Integrating Green Revolution Rht-1 Alleles in Diverse Wheat Landraces Using CRISPR/Cas9, Cytosine Base Editing and Prime Editing

Abstract

The genetic diversity preserved in wheat landraces provides an important resource for broadening the genetic basis of modern wheat breeding, but its utilisation is constrained by the limited availability of effective genome-editing technologies for diverse genetic backgrounds. Here, we developed an integrated genome-engineering framework to modify Rht-1, a major regulator of plant height, in genetically diverse bread wheat landraces from the A.E. Watkins Collection. We combined CRISPR/Cas9-mediated deletion, cytosine base editing and prime editing to create defined Rht-1 variants in landraces representing five underutilised ancestral groups (AG1, AG3, AG4, AG6 and AG7). CRISPR/Cas9 editing generated targeted deletions of the conserved DELLA motif while retaining the downstream GRAS domain in-frame, yielding altered Rht-1 variants with potential utility for functional analysis and breeding. We further developed a wheat-adapted TaCBE6b-intron system and used cytosine base editing to engineer premature-stop variants in Rht-D1. Utilising prime editing with an adapted ePPEplus system enabled precise G-to-T conversion at the Rht-D1 locus, generating the nucleotide substitution characteristic of the Green Revolution semi-dwarf Rht-D1b allele. Prime-editing activity varied substantially among Watkins genotypes, with editing detected in WATDE0394, WATDE0549, WATDE0639 and WATDE0706. At the individual-plant level, amplicon sequencing confirmed D-genome editing, with edited Rht-D1 haplotypes reaching 100% of NGS reads in an individual WATDE0394 plant, and exceeding 50% in several plants from WATDE0394, WATDE0549 and WATDE0706. The engineered Rht-D1 variants were associated with reduced plant height; notably, a WATDE0394 plant carrying a 100% edited D-genome haplotype reached 69 cm compared with approximately 131 cm for the control. All T1 progeny of this plant were homozygous for the prime edit, as assessed by KASP markers. Reducing plant height in these genetically diverse landraces addresses their tall architecture and associated lodging susceptibility, a practical constraint on their direct use in breeding populations. Together, these results demonstrate that complementary genome-editing technologies can generate defined Rht-1 variation in genetically diverse wheat landraces while introducing agronomically relevant plant-height variation. The resulting founder lines provide valuable resources for functional analysis of DELLA-mediated growth regulation and a foundation for precision pre-breeding, composite cross populations, and the development of resilient wheat.