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

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.多倍体作物的基因编辑仍存在效率偏低的问题，若要在现代育种实践中实现常规应用，仍需对该技术进行进一步优化。本研究针对异六倍体小麦（allohexaploid wheat）中影响CRISPR/Cas9介导基因编辑效率的诸多因素展开了探究。我们选取了3条向导RNA（guide RNAs, gRNAs），并评估了在幼苗期施加热激以提升转基因植株基因编辑效率的可行性。在3条gRNAs中，仅1条经热激处理后其编辑效率得到了显著提升。此外，本研究还检测了小麦叶片中响应热激的DNA修复与复制基因同源物的表达情况。热激处理后染色质重塑因子的表达失调，可能参与介导了小麦基因编辑效率的提升。总体而言，本研究中观测到的gRNA编辑效率与gRNA设计工具给出的预测得分具有相关性。通过对转基因植株进行热激处理，有望进一步提升排名靠前的gRNAs的编辑效率。