Project Details:

Title:
Improving CRISPR gene editing in soybean

Parent Project: Improving CRISPR gene editing in soybean
Checkoff Organization:Iowa Soybean Association
Categories:Breeding & genetics
Organization Project Code:021566
Project Year:2020
Lead Principal Investigator:Steve Whitham (Iowa State University)
Co-Principal Investigators:
Bing Yang (University of Missouri)
Keywords:

Contributing Organizations

Funding Institutions

Information and Results

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Project Summary

Background:
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 undoubtedly the hottest topic in germplasm improvement at the moment. In general, there have been many success stories and new developments, and because of this, the field is changing at a rapid pace. A few research groups repored that CRISPR-Cas9 works well in soybean cells. However, making soybean plants that carry useful edits that can be inherited in the next generation is still a major hurdle, and there remain just a few reports of such successes. This means that the application of powerful CRISPR-based technologies in soybean are not yet routine in the public sector, and it is still challenging to make edits that can be used in programs for breeding improved soybean. There are also successes reported from industry, of which the primary example is the editing of soybean genes encoding the enzyme acetolactate synthase to create a soybean plant that is resistant to sulfonylurea herbicides.

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. Co-PI Yang and others have found that the Cas9 protein itself has some toxicity in soybean, but we don’t know why. This toxicity limits the ability of researchers to use it to produce soybean plants with edited genes. The toxicity may be due to Cas9 amino acid sequence or the breaks it creates in soybean DNA.

There are 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. 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. We are also interested in using a different strategy to control where Cas9 and Cpf1 proteins are expressed in the soybean plant. It may be possible to increase their efficiency and reduce possible toxicity by expressing them specifically in cells that give rise to the embryo in the seed. The Cas9 base editor, Cpf1, and new expression strategies will allow us to test ideas about the toxicity of Cas9, and we expect that one or both technologies will allow us to overcome the current limitations to editing soybean genes.


Rationale:
To dramatically improve the ability of soybean researchers to discover new genes/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 developing new approaches for CRISPR-mediated gene editing in soybean that have high potential to overcome current technical hurdles and expand the uses of these technologies.

The major goals of this project are to:
1. Develop CRISPR-Cas9 base editing for soybean and demonstrate proof of principle by targeting and successfully editing two genes.
2. Develop CRISPR-Cpf1 gene editing for soybean and demonstrate proof of principle by targeting and successfully editing four genes.
3. Introduce new beneficial soybean traits through CRISPR-mediated gene editing.

Project Objectives

Objective 1: Modification of ESPS genes to confer glyphosate resistance.
Objective 2: Modification of FAD2-1 and FAD2-2 genes to alter lipid biosynthesis.
Objective 3:Characterization of edited plants

Project Deliverables

Milestones
1. Successful base editing of two soybean target genes
2. Successful CRISPR-Cpf1 editing of two soybean target genes
Deliverables
1. A Williams 82 soybean plant that is resistant to glyphosate
2. A Williams 82 soybean plant that has modified seed oil composition
3. 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.

Progress of Work

Updated April 23, 2020:
We completed the initial work to test the egg cell promoter strategy for expressing Cas9 for site-directed mutagenesis, and a manuscript describing this work was submitted to peer-reviewed journal. We are awaiting the decision.

The final conclusions are as follows:
1. An egg cell-specific promoter from Arabidopsis was identified that resulted in efficient Cas9-mediated, site-directed mutagenesis of the AGO7a gene. However, we didn’t observe a phenotype, because a second copy of the gene, AGO7b was not efficiently edited. This difference in editing was not a function of the egg cell promoter driving Cas9, but rather, the guide RNAs targeting AGO7a versus AGO7b differed in their efficacy.
2. Testing ability of guide RNAs to direct Cas9-mediated mutagenesis in hairy roots provides a reliable indication of how effective a guide RNA will be in stable transformed plants. In our work, the guide RNAs targeting the AGO7a gene worked very well in both hairy roots and in the stable transgenic plants. The guide RNAs targeting AGO7b did not work as well in hairy roots and were very inefficient in stable transgenic plants.
3. The use of the egg cell-specific promoter resulted in heritable mutations.
4. We concluded that the Arabidopsis egg cell-specific promoter with the highest activity would be useful in our subsequent studies to express different CRISPR systems including the Cpf1 site-specific nuclease and the Cas9 base editors in soybean.
5. We are taking plants of the T2 generation to seed to continue to follow inheritance of the AGO7 mutations including double mutants into the T3 generation.

Cas9 base editors to generate herbicide resistance
We made three Cas9 base editor constructs. One construct is targeting the 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) gene for glyphosate resistance, and the other two are targeting acetolactate synthase (ALS) gene for chlorsulfuron resistance. These constructs are designed to introduce site-specific point mutations (DNA base changes) into the EPSPS and ALS genes that are known to result in herbicide resistance. Success with creating the mutations and the accompanying herbicide resistance phenotype, will provide proof-of-concept that our strategy to drive expression of Cas9 base editors with an egg cell promoter is an efficient and useful approach. These constructs were submitted to the ISU Plant Transformation Facility.

Cpf1 site-specific gene editing
We have a Cpf1 system with Cpf1 under control of the egg cell-specific promoter as used for Cas9 and corresponding guide RNA under U6 promoter. The gene-specific Cpf1 construct targeting both FAD2-1A and FAD2-1B in soybean was made and submitted to the MU Plant Transformation Facility. Three regenerated plants that were observed to be positive for the GFP marker gene are currently being grown in the greenhouse. We expect to evaluate the Cpf1 editing efficiency in the progeny of these plants in the upcoming months as the plant produce seed.

Cas9+intron construct to alter seed oil composition
We also made a new version of the normal Cas9 construct that contains an intron in the coding sequence. The expression of the Cas9 is still driven by an egg cell-specific promoter, but the addition of an intron into the Cas9 coding sequence is expected to boost its expression, and thus, the activity of Cas9-directed mutagenesis. The preliminary data from the hairy roots indicates the Cas9+intron is indeed more efficient than the original Cas9. Therefore, we expect that Cas9+intron will be more active and result in more efficient editing in soybean plants. If it is successful, then all future Cas9 constructs will carry the intron. This Cas9+intron construct is designed to target the FAD2 gene. This construct was submitted to the ISU Plant Transformation Facility.

Updated October 8, 2020:
1) Test multiplex gene editing with CRISPR/Cas9 expressing Cas9+intron. We observed that the Cas9 construct that carries an intron (inCas9) is more efficient at creating mutations in the soybean hairy root system, suggesting higher levels of Cas9 protein in soybean cells. This prompted us to test the capacity of inCas9 to simultaneously edit multiple genes in Arabidopsis, which is a quicker system than soybean. Two inCas9 constructs have been made, one targeting 6 AtRBOH (respiratory burst oxidase homolog) genes and another targeting 4 AtRBOHs genes. These RBOH genes are known to be involved in biotic and abiotic stress responses. The two constructs have been introduced into Arabidopsis and seeds (T1 generation) have been harvested. The efficiency of multiplex genome editing will be determined soon.

2) Establish soybean transformation in the Yang lab. Efforts have been devoted to make improvements to soybean transformation by creating an improved vector system. It is believed that the CaMV 35S promoter does not function well in the meristematic cells. We replaced the 35S promoter with the soybean translation elongation factor (EF) gene to express the selectable marker gene, BAR. BAR confers resistance to herbicides, such as Liberty, that have glufosinate as the active ingredient. Testing of this new construct to improve stable soybean transformation is currently underway.

3) We have begun receiving plants from the ISU Plant Transformation Facility that have been transformed with one of the Cas9 base editor constructs that targets acetolactate synthase (ALS) to create an herbicide resistant plant. We have also received an inCas9 plant in which the FAD2 gene for seed oil composition is targeted. These plants are currently being grown to seed, and we are expecting more transgenic plants for these and other base editor constructs in the next few months.

4) The paper describing our initial work of using egg cell specific promoters to drive Cas9 expression and induce heritable gene edits was published in Frontiers in Plant Biology. The prior progress report summarized the major conclusions from this paper. A pdf of the paper is attached to this report.

View uploaded report PDF file

Final Project Results

Updated January 31, 2021:
1) Testing the CRISPR/Cpf1 genome editing system in soybean. Two putative transgenic lines derived from a CRISPR/Cpf1 construct produced some seeds. The progeny plants (T1 generation) are currently being grown in the greenhouse. The genotyping of those plants for presence of Cpf1 and guide RNA and for potential edits of the two targeted FAD2 genes is in progress.

2) 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 new system is currently being tested for editing in stable transgenic Arabidopsis plants with two constructs, one containing 4 guide RNAs targeting four genes of a family of 10 members and the second containing 6 guide RNAs targeting the remaining 6 genes. As controls, the original CRISPR/Cas9 was used to make two constructs with the same multiplexing guide RNAs. Transformation of Arabidopsis with the four constructs has been done and T1 plants are currently grown in the greenhouse. The preliminary genotyping results show a significant increase in the efficiency of creating edits (7 genes vs 1 gene among the 10 target genes). The results are consistent with the data from the soybean hairy root system when both the intron Cas9 was tested against the original Cas9 system. The intron Cas9 for genome editing in stable transgenic soybean plants is ongoing as plants are maturing in the greenhouse. The intron Cas9 construct targets the soybean FAD2 gene.

3) 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). These plants are being grown to maturity. Successful editing of these genes will create herbicide resistant plants, which will be tested in the progeny plants.

The main goals of this project are geared to improving gene editing in soybean and expanding the genome editing tool box for soybean.

Toward this goal, we have demonstrated that expressing the Cas9 protein specifically in the egg cells of soybean results in efficient gene editing. The egg cell-specific expression of Cas9 is significant, because this enables the CRISPR/Cas9 system to create gene edits that are more likely to be inherited to the next generation than of strategies for expression. We have also demonstrated in soybean hairy roots and Arabidopsis that the addition of an intron sequence into the Cas9 gene will result in higher expression levels of the Cas9 in plant cells. The higher level of Cas9 is important, because it increases the efficiency of gene editing. Our goal now is to combine the egg cell specific expression with the intron Cas9 to maximize gene editing in soybean. The transgenic plants necessary to test this idea are now being generated.

Towards the goal of expanding the genome editing tool box for soybean, the CRISPR/Cas9 base editor constructs are being tested, and the necessary transgenic plants are being produced. The base editors enable precise DNA changes to be made, and there functions have been tested in soybean hairy roots and Arabidopsis. If the soybean base editors work as expected, we anticipate that some of the progeny from the transgenic plants that are currently growing to maturity will be resistant to sulfonyl urea or glyphosate herbicides.

Benefit to Soybean Farmers

Successful completion of this research is expected to open up many new possibilities for gene editing in soybean. We expect that this research will help to reduce CRISPR gene editing to practice and make the technology practical for more quickly breeding new traits into soybean plants. The proposed work represents focused proof-of-concept project that could also potentially lead to a new virus resistance trait for soybean plants.

Performance Metrics

1. Now that the egg cell promoter is identified, we are proceeding with CRISPR/Cpf1 and CRISPR/Cas9 base editors. The constructs have been built.
2. Transgenic plants are being generated that express CRISPR/Cpf1 that targets the FAD1-1 and FAD1-2 genes for seed oil composition.
3. Hairy roots are being tested for function of the CRISPR/Cas9 base editors that are targeting the soybean EPSPS gene.
4. We are testing the heritability of mutations introduced by the CRISPR/Cas9 driven by the AtEC promoter.

Project Years