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.

Updated June 3, 2024:
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.
As we previously reported, the application of prime editing has proved to be more challenging in soybean than anticipated. Recently, successful prime editing was reported for two dicotyledonous plant species for the first time. These species are tomato and Arabidopsis thaliana. We have revised our approach to prime editing in soybean based on this new report, and six new prime editing constructs were designed and built for testing efficacy of editing in soybean hairy roots:

1. AtUbiPro-nCas9-RT3(M-MLV)-MASpro-eGFP-pegFAD2A
2. AtUbiPro-nCas9-RT5(M-MLV)-MASpro-eGFP-pegFAD2A
3. AtUbiPro-nCas9-RT2(SPV)-MASpro-eGFP-pegFAD2A
4. AtUbiPro-nCas9-RT2(CaMV)-MASpro-eGFP-pegFAD2A
5. AtUbiPro-nCas9-RT5(CaMV)-MASpro-eGFP-pegFAD2A
6. AtUbiPro-nCas9-RT2(SCMV)-MASpro-eGFP-pegFAD2A

These constructs have different RT (reverse transcriptase) components as defined here:
RT3(M-MLV) – Moloney Murine Leukemia Virus Reverse Transcriptase (engineered version 3)
RT5(M-MLV) – Moloney Murine Leukemia Virus Reverse Transcriptase (engineered version 5)
RT2(SPV) – Soybean Putnam Virus Reverse Transcriptase (engineered version 2)
RT2(CaMV) – Cauliflower Mosaic Virus Reverse Transcriptase (engineered version 2)
RT5(CaMV) - Cauliflower Mosaic Virus Reverse Transcriptase (engineered version 2)
RT2(SCMV) - Soybean Chlorotic Mottle Virus Reverse Transcriptase (engineered version 2)

These constructs have been made and their sequences were confirmed. Each construct has been introduced into the Agrobacterium rhizogenes strain K599, and the hairy root transformation is in progress. The next plan is to collect the GFP positive hairy roots (those roots that carry the construct), and genotype them for prime editing efficiency by deep sequencing the PCR amplicons at the target site in the GmFAD2A gene.

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.
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: 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).

We created seven constructs to knock out the selected genes in various combinations. 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.


Stable Transformation of Soybean
We have conducted stable soybean transformation for the six constructs. Five constructs are at the stage of shoot induction and will go soon to shoot elongation. Our goal is to have at least 240 explants inoculated for each construct.

Progress on Objective 3 since last report:
The progress made in the last two months after January 30, 2024, is presented below.

Previously, we reported that a T0 transgenic plant carrying a construct designed to knockout two GmWRKY genes was generated. PCR analysis using forward primer from the gene-specific and reverse pCR8-R primer from the pAtgRNA 1 and 2T vector confirmed the presence of the transgene in the transformant. We harvested 58 seeds from this plant. The first five plants (from the first two pods) were analyzed by sequencing PCR products of the target regions of two GmWRKY genes. Unfortunately, none of the five T1 plants carried any mutations at either of the two gRNA target sites. The remaining 53 seedlings are now at the unifoliate leaves and will be analyzed to determine if there is any mutation among these T1 progenies. These seedlings will first be screened for resistance to basta herbicide and the PCR analysis for the basta resistance gene to confirm that they are not generated from a non-transgenic chimeric branch. Selected progenies carrying the transgene will then be evaluated for the presence of the mutation in the Glyma.04G223200 gene.

We now have developed transformants for additional constructs, N1 (TNLR and kinase genes), N2 (eight TNLR genes), TF (six transcription factor genes) and DR1 (GmDR1).

More putative transgenic plants from additional constructs are being developed and are at different stages of transformation on plates.

View uploaded report

Updated January 30, 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.

Approach 1: Codon optimized Split PE system (sPE3)
As mentioned in the previous report, a split PE3 system was designed and tested or two different genes GmFAD2 (GmFAD2.1A and GmFAD2.1B) (edit type: stop codon at 88th and 90th positions in the amino acid sequence) and GmEPSPS1 (edit type: substitute the amino acids threonine, alanine, and proline at positions 183, 184 and 187). Different combinations of prime editing guide RNAs (pegRNAs) and nick guide RNAs (ngRNAs) were used for both genes and a total of 10 different PE constructs were tested in Agrobacterium rhizogenes K599 mediated hairy root transformation experiments. Briefly, the system was working but was less efficient than required for efficient prime editing. This conclusion was demonstrated by screening the transformed hairy roots for the desired edits by using DNA sequencing.

Approach 2: Codon optimized Fused PE system
In a different approach, a codon-optimized fused PE system was constructed and tested for its efficacy. To increase the expression of PE components, double terminator (EU terminator + pea rbcsE9 terminator) was incorporated into the PE destination vector and was compared with single terminator (pea rbcsE9 terminator). Similarly, single (HSPter) and double (EU+Rb7ter) terminators were tested for the pegRNA cassette as well. Three different PE systems (PE3, PE6c and PE7) were tested in A. rhizogenes K599 mediated hairy root systems using five different target genes (FAD2.1A and FAD2.1B for oil composition; KTI3 for trypsin inhibitor activity; SWEET3 for increase seed protein; and EPSPS1 for glyphosate resistance).

From hairy root experiments, all the tested constructs based on PE3, PE6c and PE7 showed detectable edits with an edit-specific PCR-based assay. However, genotyping based on the PCR-restriction enzyme approach showed detectable edits on GmFAD2.1A/1B and GmKTI3 only. Based on the results from hairy root experiments, some PE constructs were moved forward for soybean stable transformation and we are now obtaining plants to analyze for prime edits. The constructs for which we are obtaining plants are as follows:

1. pSoy2-AtEC-PE3-2XTer-BlpR-eGFP-GmFAD2-HSPter (Got only 1 plant, transgenic and is in greenhouse)
2. pSoy2-AtEC-PE7-2XTer-BlpR-eGFP-GmFAD2-HSPter (Got only 1 plant, transgenic and is in greenhouse)
3. pSoy2-AtEC-PE7-2XTer-BlpR-eGFP-GmKTI3-EURb7ter (Got 1 transgenic plant so far and is in greenhouse)
4. pSoy2-AtEC-PE7-2XTer-BlpR-eGFP-GmFAD2-HSP-GmKTI3-EURb7ter (Got 3 plants and yet to test for transgenics, are in small pot in growth chamber)
5. pSoy2-Atubip-PE6c-2XTer-GmEF1a-BlpR-GmScreamM4-Greenlantern-GmFAD2 (Got 2 plants so far, both transgenic. 1 is in greenhouse and 1 is small pot in growth chamber)

The stable PE constructs with AtEC as the promoter for PE components will generate edits in the T1 generation. So, the T1 generation will be genotyped for edits. However, the PE6c system targeting GmFAD2.1A/1B is driven by the Arabidopsis ubiquitin promoter (AtUbip) and hence, the edits can be detected in the T0 generation. Two transgenic plants obtained from PE6c were genotyped for the edits and the genotyping result shows that the plants carry the desired edits in the target gene. This was demonstrated by both the PCR-restriction enzyme assay and DNA sequencing.

Approach 3: Use of different reverse transcriptase (RTs) from dicot specific plant viruses
In another effort to attempt to improve PE efficiency in soybean, we engineered and tested different different reverse transcriptases (RT) from plant viruses in the A. rhizogenes mediated hairy root transformation system. The RTs used are as follows:
RT2(CaMV) – Cauliflower Mosaic Virus Reverse Transcriptase
RT5(CaMV) – modified Cauliflower Mosaic Virus Reverse Transcriptase
RT2(SCMV) - Soybean Chlorotic Mottle Virus Reverse Transcriptase
RT2(SPV) – Soybean Putnam Virus Reverse Transcriptase

The RTs were tested using GmFAD2.1A/1B as the target genes (Edit type same as above) and the PE constructs tested are as follows:
pSoy2-AtUbip-InCas9KKn-CaMV-RT2-Masp-eGFP-GmFAD2-EURb7ter
pSoy2-AtUbip-InCas9KKn-CaMV-RT5-Masp-eGFP-GmFAD2-EURb7ter
pSoy2-AtUbip-InCas9KKn-SCMV-RT2-Masp-eGFP-GmFAD2-EURb7ter
pSoy2-AtUbip-InCas9KKn-SPV-RT2-Masp-eGFP-GmFAD2-EURb7ter

From hairy root experiments, we found that none of the plant RTs were active enough to produce detectable edits. Thus, it was not possible to use these plant RTs to improve the efficiency of PE in soybean.

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 the last report, we described the development of five constructs to knockout 22 candidate resistance genes selected based on our previous studies. We aimed to knockout these genes to investigate their possible involvement in soybean immunity against Fusarium virguliforme. Among these genes, eight encode NLR-type disease resistance genes, five encode receptor-like kinases, six encode transcription factors involved in gene regulation, and three genes include GmDR1 and its two homeologs.
We generated nine independent transgenic soybean lines carrying three of the five constructs and screened their progeny (T1 generation) for the presence of mutations in the in the target genes. To date, we have identified a mutant for a construct named N1, which targets a set of genes that encode disease resistance-like proteins. A 5 base pair deletion was found in one of these genes, which was verified by sequence analysis. We have harvested seeds from this mutant plant containing a mutation in the gene with the formal name of Glyma.03G053500. A set of 32 seeds were plants for identifying the homozygous mutation.

Investigation of the NLR protein encoded by Glyma.03G053500 in the sequence data base of The National Center for Biotechnology Information (NCBI) suggested that this protein could be similar to the tobacco resistance protein N that confers Tobacco Mosaic Virus resistance. The soybean Glyma.03G053500 gene mapped closed to a region containing resistance genes against Phytophthora sojae, Soybean Mosaic Virus and soybean cyst nematode (SCN). We plan to study the T1 progenies of this mutant for segregation of the possible SCN resistance.

Our research is aimed at improving soybeans through the use of advanced gene editing techniques. Specifically, we have continued to work on a technology named Prime Editing, which is a precise method to modify specific genes. We also used CRISPR gene editing to identify genes that contribute to the ability of soybean to defend against Sudden Death Syndrome (SDS) and other pathogens.

Prime editing has proven to be more challenging in soybean than anticipated, and therefore, we have focused on approaches to try improve the efficiency of prime editing to make it feasible in soybean. We tried three different strategies in soybean hairy roots as a first test. One strategy was most promising, and so we moved forward to test the system in transgenic soybean plants. The first plants that are being produced are positive for the desired edits, and more plants are being generated. These plants will need to be taken to maturity and their progeny will be tested for inheritance of the edits. If the edits are passed on to the next generation, then it will be possible to design genetic changes to develop plants with improved traits including disease resistance, herbicide resistance, and oil composition, for example.

We have also used CRISPR gene editing to generate mutations in genes that may aid soybean in defense against sudden death syndrome and other diseases. A plant carrying a mutation in one gene was successfully identified and that plant is being taken to seed. The offspring of this plant will be tested to identify homozygous mutants that can be tested for responses to diseases.

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.