CRISPR-Cas-based gene editing technologies have revolutionized biology by enabling researchers to efficiently modify genomes at specific positions. Moreover, it is becoming increasingly feasible to control not only precisely the site where the modification occurs, but also the sequence of the DNA change. Once the DNA changes are made, the CRISPR-Cas transgenes can be removed, leaving behind the subtle DNA changes that confer desired modifications to traits in the final products that are deemed non-regulated germplasm in some countries including the US. CRISPR-Cas is increasingly being used in vegetable and crop plants to engineer them to have improved resistance to diseases, herbicide tolerance, and other agronomic traits and quality traits. CRISPR-Cas-based gene editing technologies are continually evolving, and they need to be demonstrated to work in soybean and then optimized in order to be applied in the most efficient ways and a wide spectrum of applications. One particularly powerful version of CRISPR-Cas is a new gene editing technology named Prime Editing, which was first described in late 2019, but has yet to be proven to work in soybean. This technology enables scientists to specifically re-write the genetic code within a small window at a target site within a gene. We think that Prime Editing has great promise to help the soybean research community efficiently make precise, site-specific changes in the sequence of soybean genes. We expect that Prime Editing will become a very important technology in the tool kit for precisely modifying genes controlling traits important to Iowa soybean producers, such as disease resistance and drought tolerance.

Objective 1: Develop efficient PAMless Cas9 and Prime Editing platforms for soybean.
Objective 2: Apply base editing and Prime Editing to modify genes affecting soybean responses to drought.
Objective 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 any of the six genes selected genes in this study is involved in immunity against any of the three soybean 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.

Update:
See attached file.

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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.