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
作者： Wu, Shaoping 1 ; Zhu, Haocheng 3 ; Liu, Jinxing 3 ; Yang, Qiaosong 2 ; Shao, Xiuhong 2 ; Bi, Fangcheng 2 ; Hu, Chunhua 2 ;
作者机构： 1.Hunan Agr Univ, Coll Hort, Changsha, Peoples R China
2.Guangdong Acad Agr Sci, Inst Fruit Tree Res, Minist Agr & Rural Affairs,Guangdong Key Lab Trop, Key Lab South Subtrop Fruit Biol & Genet Resourc, Guangzhou, Peoples R China
3.Chinese Acad Sci, Inst Genet & Dev Biol, Ctr Genome Editing, State Key Lab Plant Cell & Chromosome Engn, Beijing, Peoples R China
4.Univ Chinese Acad Sci, Beijing, Peoples R China
5.Univ Florida, Mid Florida Res & Educ Ctr, Apopka, FL USA
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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