CRISPR-Cas9 based editing of the susceptibility allele TaLr34 enhances leaf rust resistance in bread wheat without yield penalty.

Wheat (Triticum aestivum L.) is widely grown and consumed cereal crop around the world, but most wheat-producing regions suffer from rust diseases, especially stripe and leaf rust, which has caused a devastating global pandemic and severely reduced grain yields. The most effective way to control rust problem in wheat is to sow and breed durable, rust resistant wheat varieties. Conventional breeding for disease-resistant crops primarily relies on resistance (R) genes; however, the effectiveness of R gene-mediated resistance is often compromised by mutations in the pathogen. In this study, we employed CRISPR-Cas9-based genome editing as an advanced breeding tool to enhance rust resistance in the bread wheat cultivar Galaxy-13 by knocking out the homologs of the susceptibility allele TaLr34, specifically targeting the conserved regions within exon 11. Out of 21 transformed plants, five carried successful editing and exhibited resistance to moderate resistance against leaf rust. The TaLr34 mutants were evaluated for leaf rust resistance under both glasshouse and field conditions over three consecutive growing seasons at multiple geographical locations. Our results demonstrate that CRISPR-Cas9-mediated knockout of TaLr34 provides a robust strategy for achieving durable leaf rust resistance in the high-yielding elite wheat cultivar Galaxy-13 without compromising grain yield and agronomic performance.