Improvement of simultaneous genome editing of homoeologous loci in polyploid wheat using CRISPR/Cas9 applying tRNA processing system

Wheat (<i>Triticum aestivum</i> L.) consists of three genomes, and notable mutant phenotypes can be observed when all homoeologs are knocked out due to functional redundancy among the homoeologous gene copies. Therefore, high editing efficiency is required to rapidly obtain loss-of-function mutants in wheat. The endogenous tRNA processing system of CRISPR/Cas9 genome editing enables the expression of multiple single-guide RNA (sgRNAs) under the control of a single promoter, facilitating simultaneous multiple genome editing in an organism. Here, we evaluated the genome editing efficiency of multiple sgRNA expressions with the tRNA processing system. At first, using sgRNA of quantitative trait locus for seed dormancy 1, polycistronic tRNA-sgRNA vectors were introduced into immature embryos, and genome editing efficiency was evaluated in the transformed T₁ plants. In the use of three sgRNA modules, there was no difference in the efficiency of genome editing among the positions of the sgRNAs. We subsequently tested simultaneous genome editing of multiple homoeologous loci. Simultaneous expression of six sgRNAs per gene to target all homoeologous loci increased the editing efficiency of all homoeologous loci up to 100%. Our study indicates that the tRNA processing system is highly effective at simultaneous genome editing of homoeologous loci of wheat.