Evaluation of Factors Affecting in Planta Gene Editing Efficiency in Wheat (Triticum aestivum L.)

Abstract

Gene editing in polyploid crops still suffers from low efficiency, and further improvement is needed for its routine implementation in the modern breeding practice. Here we examined factors that affect the CRISPR/Cas9-mediated gene editing efficiency in allohexaploid wheat plants. We selected three guide RNAs (gRNAs) and evaluated the potential of using heat shock at the seedlings stage to increase editing efficiency in transgenic plants. Only one out of three gRNAs demonstrated significantly increased editing efficiency following heat shock treatment. We also examined the expression of DNA repair and replication gene orthologues in response to heat shock in wheat leaves. Misregulation of the chromatin remodelers following the heat shock treatment could potentially be involved in the increase of editing efficiency in wheat. Overall, the editing efficiency of gRNAs observed in our study correlated with predictive scores from the gRNA design tools. The editing rate of the top-ranked gRNAs could potentially be increased using heat treatment of the transgenic plants.