深度扩增测序结果表明，CRISPR/Cas9系统的编辑效率高于其他两个系统。结论PEG介导的香蕉原生质体转化系统可作为香蕉瞬时表达分析和sgRNA验证的快速有效工具。CRISPR/Cas9 RNP系统的应用使得能够通过无DNA基因编辑产生香蕉植物。
BackgroundTo 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.ResultsProtoplasts 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.ConclusionsThe 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.