Updated November 2, 2018:
Project Status - What key activities were undertaken and what were the key accomplishments during the life of this project? Please use this field to clearly and concisely report on project progress. The information included should reflect quantifiable results (expand upon the KPIs) that can be used to evaluate and measure project success. Technical reports, no longer than 4 pages, may be included in this section.
A multi-pronged approach to improve seed protein quantity was undertaken. To channel carbon into protein and oil synthesis in the seeds, a vector containing a sucrose symporter from Arabidopsis regulated by a soybean HSP.1 promoter was constructed and engineered into soybean. Progeny from one event carrying the AtSUC2 transgenic allele is growing in the greenhouse. In addition, seed-specific expression of a gene involved in programmed cell death gene is expected to prolong seed development, thus providing more time for the storage reserves to accumulate. Towards that end, plasmids harboring AtBAG4 with or without a transit peptide were used in the transformation. Primary events have been established in the greenhouse derived from transformations with both versions of the AtBAG4 transgene (±TP). Another approach used RNAi technology to down-regulate the expression of the historic protein QTL on chromosome 20. A total 12 independent events (T2 populations) was shipped to IL for characterizations.

Another goal was to alter the protein balance through the simultaneous creation of knock-outs through CRIPSPR/Cas9 technology that targets seed component combinations, such that compensation occurs. Six CRISPR targets were designed to specifically create glycinin/conglycinin knockouts. Moreover, CRISPR is also being used to remove the two KasI gene copies, which affect seed fatty acid biosynthesis and therefore seed composition. Seventeen M1 individuals in the WPT677-3 family that inherited mutations for the KasI gene and did not inherit the CRISPR/Cas9 transgene were identified. M2 families for some of these mutants are currently being grown. Six different CRISPR constructs were transformed: (1) Knock out mutations for the four glycinins; (2) Knock out mutations for the nine beta-conglycinins; (3) Deletions for eight of the nine beta-conglycinins. Putatively transformed T0 plants were identified for these constructs, some exhibiting mutations at the intended target sites. However, the transgenes and mutations did not transmit to the next generation. It was concluded that the CRISPR/Cas9 reagents are working, but germline transformation had not taken place.

An additional CRISPR knockout strategy of the major soybean seed storage proteins was implemented. Four guide cassettes were synthesized that each carry a single guide designed to edit the soybean alpha-subprime beta-conglycinin (7S), glycinin (Gly A3B4), SAM22 and P34 seed storage proteins. A binary vector carrying this 4 guide stack downstream of a Cas9 cassette was assembled and designated pPTN1454.

Four different types of CRISPR are now available, so as to enable the previous activities. The four CRISPR types were found to function in hairy roots assays, but at a low frequency. Regardless, S. pyogenes Cas9 resulted in fully edited somatic embryos, so poor editing ability seems to be limited to hairy root assays and not real-world uses in soybean.
Seed quality can be enhanced by increasing methionine content, attained by using methionine-rich proteins for protein rebalancing. As a first step, the methionine and cysteine content found in all of the 56,000 predicted soybean gene models was determined by computational methods. This produced a list of 24 genes with high methionine and 23 with high cysteine levels. Next, the expression of these genes in developing soybean seeds was determined from RNA Seq data. It turns out that proteins normally found in the seed are very low in methionine and cysteine—thus increasing existing storage proteins will not lead to increased protein quality. The proteins that have the highest level of sulfur-containing amino acids are those that play regulatory roles and that are found in very small amounts. Thus, a potential strategy is to add part of these sulfur-amino acid-containing gene sequences to the end of the seed storage proteins. Towards that end, 7 high-methionine and 3 high-cysteine soybean genes expressed in developing seed and seedling tissues were identified that appear to be most useful for overexpression of a sulfur amino acid-rich backbone under control of seed-specific promoters that vary in strength and timing. Finally, a list of genes highly expressed at different stages of seed development was produced based on RNA Seq data. The promoters of these genes may serve to allow overexpression of desired high methionine genes during various times of seed development.
A particularly promising promoter was selected from analysis of extensive RNA-Seq data available from over 100 tissues and stages of development. The GmSeed5 promoter (a conglycinin promoter) was selected based on its expression pattern during seed development, when most proteins are accumulating. High levels of seed-specific transgene expression were observed when the GmSeed5 promoter was used to regulate the visual marker, GFP, in transgenic soybean. Four additional promoters were characterized: GmSeed3 (Cysteine protease family C1-related), GmSeed6 (Glycinin Gy4 promoter), GmSeed10 (Lectin Le1 promoter) and GmSeed18 (Subtilisin-like protease). Furthermore, GmSeed promoters 3, 5, 6, and 10 were used to increase seed methionine.
The ability to enhance methionine production was explored as an alternative approach. Four seed-specific expression cassettes carrying genes of the E. coli methionine pathway were engineered into soybean. To date seven events have been established in the greenhouse and one additional event recently acclimated, but not yet screened for herbicide resistance. Progeny derived from two independent events, which carry the transgenic allele with the E. coli met pathway, are growing in the greenhouse.
Another goal was to edit the promoters of key genes to drive expression of transcription factors, amino acid biosynthetic enzymes, or seed storage protein genes. In addition to our use of Cas9 for genome editing, we also evaluated a “deactivated” Cas9 protein, which can bind to targeted DNA sequences within the promoter as a means to attenuate its function. Use of a Cas9-CRISPRi that cannot cut DNA was shown to block gene transcription. Targets near two putative enhancer motifs, as well as the transcription start site in the GmScreamM8 promoter resulted in a decrease in promoter activity during transient expression. This is the first demonstration that CRISPRi can be used to affect promoter activity. Unfortunately, activity was not highly decreased and this approach shows limited application for regulation of native promoters.
In addition to gene knockouts, an additional goal was to knock-in genes. Several strategies were employed to improve the efficiency of homology independent targeted integration (HITI) that increased CRISPR gRNA expression to allow the formation of a reconstituted GFP gene in lima bean cotyledon cells. The highest HITI efficiency (number of cells having GFP expression) resulted from the use of a strong soybean promoter with ribozymes flanking the gRNA sequence, combined with a strong soybean promoter for the tRNA flanking the gRNA sequence. The use of high temperatures for improving the efficiency of genome editing using CRISPR, reported in the literature, did not lead to any improvement in recombination, further supporting the idea that gRNA and not Cas9 levels are limiting.
New HITI promoter targets were selected for ease of observation by improving the sensitivity of measuring successful knock-ins. These were the upstream regulatory and coding sequence junction of two seed-specific glycinin genes, and two constitutive elongation factor 1-alpha genes. These promoters were isolated from previous USB-funded research and were selected because they give high gene expression and the expectation is to identify successful knock ins by observing green fluorescence. Using cotyledons from developing soybean seeds, we were able to observe, for the first time, successful knock in of a green fluorescent protein gene behind a native soybean promoter in the soybean genome. With some early optimization, the efficiency of successful knock ins varied from 2-4%; which means that DNA was stably integrated in 2-4% of the targeted cells. Even though this is early in the optimization process, 2-4% is a high stable transformation efficiency and the The Ohio State University is considering pursuing IP for use of HITI for increasing stable transformation in plants.
For all the work described thus far, the use of somatic embryos, which are basically lab-grown seeds, permits a rapid analysis of the gene’s effect on protein composition. Gene-gun versions of both the methionine and sucrose vectors were made, though no events were obtained.
As somatic embryos permit rapid testing of CRISPR-edited genes for desired effects, the ability to engineer these embryos with Agrobacterium could improve testing efficiency. While Agrobacterium tumefaciens is renowned for its ability to deliver DNA to a wide range of plants, most soybean genotypes are recalcitrant to transformation because of a host-pathogen response that elicits a hypersensitive response and death of host tissue. Evading this host defense response in soybean would facilitate a wider range of soybean genotypes suitable for use in transgenic breeding programs. A search for soybean homologs of known plant-immunity-associated pattern recognition receptors (PRRs) identified several candidates in the soybean reference genome. Because the reference genome is from an Agrobacterium-resistant cultivar, re-sequencing data from an Agrobacterium-susceptible variety (Peking) was mapped to the reference genome and analyzed for differences among the candidate genes. A PRR, Glyma.09g216400, is absent in the Agrobacterium-susceptible variety, Peking. Furthermore, this gene is the most similar homolog in soybean to the Arabidopsis Elongation Factor – Thermo unstable receptor (AtEFR), known to restrict transformation in Arabidopsis by recognizing the bacterial Elongation Factor – Thermo unstable (EF-Tu) protein of Agrobacterium. Peking becomes resistant to Agrobacterium that has had its EF-TU replaced with that of Bradyrhizobium USDA 110, indicating that more versions of EF-Tu need to be tested for compatibility with soybean. Two F5 NIL populations, developed in order to map genes that inhibit Agrobacterium-mediated transformation of soybean, and containing 220 lines each, were harvested. Each population had an Agrobacterium-susceptible parent and an Agrobacterium-resistant parent (Jack x Peking and Jack x [Peking x Century (5)]). These lines were selected for loss of the GmEFR receptor (Glyma.09g216400) in the F2 generation, and are mostly susceptible to Agrobacterium. To assist with the identification of other genes involved with susceptibility to Agrobacterium, Peking (PI 548402) was sequenced with 10X Genomics and a de novo genome was assembled and mapped to the reference. (Note: there is a sequence for PI 548402 on SoyKB, but it is mislabeled as such, and is actually something else.)
Did this project meet the intended Key Performance Indicators (KPIs)? List each KPI and describe progress made (or not made) toward addressing it, including metrics where appropriate.
FY2017 KPIs
• A set of new CRISPR vectors optimized for soybean are developed and available to the soybean research community and beyond -- done
• The first set of transformed plants with altered protein will be initiated. When completed (in year 2) the plants and/or results are made available and used by at least 3 public researchers breeding for protein quality – plants still need to be analyzed.
FY2018 KPIs
• Three seed specific promoters are characterized for seed specificity and timing of expression using transgene expression in both somatic and zygotic embryos. Done
• Gene edited promoter knock outs are generated using CRISPR/Cas targeting cis-regulatory elements in seed-specific promoters. Done
• The first set of glycinin/beta-conglycinin knockout mutations are identified and evaluated for protein and amino acid compensation profiles—Done but not evaluated.
• One or more genes specifically involved in protein synthesis and storage has been discovered and markers for those genes have been developed and are available for use by soybean breeders by 8/18 – Identified and supplied but need different alleles before a marker can be made.
Expected Outputs/Deliverables - List each deliverable identified in the project, indicate whether or not it was supplied and if not supplied, please provide an explanation as to why.
FY2017
Report on first successful alteration of seed protein gene 04/17. Supplied.
Report on first successful ¬¬alteration of a seed protein profile 08/17. Not been supplied because there have been no materials to analyze because of the time it takes to modify soybean.
Report on first successful seed-specific promoter targeting 04/18. Supplied.
FY2018
Report on first identification of CRISPR-based knockout mutations in the glycinins/beta-conglycinin gene families (T0 generation); 9/18. Supplied, but not heritable changes.
Finalize CRISPR genome editing tools, 10/17. Supplied but not fully tested.
Provide target genes for construction of a high sulfur amino acid (methionine or cysteine) construct with seed specific promoters and designed for storage protein body localization. 9/18 - Genes have been supplied but constructs not assembled.
Provide transcription factor or other metabolic targets designed to influence protein quantity from analyses of genome and transcriptome data of naturally occurring or transgenic lines. 9/18 Targets have been supplied but plants are not ready for analysis.
One or more genes specifically involved in protein synthesis and storage has been discovered and markers made 8/18 Gene discovery has been supplied but markers will not be available until there are different gene alleles.
Describe any unforeseen events or circumstances that may have affected project timeline, costs, or deliverables (if applicable.)
Staff departures & replacements
What, if any, follow-up steps are required to capture benefits for all US soybean farmers? Describe in a few sentences how the results of this project will be or should be used.
The goal of this work was to identify and assess biotechnological approaches that can increase protein content and quality in soybean. Several engineered and edited soybean lines are now available for analysis. The vectors and other tools developed during this proposal are also available and ready for future use.
List any relevant performance metrics not captured in KPI’s.