Abstract
The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR–Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR–Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR–Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.
Highlights
Publisher’s Note: MDPI stays neutralCultivated peanut or groundnut (Arachis hypogaea L.) is an allotetraploid (2n = 4x = 40)with a large reservoir of seed oil (~46–58%) and high-quality protein (~22–32%) [1]
Protoplast transformation is a convenient and reliable system to optimize gene editing in plants [27]
The results showed that the yield of protoplasts isolated from 5 days old seedlings was higher than that of the 10 days old peanut seedlings (Figure 1C,D)
Summary
Traditional peanut breeding has been a lengthy process with difficulties due to polyploidy and sterility barriers [2]. The implementation of functional genomics combined with biotechnology, especially DNA recombinant technology, will serve as an essential tool to further enable the discovery and characterization of genes of agronomic importance and speed up the progress in peanut breeding efforts. Unlike Arabidopsis and rice, making transgenic peanut plants through Agrobacterium transformation is more challenging and has a lower efficiency [15]. In this case, Agrobacterium rhizogenes has been frequently used with regard to jurisdictional claims in published maps and institutional affiliations
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