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.
Three prime editing systems have been made for soybean hairy root and stable transformation and genome editing based on two different variants of SpCas9 nickase and reverse transcriptase of M-MLV. The three systems, which are named PE1, PE2, and PE3 were used separately to make prime editing constructs targeting soybean genes encoding CDPK47, CDPK48, CDPK49 and CDPK50. The PE1 and PE2 systems were compared to determine which one is best for creation of precise genetic changes for improved traits in soybean. Unfortunately, these two systems were not effective in creating mutations in the four CDPK genes in hairy roots. Therefore, we decided to test additional genes, FAD2 and EPSPS, using the PE2 system, and again, did not find evidence that the target genes were modified. A third prime editing version, named PE3, was also tested for ability to edit the FAD2 and EPSPS genes in hairy roots and this was also unsuccessful.
The PE1, PE2, and PE3 prime editing constructs do not appear to be functional in soybean, so we are taking alternative approaches to modify the vectors to produce the prime editing guide RNAs using different strategies. These constructs will be tested during the next reporting period. In summary, the application of prime editing in soybean is not efficient using strategies that have worked in other plants. We continue to work to identify a prime editing strategy that will be efficient in soybean.
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. We have obtained a total of four transgene positive plants for the NK44 construct. We obtained a total of seven transgene positive plants for the NK46 construct. Seeds were harvested from these 11 transgenic plants, and we refer to these seed as the T1 generation. At least 24 T1 seedlings were germinated for each line, and we conducted PCR to first establish that the NK44 or NK46 constructs were inherited, and we tested if any of the plants carried mutations in the target genes. Unfortunately, we found that the constructs were either not inherited or if they were inherited they carried deletions that rendered them ineffective. In line with these observations, no mutations were detected in the target genes. We hypothesize that soybean may not tolerate loss of function of these genes, and we are investigating alternative approaches to test roles of these genes in soybean defense and stress responses.
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).
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).
Progress on Objective 3 since last report:
We were able to develop a transgenic soybean plant carrying the construct #3 designed to knockout two GmWRKY genes (Table 1). This plant is now growing in the growth chamber and being analyzed for the presence of any possible mutations in the GmWRKY factor gene. Genomic DNA was extracted from the leaves of the candidate transgenic line and analyzed by conducting PCR. The forward primer from the gene and reverse pCR8-R primer from the pAtgRNA 1 and 2T vector were used in PCR (Table 3). The presence of PCR products demonstrated the presence of the intact construct #3 and thus successful transformation. We also conducted the restriction digestion of the PCR products of the target regions of two GmWRKY genes amplified by gene-specific primers. Digestion was done either with AluI or with MspJI. The observed bands indicated that there might be a mutation in gene 1 (Glyma.04G223200) because there is a faint un-digested larger band for one of the WRKY genes. The promoter fused to Cas9 is egg-cell specific and therefore we expect to have mutations in the WRKY genes in the seeds that are being developed in the transgenic plant.
We have developed additional gene editing constructs, which are summarized below in Table 4. We created 5 constructs in total (N1, N2, N3, TF, and DR1) (Table 4). These constructs are being used in creating transgenic soybean lines (Table 5) using recently harvested soybean seeds. We have observed that the new seeds are highly responsive, and we already have several explants in the “Shoot Elongation 3” medium (Table 5). In summary, we have developed a transgenic soybean line using old seeds that were not very responsive to the transformation process and were frequently contaminated. Recently harvested soybean seeds are showing good responses to the transformation process, and we expect to obtain mutants for most of the target genes selected for this study.
View uploaded report 
In the second year of this project, we achieved the following results in our technology development objective, drought tolerance objective, and SDS resistance objective.
1. Technology development: Three different prime editing systems were tested on multiple genes in soybean hairy roots, and we found that the prime editing strategies that work in other plant species do not appear to work effectively in soybean. It is necessary to test new prime editing strategies in order to produce a system that works efficiently in soybean.
2. Drought tolerance objective: We produced plants carrying CRISPR-Cas constructs designed to knock out four target genes (CDPK47, 48, 49, and 50). Seeds were collected from the transformed plants, and subsequently, seedlings were tested for edits in the four target genes. Unfortunately, no plants carrying the edits were obtained. We are evaluating why no edits occurred and are trying alternative approaches to edit these genes and investigate their potential roles in protecting soybean from drought and other stresses.
3. SDS resistance objective: Six CRISPR-Cas9 constructs specific to each of the six target genes and combination of all six signaling and regulatory genes were generated, and transgenic plants carrying these constructs are being produced. In addition, five more constructs were designed and these are also being transformed into soybean.