Update:
To lay a foundation for improving gene editing in soybean, we have been building up new tools and resources for gene editing in soybean and initiating tests of their feasibility. The major activities in months seven to 12 of year 2 plus the 3 months of no-cost extension of the project are described here.

1. Building a CRISPR/Cas9 for genome editing in soybean

In this reporting period, we continued to test the activity of different egg cell promoters for driving expression of Cas9 proteins in stably transformed Williams 82 soybean. We have now completed testing of four different promoters: Arabidopsis egg-cell specific gene promoters (P5p and AtEC) and two soybean egg-cell specific gene promoters (GmEC1.1 and GmEC1.2). These constructs were also designed to express CRISPR RNAs targeting two genes that affect small RNA regulation in soybean (AGO7a and AGO7b).

P5p-Cas9/gRNAGmAGO7 yielded 5 T0 plants (n=5) that were tested to be Cas9 and gRNA positive and grown to maturity. A total of 128 progeny plants were obtained from the five T0 plants. A simple and quick mutation test assay (so-called T7 endonuclease I assay, T7E1) was used to analyze the PCR products derived from the CRISPR target regions of those 128 plants. No mutation could be detected in any of the 128 plants, suggesting the CRISPR construct with the Arabidopsis P5 promoter is not efficient to induce gene editing in soybean.

AtEC-Cas9/gRNAGmAGO7 yielded 12 T0 plants (n=12) that were tested to be Cas9 and gRNA positive and grown to maturity. A total of 61 progeny plants (T1) were tested to be Cas9 and gRNA positive. A total of 28 plants from 4 T0 lines (ST410-4, ST410-25, ST410-36, and ST410-44) were subjected to T7E1 assay to determine the efficiency of gene editing. Out of the 28 plants tested, 16 plants (~60%) were positive for gene edits in the T7E1 assay. PCR products from the 16 plants were Sanger sequenced, and 6 of them (6/28 = ~25%) were genuine gene edited plants. The results indicate that the AtEC-Cas9 system is efficient to generate gene edited soybean. Currently, 25 T1 plants are being grown to maturity in the greenhouse, and their progeny plants (the T2 generation) will be tested for inheritability of their edits.
Based on this success with the AtEC promoter, we designed a new CRISPR/Cas9 construct aiming to edit the soybean omega-6 fatty acid desaturase (FAD2) genes (FAD2-1 and FAD2-2). We expect that edited plants will produce high oleic acid (healthy oil). The construct is currently in the soybean transformation pipeline.

GmEC1.1-Cas9/gRNAGmAGO7 yielded 2 T0 plants and GmEC1.2-Cas9/gRNAGmAGO7 yielded 1 T0 plant that were Cas9 and gRNA positive and grown to maturity. DNA was extracted from a total of 12 T1 progeny plants and the T7E1 assay was performed to test them for gene edits. None of plants were positive for edits.

Conclusion: We developed a CRISPR/Cas9 gene editing system driven by an Arabidopsis egg cell promoter that can be used to generate edited soybean.


2. Construct CRISPR/Cpf1 for soybean genome editing.

We have built six new constructs for Cpf1-mediated gene editing in soybean: 2x35S-Cpf1, CMVe+CMV-Cpf1, AtUbi-Cpf1, CMVe+AtUbi-Cpf1, CMVe+GmUbi-Cpf1, and AtEC-Cpf1 (see Figure 1). The difference between each of these six constructs is the gene promoter that is being used to drive expression of the Cpf1 gene. The CMV promoter is a virus gene promoter, CMVe is the CMV gene enhancer, AtUbi is the Arabidopsis Ubiquitin promoter, and GmUbi is the soybean Ubiquitin promoter. We first tested the gene editing activities of Cpf1 in soybean hairy root system by expressing it under different promoters (except the egg-cell specific gene promoter, AtEC) and under different temperatures. The preliminary results indicate that Cpf1 works in hairy roots based on editing of the FAD2 genes, and the Cpf1 gene editing activities increase at higher temperatures (e.g., 28 or 30 ?C) compared to 25?C. The construct expressing Cpf1 under the AtEC promoter and guide RNAs to target FAD2-1 and FAD2-2 is in the soybean transformation pipeline. We decided to use the AtEC promoter, because of the success observed in the CRISPR/Cas9 system described in the previous section of this report.

3. Construct of CRISPR/Cas9 based base editors.
The constructs described in sections 1 and 2 above are designed to cause edits in the target genes, but we cannot control the final sequence of the edits. To improve our ability to control the sequence of the final edits, we are working on a set of base editors. We have built four versions of base editors (see Figure 2). The versions for causing C to T mutations are called C Base Editors (CBE) and the version for causing A to G mutations are called A Base Editors (ABE). The two versions of CBE and two versions of ABE have been individually cloned to be under control of the AtEC promoter and 2x35S promoter. The AtEC-CBE and -ABE along with the guide RNAs targeting soybean 5-enolpyruvyl-shikimate synthase (EPSPS) gene for glyphosate resistance will be used for stable soybean transformation. The 2x35S-CBE and -ABE along with the guide RNAs targeting GmEPSPS were tested for their base editing activities in the soybean hairy root system, which was faster and easier than making stable transgenic plants. The editing activities of these constructs was confirmed in hairy roots, and so the subsequent experiments were initiated to make transgenic plants with stable mutations induced by the AtEC promoter driving expression of the CBE and ABE base editors.

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In the second year of the project, the final resuls and major conclusions are:

1. We have established a new CRISPR/Cas9 system for soybean using a promoter to drive expression of Cas9 in soybean egg cells. This system results in gene edits in soybean genes that are inherited to the next generation and establishes a foundation for other new gene editing technologies to be deployed in soybean that include the CRISPR/Cpf1 and Cas9 base editor systems.

2. We established that the CRISPR/Cpf1 system works in soybean hairy roots, and we are now waiting for stable transgenic soybean lines that will be tested for edits caused by Cpf1. CRISPR/Cpf1 is of interest to us, because it can be used to generate a different spectrum of gene edits than CRISPR/Cas9. The offspring of these transgenic plants will be screened for mutations in the FAD2 gene in the next year, and the seeds of lines carrying mutations will be tested for altered fatty acid profile, but this will not be possible before the end of year 3.

3. The Cas9 Base Editors were tested in soybean hairy roots and demonstrated to be functional, and the constructs were then made to express the Cas9 base editors under control of the egg cell promoter in transgenic soybean plants. The Cas9 Base Editors are of interest to us, because we can control the specific mutation that is made in the target gene, and is thus more precise than standard Cas9 or Cpf1 editing. The seedlings derived from the transgenic plants that will be produced in the next year will be screened for the site-specific mutations and the ability to tolerate glyphosate application.