The development of CRISPR-based technologies has created unprecedented opportunities for “editing” the genes of crop plants to create optimized versions of genes that can be used in breeding programs. The ability to edit the genes of crop plants with CRISPR-based technologies is the hottest topic in germplasm improvement. There have been many success stories of using CRISPR-Cas9 for targeted mutagenesis. Targeted mutagenesis results from Cas9 cutting the DNA at the specified position followed by repair of the broken DNA by the cell. The result of the repair is a collection of mutations at the target site, but the sequence of the mutations cannot be controlled. This targeted mutagenesis is very powerful on its own and there are several examples of its use in soybean, but it represents just one CRISPR technology for the soybean gene editing toolkit. Other technologies have been developed that provide the opportunity to expand the spectrum of sites that can be targeted for mutagenesis or that increase the specificity of gene editing so that it is possible to exactly change a specific base in the DNA so that a single amino acid is changed in an encoded protein that modifies its function. Modifying gene function is one way to extrapolate superior traits from nature for germplasm improvement. This project directly addresses the development and demonstration of CRISPR technologies to expand the sites that can be targeted for mutagenesis in the soybean genome or that can increase the precision by which we can edit the target genes. In short, our goal is to expand the CRISPR toolkit for soybean.

State of the art: The primary form of the CRISPR technology that has been used so far is called CRISPR-Cas9. Cas9 refers to the enzyme that is guided by a CRISPR RNA, which carries the genetic information that guides Cas9 to make a cut in a specific place in a target gene. The sequence of the CRISPR guide RNA is easily changed, so that the Cas9 protein can be targeted to almost any gene. After the cut is made, the DNA is repaired by the plant cell. The repair process is imprecise unless a repair template is provided. In the absence of a repair template, random mutations occur in the gene, some of which may produce the desired change in DNA sequence. In the presence of a repair template, the gene is precisely edited, so that the expected outcome is predictable and produces the desired effect.

There are also new ways to use CRISPR-Cas9 to edit genes that do not require Cas9 to break the soybean DNA strands. One new technology is to make Cas9 into a CRISPR-guided DNA base editor. The CRISPR RNA still guides the Cas9 enzyme to the desired location in a target gene, but the Cas9 used has been first modified so that it can no longer cut the soybean DNA to create a break. Instead, Cas9 carries a second modification that allows it to chemically change the DNA bases at that location. The DNA genetic code is made of four bases A, G, C, and T. The Cas9 base editor can simply make a chemical modification to the DNA that converts one base to another, for example C to T or A to G. Another recent advance is the discovery that other enzymes similar to Cas9 exist, and they can also be used for gene editing in plants. The best studied of these is Cpf1, which uses a similar CRISPR guide RNA to create breaks in DNA. Cpf1 enables different sites within genes to be targeted than Cas9, and thus expands our ability to do targeted mutagenesis.

To test our ideas about base editors, we are targeting the soybean acetolactate synthase genes (Als1 and Als2) and the soybean 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) genes for base editing. We know the precise mutations that are needed in these genes to produce sulfonylurea or glyphosate herbicide resistance, so these will be ideal genes for proof-of-concept studies. We expect that soybean lines generated will be resistant to the respective herbicide. To test our ideas about Cpf1, we are targeting the FAD2-1 and FAD2-2 genes as a proof-of-concept. We expect that homozygous mutants will have altered seed oil composition.

1. Develop CRISPR-Cas9 base editing for soybean and demonstrate proof of principle by targeting and successfully editing two herbicide resistance targets.
2. Develop CRISPR-Cpf1 gene editing for soybean and demonstrate proof of principle by targeting and successfully editing two genes that control soybean oil composition.
3. Increase the efficiency of CRISPR systems in soybean reproductive cells.
4. Expand the toolkit for introducing new and beneficial soybean traits through CRISPR-mediated gene editing.

Milestones
1. Successful base editing of the ALS1 and ALS2 genes (sulfonylurea resistance)
2. Successful base editing of the EPSPS genes (glyphosate resistance)
3. Successful CRISPR-Cpf1 editing of the FAD2-1 and FAD2-2 genes
4. Higher frequency of CRISPR-Cas9 gene editing in reproductive cells

Deliverables
1. A Williams 82 soybean line that is resistant to a sulfonylurea herbicide
2. A Williams 82 soybean line that is resistant to glyphosate
3. A Williams 82 soybean line that produces modified oil content in seeds
4. A protocol/method for CRISPR-Cas9 base editing in soybean that will be shared with the soybean research
community for their research purposes
5. A protocol/method for CRISPR-Cpf1 gene editing in soybean that will be shared with the soybean research
community for their research purposes
6. A method for increasing gene editing efficiencies in soybean reproductive cells

Update:
1) Improving the efficiency of CRISPR/Cas9 genome editing. The CRSPR/Cas9 system has been modified into intron Cas9 (Cas9 gene containing an intron), which is expected to produce higher expression levels of Cas9 protein in plants. The intron Cas9 for genome editing in stable transgenic soybean plants is ongoing. Four transgenic lines were received from the ISU Plant Transformation Facility. The first plant has produced seeds, and they are being planted to test for presence of the Cas9 + intron construct and the presence of inherited mutations at the target site. We will also determine if edits are heterozygous, homozygous, or biallelic. The intron Cas9 construct targets the soybean FAD2 gene.

2) Testing CRISPR/Cas9 base editors in soybean. We have received plants from the ISU Plant Transformation Facility that have been transformed with three Cas9 base editor constructs, two of which target the genes encoding acetolactate synthase (ALS), and one that targets the gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). For the Cas9 targeting EPSPS, we received a total of seven transgenic lines, and six of these have survived to maturity. For the Cas9 targeting ALS, we received a total of three transgenic plants for construct 1 and six plants for construct 2. We are expecting eight of these plants to reach final maturity. Seeds from these plants are being collected. We are starting to plant seeds to test for presence of base edits at the target sites in the progeny of these transgenic plants. Successful editing of these genes will create herbicide resistant plants, which will be tested in the progeny plants after they have been genotyped.

Update:
1) Improving the efficiency of CRISPR/Cas9 genome editing. The backbone of the soybean transformation vector has been modified to express the selection marker gene (BAR) by replacing the 35S promoter with a strong soybean gene promoter. The Cas9 for genome editing has been modified to contain an intron to increase the expression of Cas9, and thus, the abundance of editing reagents. With the new system, which is named pSoy2-inCas9, the transformation efficiency of Williams 82 reached up to 18% as demonstrated with two replicated transformation experiments. A total of 48 transgenic plants (T0 generation) are currently growing in the greenhouse. These plants are going to maturity so that seed can be harvested. The editing efficiency mediated by the Intron Cas9 (inCas9) under the egg cell specific promoter for the soybean FAD2A and FAD2B genes will be determined in T1 progeny of those plants in November and December of 2021.

We are also continuing to follow inheritance of intron Cas9 and mutations induced by it from the original version of the construct. We now have T2 seed from one event and are in the process of genotyping seeds to identify lines that are homozygous for FAD2 mutation and lack the intron Cas9 construct. We also harvested T1 seeds from a second T0 plant that have been planted and we are in the process of testing for the frequency of edits in the FAD2A and FAD2B genes.

2) Testing CRISPR/Cas9 base editors in soybean. We received plants from the ISU Plant Transformation Facility that were transformed with three Cas9 base editor constructs, two of which target the genes encoding acetolactate synthase (ALS), and one that targets the gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). For the Cas9 targeting EPSPS, we received a total of seven transgenic lines, and six of these have survived to maturity. For the Cas9 targeting ALS, we received a total of three transgenic plants for construct 1 and six plants for construct 2. Seeds were collected from these plants, and progeny were screened for presence of base editor constructs and for the expected edits in the target genes. We did not detect any successful base edits in any of the plants tested, and for this reason did not conduct herbicide resistance testing. Therefore, we saved three plants from each construct, and took them to maturity to test for the presence of edits in the next generation (T2 generation). Seeds were harvested and will soon be tested for the presence of expected base edits.

The results suggest that the base editor constructs as originally constructed are not sufficiently active in soybean. Therefore, the C to T conversion base editor was redesigned to include the Cas9 version that carries an intron and new vector (i.e., pSoy2-CBE) has been developed and applied to generate the herbicide resistant soybean by editing the GmALS and GmEPSPS. Two CRISPR constructs were submitted to the MU Plant Transformation Facility in August and are in the process of selection.

Update:
1) Improving the efficiency of CRISPR/Cas9 genome editing. We harvested seed (T1 generation) from 48 T0 generation plants that were transformed with the new system named pSoy2-inCas9. We are currently testing 24 T1 seedlings from each of the T0 plants for the presence of edits in the soybean FAD2A and FAD2B genes.

We are also continuing to follow inheritance of intron Cas9 and mutations induced by it from the original version of the construct. T2 seedlings from one event are being screened to identify lines that are homozygous for FAD2A and FAD2B mutations and lack the intron Cas9 construct. We also tested T1 seeds from a second T0 plant transformed with this construct and found that there were no edits in the FAD2A and FAD2B genes, so this line will not be further pursued. We expect differences from one transgenic line to another, so it is not unexpected that the second line could not produce edits. This result further highlights the advantages of the newly improved pSoy2-inCas9 system, because we are now able to generate more T0 lines, and so, there is a higher probability of recovering multiple lines that are able to produce the gene edits that are of interest.

2) Testing CRISPR/Cas9 base editors in soybean. We previously reported that the transgenic lines carrying the first version of base editors did not produce T1 progeny that carried base edits. We were going to take these to the T2 generation and test for the presence of edits, but decided to stop work with these lines after the seeds were harvested. The success of the improved transformation vector coupled with the introduction of the intron in the pSoy2-inCas9 system caused us to rethink the strategy. A new set of transgenic plants that applies the features of the pSoy2-inCas9 system to base editing is being produced. We expect that we will be able to recover many more transgenic lines with more active base editors, and so, the probability of recovering T1 plants with the desired base edits will be higher.

We have modified the CRISPR-Cas9 system to make it more efficient for use in soybean. This was accomplished in two steps. The first was that the process of soybean transformation was significantly improved by making regeneration of transgenic plants more efficient. One of the challenges of soybean transformation is that usually only a few plants are recovered that carry the desired transgene. The improved system allows us to recover many more transgenic plants, which means that we increase the likelihood of recovering plants that not only carry the desired transgene, but also produces more lines to choose from in order to select the best one(s). The second improvement was the addition of an intron sequence into the Cas9 gene. This enabled us to increase the frequency of edits induced by the CRISPR-Cas9 system in the offspring of the transgenic lines. These improvements are now being extended to base editing and other CRISPR-Cas applications in soybean.

To dramatically improve the ability of soybean researchers to associate genes with traits, accelerate production of novel germplasm, and broaden the utility of the CRISPR-based gene editing technologies, more research and development (R&D) is needed. The proposed research is aimed at demonstrating the utility of new approaches for CRISPR-mediated gene editing in soybean that have high potential to expand the gene editing toolkit for soybean.