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.

Update:
Objective 1: Develop efficient PAMless Cas9 and Prime Editing platforms for soybean.
This is a gene editing tool development objective that builds upon the CRISPR-Cas9 gene editing platform that we previously developed.

1. Genotyping of T1 progeny plants derived from the improved soybean CRISPR-Cas9 constructs targeting GmFAD2 for mutagenesis. A total of 450 plants were grown, genomic DNA was extracted, and then genotyping was performed to determine the presence of CRISPR transgenes and edits for GmFAD2a and GmFAD2b. The results demonstrate that about 50% of T0 transgenic lines (34 lines positive for CRISPR constructs) carried CRISPR constructs that were inherited into their progeny. The lines that did not inherit a CRISPR transgene produced no progeny plants that had edits. For the lines where CRISPR constructs were inherited, editing efficiencies range from 15% to 90% with the majority of lines having 80% of progeny plants containing edits for both genes. The results demonstrate that our Soy CRISPR-Cas9 system is superior to other previously reported systems.

2. Building a Prime Editing system for soybean. We analyzed the most recent prime editing systems developed in animals and plants, and designed one architecture featuring all improvements from the literature and from our prior work. The backbones of plasmids and gene cassettes are currently being designed and synthesized. Two soybean prime editing systems will be developed – one for hairy roots and another for stable transformation.

3. Soybean transformation improvement. Because stable plant transformation continues to be a critical bottleneck for genome editing in soybean, the Yang lab continues to do research on improvements. They have started to build the soybean transformation capacity with one graduate student who is working on establishing a transformation protocol for elite soybean genotypes.

Objective 2: Apply base editing and Prime Editing to modify genes affecting soybean responses to drought.

1. Designing and constructing CRISPR-Cas9 constructs to mutagenize soybean calcium-dependent protein kinase (GmCDPK) genes. The CRISPR guide RNA design for one set of the closely related GmCDPK family members (GmCDPK49 and GmCDPK50) has been done and construction of the corresponding CRISPR-Cas9 plasmid is in progress.

2. Developing soybean immune and drought response assays. The Whitham lab has initiated experiments to establish protocols for soybean immune and drought responses that will be needed when the CRISPR mutant lines are ready. These assays will allow us to determine if the mutations have significant effects on soybean defense against pathogens and tolerance to drought.

3. Virus-induced gene silencing of GmCDPK genes. We have made a set of bean pod mottle virus constructs designed to silence different groups of GmCDPK genes. The reason we are doing this is to speed up identification of the GmCDPKs that will be edited. If we observe effects on soybean immune and drought responses when the genes are silenced, then this will provide more confidence to move them into the gene editing pipeline, which takes much more time and effort. The virus-induced gene silencing will also be coupled with the immune and drought response assays. A first/pilot round of gene silencing experiments is underway with one bean pod mottle virus construct designed to silence one group of four GmCDPKs that are most similar in sequence to each other.

Objective 3: Application of CRISPR-Cas-based gene editing to identify genes that are critical for SDS resistance in soybean.

The goal for the end of year 1 is that we will have completed the seven CRISPR-Cas9 constructs needed for this objective, and they will be transformed into Agrobacterium rhizogenes and A. tumefaciens for hairy root assays and stable transformation of soybean, respectively. These constructs will target nine soybean genes that were previously associated with soybean resistance against Fusarium virguliforme, spider mites, soybean aphids, and soybean cyst nematode. The 9 genes are: (i) two LRR-receptor kinase genes; (ii) four NB-LRR-type disease resistance genes; (iii) one WRKY transcription factor gene; (iii) one NAD(P)-linked oxidoreductase gene and (iv) one F-box family protein gene.

The following progress was made in building the CRISPR-Cas9 constructs. The CRISPR-Cas9 technology is composed of the three cloning steps, and the progress made to date is described under each of the following three steps.

1. Cloning each CRISPR guide RNA into the specific pAtgRNA expression vector.
For each gene one or two guide RNAs were designed and cloned into the pAtgRNA expression vector. The resultant plasmids were transformed into Escherichia coli and colonies were initially analyzed by polymerase chain termination reaction (PCR) and then confirmed by sequencing. We have completed cloned gRNA-specific sequences for all nine genes and confirmed by sequencing.

2. Golden Gate-cloning of all units of each construct into the pENTR4-ccdB.
In the second step, we incorporate multiple gRNA sequences of Step 1 in a single pENTR4-ccdB plasmid vector. For example, we have already completed cloning gRNA molecules specific to two LRR-receptor kinase genes; two gRNA molecules specific to one WRKY factor gene and two gRNA molecules specific to one F-box family protein gene individually into this vector. The pENTR4-ccdB plasmids for the other four CRISPR-Cas9 constructs are being constructed.

3. Cloning the pENTR4-gRNA into the destination vector P1300-AtEC-Cas9-GFP-ccdB. This is the last step of cloning gRNA molecules into a binary plasmid vector containing Cas9 gene. The destination vector P1300-AtEC-Cas9-GFP-ccdB is used to incorporate the gRNA molecules cloned in the pENTR4-ccdB plasmid in Step 2. We already have incorporated the three pENTR4-ccdB plasmid constructs generated in Step 2 in the P1300-AtEC-Cas9-GFP-ccdB plasmid. The resultant three P1300-AtEC-Cas9-GFP-ccdB plasmids that carry: (i) gRNA molecules specific to two LRR-receptor kinase genes, (ii) two gRNA molecules specific to one WRKY factor gene, or (iii) two gRNA molecules specific to one F-box family protein gene. The three P1300-AtEC-Cas9-GFP-ccdB plasmids will be transformed into A. tumefaciens for transformation of soybean cotyledonary explants.

Update:
Objective 1: Develop efficient PAMless Cas9 and Prime Editing platforms for soybean.
This is a gene editing tool development objective that builds upon the CRISPR-Cas9 gene editing platform that we previously developed.

Building a Prime Editing system for soybean.
Two separate Prime Editing systems have been built for soybean based on our highly efficient Prime Editing system in rice. The systems have been used to make constructs to designed to edit calcium dependent protein kinase (CDPK) family genes (CDPK47, 48, 49, and 50).

a. Construct NK124: pAtEC-ePE1-CDPK49-50 (Targets CDPK49 and CDPK50). We are in the process of producing transgenic plants that carry this construct. Currently, four regenerated plants are in soil, and three additional shoots are in rooting medium.

b. Construct NK135: pAtEC-ePE1-CDPK49-50 is ready for transformation, which will begin soon.

c. We are in the process of making a few additional constructs to also target the CDPK47 and CDPK48 genes.

Objective 2: Apply base editing and Prime Editing to modify genes affecting soybean responses to drought.

1. We have designed two different CRISPR-Cas9 constructs to knockout the function of CDPK genes that are predicted to affect soybean responses to drought.

CRISPR-Cas9 based gene knockout of the soybean CDPK family genes (CDPK47, 48, 49, and 50)
Two CRISPR constructs (NK44, and NK46) have been built to knockout two combinations of CDPK genes.

a. NK44: pAtEC-Incas9-gCDPK49-50 (Targeting CDPK49 and CDPK50)

b. NK46: pAtEC-Incas9-gCDPK47-50 (Targeting CDPK47, CDPk48, CDPK49 and CDPK50)

Soybean transformation was performed with these two constructs and regenerated plants were genotyped for the presence of the transgenes. The resulting plants are now being grown in the greenhouse, and they have started flowering and setting seed.

So far, we have obtained three transgene positive plants for the NK44 construct with a few more awaiting genotyping. We have obtained seven transgene positive plants for the NK46 construct. After seeds are obtained from these plants, we will test the progeny for the presence of mutations that disrupt the target genes.

Objective 3: Application of CRISPR-Cas-based gene editing to identify genes that are critical for SDS resistance in soybean.
We have reported earlier that overexpression of GmDR1 enhances broad-spectrum resistance against two soybean pathogens and two pests including Fusarium virguliforme that causes sudden death syndrome (SDS). Our results suggested that enhanced resistance against F. virguliforme in plants overexpressing GmDR1 is mediated by a number of genes including those that encode disease resistance-like receptors, receptor-like kinase, and WRKY transcription factor. The rationale of the proposed study is that once we establish that overexpressed GmDR1 mediates defense functions by regulating the expression of genes encoding disease resistance-like receptor proteins, receptor kinases and a transcription factor, it will be feasible to utilize these genes in enhancing SDS resistance in soybean. At the end of the three-year project period, we expect to establish the defense functions for six signaling and regulatory genes. Once we establish the role of these genes in SDS resistance, one could use these as markers in breeding soybean for SDS resistance. We have generated CRISPR-Cas9 DNA constructs, using resources optimized for soybean, to knockout six target genes for determining their role in defense responses. The egg cell-specific promoter that we demonstrated to work well in expressing Cas9 in soybean has been used in generating the constructs. The constructs will be evaluated in hairy root assays prior to time consuming stable soybean transformation. It has been shown that multiple genes can be mutated simultaneously in one plant through CRISPR-Cas9 system. We will determine if all six genes can be knocked out in hairy root assays. If we are successful, then we will generate stable transgenic soybean lines to knock-out all six target genes. The stable transgenic mutant plants will be evaluated for responses to F. virguliforme, P. sojae, and SCN infections.

Selected genes and construction of CAS9 vectors
Based on our earlier RNA-seq and qRT-PCR results, nine genes were selected for being knocked out to investigate their involvement in soybean immunity against F. virguliforme (Table 1): four encode disease resistance-like receptors leucine-rich repeat (LRR), two encode the LRR receptor kinases, and 3 are encode regulatory genes. Next, primers were designed for the guide-RNA (gRNA) of each of the selected genes using the Iowa State University Crop Bioengineering Consortium's CRISPR Genome Analysis Tool http://cbc.gdcb.iastate.edu/cgat/ (Zheng et al., 2020).

Table1. Constructs created for selected genes
Construct # Genes # of units pAtgRNA expression vector used (each gene in 1 vector)
1 4 LRR (all As) 4 pAtgRNA1, 2, 3, 4T
2 2 kinases (all Bs) 2 pAtgRNA1 and 2T
3 WRKY DNA -binding domain (C1) 2 pAtgRNA1 and 2T
4 NAD(P)-linked oxidoreductase (C2) 2 pAtgRNA1 and 2T
5 F-box family protein (C3) 2 pAtgRNA1 and 2T
6 3+4+5 6 pAtgRNA1, 2, 3, 4, 5, 6
7 1+2 6 pAtgRNA1, 2, 3, 4, 5, 6

Progress Report:
We created seven constructs to knock out the selected genes in various combinations as shown in the Table 1. After cloning each individual CRISPR guide RNA spacer sequence into pAtgRNA expression vector, the constructs were assembled into pENTR4-ccdB vectors using the Golden Gate-cloning technology. Each of the constructs were sequenced to confirm the identity of each of the seven constructs. Each construct was transferred into two different binary vectors using the LR Gateway cloning system to obtain the following two plant expression vectors:
1. P1300-2X35S-Cas9-ccdB (vector A) for the generation of soybean hairy roots in order to check the success of knocking out the genes.
2. P1300-AtEC-Cas9-GFP-ccdB (vector B) for the generation of stable soybean transgenic lines.
Except for construct # 7, all the 6 other constructs have been cloned in both binary vectors and transferred to Agrobacterium rhizogenes k599 for soybean hairy roots, and to A. tumefaciens EH105 for production of stable soybean transgenic plants (Table 2). We are preparing the media and reagents to initiate the soybean transformation, which will be started soon.

Table 2. Status of the constructs
Constructs # # of units In pENTR4-ccdB In vector A In vector B In A. rhizogenes In A. tumefaciens EH105
1 4 yes yes yes yes yes
2 2 yes yes yes yes yes
3 2 yes yes yes yes yes
4 2 yes yes yes yes yes
5 2 yes yes yes yes yes
6 6 yes yes yes yes no
7 6 no no no no no

In the first year of this project, we achieved the following results in our technology development objective, drought tolerance objective, and SDS resistance objective.

1. Technology development: New Prime Editing constructs were built, and we are in the process of producing stable transgenic plants from which seeds will be obtained. The seeds from these plants will be germinated and seedlings will be tested for the presence of the desired specific changes in target genes in year 2. These expected results will demonstrate the functionality of the new Prime Editing system.
2. Drought tolerance objective: We have produced four different constructs so far that will create either specific DNA changes or knockout of function in four genes that we are targeting to determine if they have a role in drought stress and and be modified to improve soybean drought stress.
3. Drought tolerance: Transgenic plants carrying Prime Editing constructs are in the process of being generated. We have also generated transgenic plants of CRISPR-Cas constructs designed to knock out four target genes (CDPK47, 48, 49, and 50).
5. Drought tolerance: We have begun genotyping T0 to determine presence of CRISPR-Cas constructs and seed are being produced to generate progeny plants in which we will determine if edits can be identified in the target genes.
6. SDS resistance objective: Seven CRISPR-Cas9 constructs specific to each of the six target genes and combination of all six signaling and regulatory genes were generated, and we are ready to begin producing transgenic soybean plants carrying each of these seven constructs.

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.