Engineering the key domains of starch synthases and branching enzyme to balance the amylose increase and yield loss in maize kernels.

Unlabelled: Amylose content and resistant starch (RS) are of great importance due to their multiple functionalities in the food and pharmaceutical industries and their benefits for human health. However, breeding high-amylose maize remains challenging because of the trade-off between amylose content and yield loss. Here, we report targeted mutagenesis of the key domains of starch synthases and branching enzyme including , and via a CRISPR-Cas9 technology; this generated 15, 21, and 14 novel alleles, respectively, in the maize inbred line LH244. Except for mutants, the and mutants had significantly greater apparent amylose content (AAC) and RS content compared with wild-type kernels. Although most mutants had reduced hundred-kernel weight (HKW) relative to wild-type plants, some mutants had only a small HKW reduction. Investigation of six representative mutants revealed that mutants of and with higher AAC and RS content were accompanied with reduced starch content viscosity, increased content of reducing sugars and soluble sugars, and yet no apparent trade-off with agronomic traits. These findings offer a promising path for high-amylose maize breeding, accelerating the development of germplasms with enhanced RS content for the benefit of both global health and industry.

Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-025-01559-z.