Updated November 14, 2018:
A. 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.
The main goal of this proposal is create soybean genotypes with increased seed methionine (Met) levels by engineering pathways for increased production of free Met and Met-enhanced storage proteins. We completed the construction CRISPR/Cas9 vectors and initiated stable transformations in Year 1. We also designed GY1 genes with increased Met codons, cloned them in front of a seed specific promoter and performed soybean transformations. In Year 2, we genotyped transgenic plants for mutations in the genes we targeted. We are happy to report that targeted mutagenesis worked very well in our hands and were able to obtain new soybean genotypes with knock-out mutations in genes encoding the 7S a, a’ and/or ß subunits, as well as in three genes encoding the Met catabolic enzyme Methionine-gamma-lyase (MGL). We also obtained T0 plants overexpressing Met-rich GY1 genes and putative 11S gene knock-outs. Lastly, we developed two methods of amino acid analysis and identified regulatory networks that are operable during soybean seed development. Detailed technical reports on these activities and the results we obtained were given in our quarterly reports. Given our budget and the challenges with CRISPR technology and soybean transformation, we accomplished almost all of the KPIs and deliverables that we proposed (see below).
B. 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.
1. Knock-out plants (T0 and/or T1) for 7S and at least three methionine catabolic genes with increased production of 11S proteins and free methionine are obtained by the end of Year 2.
? 7S knockout (KO) plants for a, a’ and/or ß subunits were obtained.
? Created KOs in three Methionine-gamma-lyase (MGL) genes that are involved in Met catabolism.
? PCR-based genetic markers for 7S and MGL mutant alleles were developed.
? Methods to measure free and protein-bound amino acids were developed.
? Construction of CRISPR/Cas9 vectors for KO of 7S in combination with additional Met catabolic genes were completed. However, Dr. Robert Stupar’s Lab was not able to generate transgenic plants for most of the constructs due to difficulties during transformation.
2. Knock-in plants containing additional methionine codons in 11S proteins are obtained by end of Year 3. (Note this report is for Year1-2)
? Three engineered GY1 constructs with increased Met codons were created (2, 4 and 7% Met codons compared to 1.6% in wildtype GY1)
? Transgenic plants expressing pGY1-GY1mod1 (7% Met codons) were created and grown in the field in summer 2018.
? PCR-based genetic markers for high-Met GY1 were developed.
? CRISPR knock-in vectors containing viral replicons were constructed, but we failed to obtain knock-in plants (see explanation below).
3. Key transcription factors and regulatory networks operable during seed development, specifically those involved in seed quality, are defined by the end of Year 2.
? Co-expression modules were identified using RNA-seq data from nine stages of soybean seed development.
? Several transcription factors that are differentially expressed during various stages of seed development were identified.
? Developmental expression of key genes involved in oil, protein, RFOs and amino acids
was characterized.
4. Knock-out plants (T0) in at least two key transcription factors (TFs) governing soybean seed quality are obtained and characterized by the end of Year 3. (Note this report is for Year1-2)
? Several TFs oprating in seed development, but so far, we have not identified
transcription factors that are specific to methionine or protein production.
? CRISPR-Targeted editing of relatively high numbers of TFs is not feasible given the
requested budget and the time it takes to obtain transgenic plants.
5. At least one of the new genetic variations for increased soybean seed methionine content developed in the project is adopted by at least two breeders and/or seed companies for soybean meal improvement.
? Too early to assess this KPI.
6. Basic knowledge, techniques and genetic materials (including plasmid vectors) developed in this project are utilized by at least five soybean researchers to further the goal of improving soybean seed quality.
? Too early to assess this KPI.
C. 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.
1. Soybean cultivars with new genetic variations in genes encoding ß-conglycinin, glycinin and enzymes involved in methionine and protein metabolism.
?Soybean germplasms with knock-out mutations in a, a’ and/or ß subunits of conglycinin storage proteins (7S) were developed.
?Soybean germplasms with knock-out mutations in three MGL genes were developed.
? T0 plants with putative knockout mutations in glycinin proteins (11S) are available and will be characterized to confirm the mutations.
2. Genetic markers for each of the new genetic variations mentioned in above.
? PCR-based genetic markers were developed for mutant alleles in 7S and MGL genes.
3. Efficacy of suppressed methionine catabolism combined with increased production of methionine-rich storage proteins in improving the amino acid balance in soybean seeds.
? Soybean germplasms expressing Met-rich GY1 proteins were developed.
? Met-rich GY1 expressing germplasms will be combined to MGL catabolic mutants by genetic crosses or re-transformation with CRISPR constructs targeting MGL genes.
4. Genes encoding key transcription factors and enzymes involved in seed development, specifically those governing protein production and quality.
? Based on our RNA-seq data, we identified several key TFs during seed development as well as genes encoding the major storage proteins and enzymes involved in Met, Lys, and Thr biosynthesis.
5. A newly developed algorithm and publicly available online tool for identifying gene regulatory networks operable during seed development that can aid the soybean community’s efforts to develop a better soybean crop.
? We performed bioinformatics analyses and obtained co- expression networks
operable during seed development. Identification of key transcription factors
associated with the expression networks is on-going.
D. Describe any unforeseen events or circumstances that may have affected project timeline, costs, or deliverables (if applicable.)
? We were not able to obtain “knock-in” plants. We included viral replicon sequences in our CRISPR knock-in vectors to increase the frequency of insertions, but this approach was not successful. This failure is largely due to poor knock-in frequency in plants in general, and the time it takes to do soybean transformation. Creating knock-in soybean lines would require more dedicated efforts and budget than we proposed.
? Dr. Stupar’s lab was unsuccessful in obtaining germplasms with knock-out mutations in both 7S and Met catabolic genes (combination mutants) due to contamination and die off of shoots. Developing combination mutants is on-going.
E. 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.
At this point, more experiments to determine if our proposed “push-pull” approach will increase Met levels in soybean seeds. Specifically, we need to obtain combination mutants to combine two traits namely high free Met production and expression of Met- enriched storage protein. Once confirmed, we can deploy CRISP/Cas targeted mutagenesis in elite or commercial cultivars.
List any relevant performance metrics not captured in KPI’s.
Methods to measure free and protein-bound amino acids were developed. This aspect of the proposal is critical in assessing the various germplasms we developed and are developing for increased seed Met levels, as well as other amino acids.