Progress 06/01/24 to 05/31/25

Outputs
Target Audience:The target audience includes the plant science community at large, plant biologists, and wheat scientists and wheat growers. Changes/Problems:The project started late due to a protracted process to recruit a postdoc. We will catch up on the progress with an additional effort from a graduate student. What opportunities for training and professional development has the project provided?This project trained one research scientist and a graduate student in genome editing, wheat genetics, and molecular biology. Post-doctoral scientist Dr. Xi Chen originally worked on other projects and was reassigned this project in Dec. 2024. Xi Chen, previously trained in rice biology, worked on molecular cloning, transformation, tissue culture, and mutation genotyping. PhD graduate student Wei Jiang was trained in DNA extraction and amplicon sequencing library construction. How have the results been disseminated to communities of interest?News release. https://www.sdstate.edu/news/2024/07/sdsu-researchers-improve-heat-tolerance-wheat-crops. Conference presentation. The results from the project were presented at the Midwest ASPB conference held in March at University of Nebraska-Lincoln (product table). What do you plan to do during the next reporting period to accomplish the goals?Goal One: Create novel TaRca2 alleles for enhanced thermostability. Continuing wheat transformation with the three systems. Screen the mutagenesis populations for the I159M/N161K mutation by amplicon sequencing. Select for homozygous mutations at the homoeologous loci and against transgenes. Goal Two: Evaluate mutation effect on photosynthetic efficiency and heat tolerance. Evaluate the leaf elongation and photosynthesis rate at different temperature regimes. Goal Three: Develop breeding-ready germplasm. Cross the Rca2 mutant lines with SD spring wheat cultivars. Develop mutation-specific primers for PCR markers.

Impacts
What was accomplished under these goals? Goal One: Create novel TaRca2 alleles for enhanced thermostability. 25% Accomplished. Identification is a highly regenerating genotype. During our routine screening Fielder-derived populations for high callus regeneration, we identified a line (pedigree 24-144) with a regeneration rate of ~80%, which is almost as double as that of Fielder. With the improved regeneration rate, 24-144 will empower wheat transformation-based research including gene editing. Thus, it is used in the current project. Construction of a SpRY-based prime editing system. The pB-CMPE-ePPEplus is an improved prime editing (PE) system for wheat requiring the canonical PAM (protospacer adjacent motif) sequence NGG for its nickase Cas9 (nCas9) activity (Ni et al 2023, Genome Biology 24:156). The nearest PAM in the Rca2 is 17 bp away from the target site, which would affect the PE efficiency. To apply the PE for the current project, we modified the PE system by replacing the canonical nCas9 with a PAMless nCas9 derived from SpRY Cas9. To do so, we introduced four mutations into SpRY Cas9, including H840A for nickase activity, N863A for increase nickase specificity, and R221K and N394K for increasing the nuclease activity. At the same time, we designed an epegRNA for the installation of the I159M and N161K substitutions to Rca2 and integrated it into the binary vector with the PAMless nCas9. The resultant binary vector has been used for wheat transformation via Agrobacterium-mediation and ~300 calluses are generated at the selection medium. RNP-mediated sequence replacement via TR-HDR. We previously used plasmid constructs and donor DNA for Biolistic transformation to achieve the targeted gene replacement mediated by tandem repeat-homology-directed repair (TR-HDR). A drawback is that the final products (mutants) are transgenic, and the transgene needs to be eliminated before deploying the mutant in breeding. To avoid this drawback, we adopted a CRISPR-Cas9/sgRNA ribonucleoprotein (RNP)-mediated approach, which has been used for knockout mutagenesis. We modified the procedure by including donor DNA and successfully inserted a 50-bp sequence in a target site in the barley genome. For precision editing Rca2 with the I159M and N161K substitutions, we designed a sgRNA and a 116-bp donor DNA. More than 1,000 immature embryos of line 24-144 were transformed using particle bombardment, from which 814 seedlings were regenerated. The first batch of PCR products from 72 plants were Sanger-sequenced. Sequence analysis detected deletions but not the substitution, suggesting CRISPR-Cas9 RNP worked but insertion of the donor DNA probably occurred at very low frequency. For the cost-effectiveness, we adopted the Hi-TOM pipeline for amplicon deep sequencing to detect the mutations from the remaining plants while improving this RNP-mediated precision editing system by increasing donor DNA amount. A pool of 8 plates of indexed PCR products has been submitted for sequencing. Impact. The dentification of the high regeneration genotype significantly increased wheat transformation efficiency. The development of the PAMless PE system expands the target scope. The RNP-based approaches would directly generate transgene-free mutations. All this laid a foundation for the current project and helps advancing wheat genome editing. Goal Two: Evaluate mutation effect on photosynthetic efficiency and heat tolerance. There was no activity to report under this goal during this period. Goal Three: Develop breeding-ready germplasm. There was no activity to report under this goal during this period.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Chen X, Li W. 2025. Precision editing of Rca2 for enhanced heat tolerance in wheat. Midwest ASPB Conference. Mar 21  23, 2025. Lincoln, NE. Poster: 121. (2025 Midwest ASPB Abstract Book.docx - Google Docs; https://docs.google.com/document/d/1lL5c8ekwHpRNz09J0QjAp5Ve1VxLWQkp/edit?tab=t.0)