Abstract
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 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.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.
Highlights
To date, Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 RNP-Phytoene dehydrogenase (PDS) complex (RNPs) editing tools have not been applied to the genetic modification of banana
Establishment of a Polyethylene glycol (PEG)-mediated protoplast transformation system in banana To establish a PEG-mediated banana protoplast transformation system, we optimized the transformation method based on transformation protocols for rice and wheat
The rice and wheat protocols are quite effective in rice and wheat protoplast transformation, and transformation efficiency reaches 58.4 and 64.5%, respectively, confirmed by flow cytometry detection [24]
Summary
CRISPR/Cas RNP editing tools have not been applied to the genetic modification of banana. In 1960, Cocking successfully isolated tomato root tip protoplasts for the first time by enzymatic hydrolysis [2] This method was widely used because of its high yield, high activity, easy operation and wide adaptability. The PEG-mediated method is widely used due to its easy operation, low cost, lack of requirements for specific equipment and generation of stable results. To this date, mature and stable genetic transformation systems for protoplast transient expression have been established in Arabidopsis [11,12,13,14], wheat [15], rice [16], maize [17] and other species
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