The problems of SDS resistance and drought tolerance have been difficult to tackle due to their complexity. We are now at a time where our basic knowledge of genes that contribute SDS resistance and drought tolerance are converging with technological developments needed to modify genes in a rational way to generate novel plant genotypes possessing improved resistance to SDS or tolerance to drought. We now have the capability to introduce a range of different types of edits to genes that are required for the successful completion of this project and realization of our goals. The technologies are available to selectively knock out genes or make specific and subtle changes in DNA sequence in order to modify gene functions. The proposed project will apply all of these technological capabilities to soybean with the goal of improving disease resistance and drought tolerance.

1. Continued technology development that adds PAMless Cas9-based gene editing and Prime Editing to the previously demonstrated site-directed mutagenesis and base editing technologies.
2. Application of CRISPR-Cas9 site-directed mutagenesis, base editing, and Prime Editing to engineer soybean plants with enhanced tolerance to drought stress and possibly other stresses.
3. Application of CRISPR-Cas based gene editing to identify genes that are critical for SDS resistance in soybean.

1. Protocols and DNA constructs for base editing, Prime Editing, PAMless Cas9 editing, and site-directed mutagenesis in soybean
2. Methods for modifying soybean genes to produce drought tolerant plants
3. Soybean lines that perform better under drought stress in growth chamber and greenhouse tests. Once we have genetically separated the CRISPR-Cas9 constructs from the target mutations, this will set the stage for future field tests of the lines.
4. It will be known if the six genes selected in this study are involved in immunity against three pathogens, F. virguliforme, P. sojae, and SCN.
5. Tools, resources, and protocols for the soybean research community that facilitate new and improved methods for gene editing in soybean. These will be shared through presentations at major soybean meetings and publications in journals and books.

Updated September 9, 2025:
Objective 1: Develop efficient PAMless Cas9 and Prime Editing platforms for soybean.

This is a gene editing tool development objective that seeks to develop a Prime Editing system for making specific mutations in the soybean genome.
We previously reported promising data for successful prime editing in soybean based on preliminary tests in hairy roots. We were in the process of producing transgenic plants carrying five different versions of prime editing constructs. We now have multiple transgenic plants for each of these constructs that are now maturing in the greenhouse. As soon as seeds can be harvested, they will be planted, so that we can perform genotyping analyses to determine if the specified gene edits are inherited and if the transgene constructs were also inherited.

Objective 2: Apply base editing and Prime Editing to modify genes affecting soybean responses to drought.
No progress to report this period, because we are revisiting the prime editing approaches needed to carry out this objective.

Objective 3: Application of CRISPR-Cas-based gene editing to identify genes that are critical for SDS resistance in soybean.
In our last report, we presented data related to the screening of T1 plants from eight events generated from two of the five constructs created in this project, DR1 and N1. For the DR1 construct, 121 T1 plants were screened and none of them carried mutations in either of the two genes that were targeted. For the N1 construct, we screened 73 progeny plants for mutations in the eight genes that were targeted. We identified one heterozygous mutation in Glyma.03G053500 carrying a deletion of five nucleotides in the protein coding sequence (Figure 1, see attached file).
We also reported that seeds were harvested from this heterozygote plant. As of today, we have analyzed the T2 generation of the identified mutant. Out of 30 plants screened, 6 were homozygous for the mutation, 14 were heterozygous, and 10 possessed only the wild-type genotype (Figure 2, see attached file). The numbers of homozygous mutants, heterozygotes and wild type progeny plants are consistent with the expected ratio indicating that the mutation was inherited as expected.
We sequenced the PCR products from the six homozygous mutant progenies and the wild type. All six homozygous mutant progenies possessed the same deletion of the five nucleotides (Figure 3, see attached file). These plants are being grown in the growth chamber. The seeds of these six homozygous plants will be grown to evaluate for the possible role of the disease resistance NLR protein encoded by Glyma.03G053500 gene in disease resistance against Fusarium virguliforme and soybean cyst nematode.
All 30 plants were evaluated also for possible mutations in any of the other seven disease resistance genes, for which gRNAs were included in the construct for soybean transformation. However, no additional mutations were detected among these plants.

View uploaded report

Updated September 9, 2025:

We expect that the gene editing technologies that will be tested and applied will become very important additions to the tool kit for precisely modifying genes controlling traits important to Iowa soybean producers, such as disease resistance and drought tolerance. In this project, the gene editing technologies will be applied to SDS resistance and drought tolerance. In the U.S., the total annual soybean yield suppression from SDS is approximately $600 million. Even if we can reduce the SDS incidence by 20% through cultivation of novel SDS resistant cultivars to be generated from the outcomes of this project, eventually we can expect to have significant increase in the annual soybean yield values close to $120 million in U.S. and approximately $17 million in Iowa. A 20% reduction in yield suppression by F. virguliforme will be translated to an extra $80 million in farm income for soybean growers of the U.S. and will significantly contribute towards for sustainability of soybean industry. Severe drought does not occur frequently, but when it does, it can cause major losses in productivity. Most recently, the drought of 2012 reduced soybean yields across the state of Iowa by an average of 5 – 6 bushels/acre compared to 2011, and for the US, soybean yields were estimated to be reduced by 9% on average for a total reduction of 170 million bushels.