Optimization of gene editing in cowpea through protoplast transformation and agroinfiltration by targeting the phytoene desaturase gene.

Cowpea (Vigna unguiculata) is a legume staple widely grown across Sub-Saharan Africa and other tropical and sub-tropical regions. Considering projected climate change and global population increases, cowpea's adaptation to hot climates, resistance to drought, and nitrogen-fixing capabilities make it an especially attractive crop for facing future challenges. Despite these beneficial traits, efficient varietal improvement is challenging in cowpea due to its recalcitrance to transformation and long regeneration times. Transient gene expression assays can provide solutions to alleviate these issues as they allow researchers to test gene editing constructs before investing in the time and resource- intensive process of transformation. In this study, we developed an improved cowpea protoplast isolation protocol, a transient protoplast assay, and an agroinfiltration assay to be used for initial testing and validation of gene editing constructs and for gene expression studies. To test these protocols, we assessed the efficacy of a CRISPR-Cas9 construct containing four multiplexed single-guide RNA (sgRNA) sequences using polyethylene glycol (PEG)-mediated transformation and agroinfiltration with phytoene desaturase (PDS) as the target gene. Sanger sequencing of DNA from transformed protoplasts and agroinfiltrated cowpea leaves revealed several large deletions in the target sequences. The protoplast system and agroinfiltration protocol developed in this study provide versatile tools to test gene editing components before initiating plant transformation, thus improving the chance of using active sgRNAs and attaining the desired edits and target phenotype.

Optimization of Prime Editing in Rice, Peanut, Chickpea, and Cowpea Protoplasts by Restoration of GFP Activity.

Precise editing of the plant genome has long been desired for functional genomic research and crop breeding. Prime editing is a newly developed precise editing technology based on CRISPR-Cas9, which uses an engineered reverse transcriptase (RT), a catalytically impaired Cas9 endonuclease (nCas9), and a prime editing guide RNA (pegRNA). In addition, prime editing has a wider range of editing types than base editing and can produce nearly all types of edits. Although prime editing was first established in human cells, it has recently been applied to plants. As a relatively new technique, optimization will be needed to increase the editing efficiency in different crops. In this study, we successfully edited a mutant GFP in rice, peanut, chickpea, and cowpea protoplasts. In rice, up to 16 times higher editing efficiency was achieved with a dual pegRNA than the single pegRNA containing vectors. Edited-mutant GFP protoplasts have also been obtained in peanut, chickpea, and cowpea after transformation with the dual pegRNA vectors, albeit with much lower editing efficiency than in rice, ranging from 0.2% to 0.5%. These initial results promise to expedite the application of prime editing in legume breeding programs to accelerate crop improvement.

Homology-based identification of candidate genes for male sterility editing in upland cotton (Gossypium hirsutum L.).

Upland cotton (Gossypium hirsutum L.) accounts for more than 90% of the world's cotton production, providing natural material for the textile and oilseed industries worldwide. One strategy for improving upland cotton yields is through increased adoption of hybrids; however, emasculation of cotton flowers is incredibly time-consuming and genetic sources of cotton male sterility are limited. Here we review the known biochemical modes of plant nuclear male sterility (NMS), often known as plant genetic male sterility (GMS), and characterized them into four groups: transcriptional regulation, splicing, fatty acid transport and processing, and sugar transport and processing. We have explored protein sequence homology from 30 GMS genes of three monocots (maize, rice, and wheat) and three dicots (Arabidopsis, soybean, and tomato). We have analyzed evolutionary relationships between monocot and dicot GMS genes to describe the relative similarity and relatedness of these genes identified. Five were lowly conserved to their source species, four unique to monocots, five unique to dicots, 14 highly conserved among all species, and two in the other category. Using this source, we have identified 23 potential candidate genes within the upland cotton genome for the development of new male sterile germplasm to be used in hybrid cotton breeding. Combining homology-based studies with genome editing may allow for the discovery and validation of GMS genes that previously had no diversity observed in cotton and may allow for development of a desirable male sterile mutant to be used in hybrid cotton production.