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. 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 the PDS gene 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. 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.

中文翻译：

---

基于DNA和CRISPR / Cas9核糖核蛋白复合物的香蕉PEG介导原生质体转化系统的建立。

迄今为止，CRISPR / Cas9 RNP编辑工具尚未应用于香蕉的基因修饰。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为定点诱变系统建立一种有效的无DNA方法。原生质体构成了植物科学中瞬时表达的通用平台。在这项研究中，我们建立了PEG介导的香蕉原生质体转化系统。该系统经过进一步优化，可成功递送CRISPR / Cas9和CRISPR / Cas12a质粒以及CRISPR / Cas9核糖核蛋白（RNP），以将PDS基因靶向递送至香蕉原生质体中。在PCR限制性酶切（PCR-RE）分析中观察到特定条带，单个克隆的Sanger测序进一步证实了目标位点的插入缺失。深度扩增子测序结果表明，CRISPR / Cas9系统的编辑效率高于其他两个系统。PEG介导的香蕉原生质体转化系统可作为快速有效的工具用于香蕉中的瞬时表达测定和sgRNA验证。CRISPR / Cas9 RNP系统的应用使无DNA基因编辑工程改造的香蕉植株得以生成。