CRISPR-Cas9-Mediated Gene Editing in Wheat: A Step-by-Step Protocol

The CRISPR-Cas9-mediated gene editing has sparked a new revolution in biological and agricultural research. This innovative technology is not only valuable for understanding the function of genes but also offers an efficient approach for genetic manipulation and crop improvement. However, the efficiency of gene editing in wheat (Triticum aestivum L.) is highly variable because of the genetic complexity of this plant species including 17 gigabases of genetic code, hexaploid genome, 80% genome with repetitive DNA, and large gene families. Delivery of gene editing reagents into wheat relies on optimization of transformation and plant regeneration methods. Currently available systems of wheat transformation are Agrobacterium-mediated transformation and biolistic bombardment. Recently, potential application of haploid microspore-based gene editing system in wheat has also been described. Here, we provide a step-by-step protocol for the implementation of CRISPR-mediated wheat gene editing and trait improvement. We begin with gRNA designing using online tools, followed by assembling CRISPR constructs or ribonucleoprotein (RNP) complex and in vitro and in vivo gRNA validation using wheat mesophyll protoplasts. Furthermore, we provide details of the tissue culture protocols for a stable wheat transformation using the most efficient gRNA for editing the gene of interest. Plant selection and regeneration protocols are also provided. We conclude this chapter with the mutation detection methods before phenotyping for the desired traits.