EDGE-FGT: Genotype-independent Transformation and Genome Editing in Diploid Wheat via Shoot Apical Meristem (SAM)-based Delivery

Wheat is the most grown crop in the world. It is one of the most versatile human food crops, contributing to bread, pasta, noodles, cakes, pastries, and many other foods. However, genetic and genomic research in wheat has been hindered by the large and complex genome of common bread wheat, where functional redundancy makes it difficult to determine the role of individual genes. Einkorn wheat is a diploid wheat species and the first domesticated wheat. Because its genome resembles the A genome of bread wheat and high-quality genomic resources are becoming available, einkorn is emerging as an attractive model for wheat functional research. Yet reliable transformation and genome editing tools have not been established in einkorn, creating a major bottleneck for gene discovery and trait development. This project will address this need by developing genotype-independent transformation and efficient genome editing systems in einkorn wheat. The resulting tools will enable genetic studies directly in a diploid wheat model and support rapid translation of discoveries in gene function to wheat improvement. The project will advance plant biotechnology, benefit a broad community of wheat and grass researchers, train postdoctoral, graduate, undergraduate, and high school students. This project will also disseminate protocols, reagents, and know-how through publications, Addgene vector distribution, hands-on workshops, and public outreach activities. This project will develop a suite of genetic transformation and CRISPR-Cas genome editing tools for einkorn wheat. First, the project will establish an optimized shoot apical meristem (SAM)-based transformation method in multiple wild and domesticated einkorn accessions, using GFP and RUBY reporter constructs to evaluate delivery efficiency, germline transmission, and genotype flexibility. Second, the project will develop multiplexed CRISPR-Cas9 and CRISPR-Cas12a systems in einkorn. These two nuclease platforms will be pursued because they generate different editing outcomes and support complementary applications, including gene knockout, validation of mapped candidate genes, and promoter editing to fine-tune gene expression. The project will test these systems using target genes such as MLO and candidate genes controlling coleoptile color, with editing outcomes measured by PCR amplicon sequencing and downstream phenotyping. Third, the project will develop DNA-free genome editing through ribonucleoprotein (RNP) delivery of CRISPR-Cas9 and CRISPR-Cas12a into einkorn SAMs. This approach may increase editing efficiency, reduce potential off-target effects, and facilitate the use of edited plants without integrated transgenes. Collectively, this work will create enabling biotechnology tools for einkorn as a genetically tractable wheat model, while generating methods that may be further adapted to other diploid wheat systems, durum wheat, bread wheat, and diverse grass species. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.