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Summary  Foods high in amylose content and resistant starch (RS) offer great potential to improve human health and lower the risk of serious noninfectious diseases. Common wheat ( Triticum aestivum  L.) is a major staple food crop globally. However, the RS contents in the grains of modern wheat varieties are low. Here, we report the generation of high‐amylose wheat through targeted mutagenesis of  TaSBEIIa  in a modern winter wheat  cv  Zhengmai 7698 (ZM) and a spring wheat  cv  Bobwhite by CRISPR/Cas9, respectively. We generated a series of transgene‐free mutant lines either with partial or triple‐null  TasbeIIa  alleles in ZM and Bobwhite, respectively. Analyses of starch composition, structure and properties revealed that the effects of partial or triple‐null alleles were dosage dependent with triple‐null lines demonstrated more profound impacts on starch composition, fine structures of amylopectin and physiochemical and nutritional properties. The flours of triple‐null lines possessed significantly increased amylose, RS, protein and soluble pentosan contents which benefit human health. Baking quality analyses indicated that the high‐amylose flours may be used as additives or for making cookies. Collectively, we successfully modified the starch composition, structure and properties through targeted mutagenesis of  TaSBEIIa  by CRISPR/Cas9 in both winter and spring wheat varieties and generated transgene‐free high‐amylose wheat. Our finding provides deep insights on the role of  TaSBEIIa  in determining starch composition, structure, properties and end‐use quality in different genetic backgrounds and improving RS content with multiple breeding and end‐use applications in cereal crop species through genome editing for health benefits.

Modification of starch composition, structure and properties through editing of TaSBEIIa in both winter and spring wheat varieties by CRISPR/Cas9