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  • Title: Establishment of a PEG-mediated protoplast transformation system based on DNA and CRISPR/Cas9 ribonucleoprotein complexes for banana.
    Author: Wu S, Zhu H, Liu J, Yang Q, Shao X, Bi F, Hu C, Huo H, Chen K, Yi G.
    Journal: BMC Plant Biol; 2020 Sep 15; 20(1):425. PubMed ID: 32933485.
    Abstract:
    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 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. 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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