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Establishment of a PEG-mediated protoplast transformation system based on DNA and CRISPR/Cas9 ribonucleoprotein complexes for banana
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Establishment of a PEG-mediated protoplast transformation system based on DNA and CRISPR/Cas9 ribonucleoprotein complexes for banana
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Journal Article
Establishment of a PEG-mediated protoplast transformation system based on DNA and CRISPR/Cas9 ribonucleoprotein complexes for banana
2020
Overview
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.
Publisher
BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
Subject
/ bananas
/ Biomedical and Life Sciences
/ Cloning
/ CRISPR
/ CRISPR-Associated Protein 9 - genetics
/ CRISPR-Associated Protein 9 - metabolism
/ Crops, Agricultural - genetics
/ Crops, Agricultural - metabolism
/ DNA
/ DNA-free
/ Fruits
/ Gene Expression Regulation, Plant
/ genes
/ Genetically modified organisms
/ Genetics and crop biotechnology
/ Genomes
/ Genomics
/ Mutagenesis, Site-Directed - methods
/ Mutation
/ Pds gene
/ Plasmids
/ Polyethylene Glycols - metabolism
/ Ribonucleoproteins - genetics
