Progress 09/01/20 to 04/30/21
Outputs
Target Audience:The target audience is the agricultural seed industry, which is keen to pursue hybrid wheat as one of their critical business goals. Our potential customers/collaborators are major seed companies such as Bayer, Corteva and Syngenta, that are aggressively looking for a robust and elite hybrid wheat seed production system. Efforts to reach the target audience include press releases, publication of our research data in peer-reviewed journals, presentation of our research data at relevant scientific and trade conventions, and licensing negotiations regarding patent application(s) on our technologies. Changes/Problems:Our initial Phase I Project had the following goal: Transformation of wheat mitochondria. However, we encountered several challenges to this goal as described below. To circumvent these challenges, we revised this Phase I Project goal to be the following: Enabling of mitochondrial transformation in rice as a model for cereal crop plants using a selectable marker instead of a screenable fluorescence marker. This change was communicated to the Program Director and accepted. To achieve our original first goal, transformation of wheat mitochondria, our initial proposal was to use the method of directly transforming DNA into meristematic cells of wheat embryos. This method, which introduces a gene encoding a fluorescent protein, has been successfully developed in wheat for nuclear gene transformation. To enable visual screening with the fluorescence protein, we proposed several expression elements to allow for gene expression specific to mitochondria. In our initial attempts at monitoring mitochondrial expression, we faced an unexpected and daunting challenge. Fluorescence signals were detected in control samples that lacked the fluorescent reporter gene. Stresses to the plant cells, presumably produced by biolistic transformation, appeared to create emission of light similar to that of the fluorescence proteins, which seemed to co-localize with mitochondria. To circumvent this technical challenge, we revised our original Phase I Project first goal to be the enabling of mitochondrial transformation in rice as a model for cereal crop plants using a selectable marker instead of a screenable fluorescence marker, i.e., transformed cells would be identified by their ability to grow in the presence of a selective agent/chemical. The selection of cells with mitochondria transformed with DNA was to be performed on callus tissues.Since wheat callus induction requires immature embryos, i.e., flowering plants, which we did not have, we chose rice as a model plant.Rice callus tissues are easily produced from embryos of mature seed using existing protocols.Rice is an excellent model for wheat as the two crop plants belong to the same evolutionary phylogenic group, Pooideae, the subfamily of the grass family, Poaceae. What we demonstrate in rice should be applicable to wheat with only few complications. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?1. NAPIGEN was featured in a Delaware Sustainable Chemistry Alliance blog article (October 2020). 2. NAPIGEN was featured in an article on October 2, 2020, in the journal, "Genetic Engineering & Biotechnology News". 3. NAPIGEN won the best startup company in the Industrial Biotechnology category at the world-wide competition, Hello Tomorrow Global Challenge, organized in Paris, France, on November 20, 2020. 4. Hajime Sakai, CEO & Co-founder at NAPIGEN, presented the latest advancements of NAPIGEN's science as an invited speaker at the 4th CRISPR AgBio Congress on December 1, 2020. 5. Dr. Hajime Sakai, Co-Founder & CEO, discussed NAPIGEN's gene editing technology as a featured speaker at the 8th Plant Genomics & Gene Editing Congress on March 3-4, 2021. 6. NAPIGEN was selected as one of sixteen finalists to pitch their technology at the Startup Stadium held during the BIO International Convention on June 10-18, 2021. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The major goal of this Phase I project was to provide an efficient method to gene edit plant mitochondrial DNA. A specific use of this technology would be the development of cytoplasmic male sterile (CMS) elite lines of wheat to enable the development of an efficient wheat hybrid seed production system. Hybrid wheat plants have the potential to create 15% more yield with the current germplasm. In addition to enhanced yield, hybrid plants are known for better tolerance to biotic and abiotic stresses than inbred plants. The introduction of hybrid seed into a traditionally non-hybrid crop would dramatically increase the market potential for agricultural seed companies. Farmers would benefit by having hybrid seed that would produce higher yield and have better tolerance to biotic and abiotic stresses. For the consumer, increased yield will make wheat more affordable across the world. Additionally, such breakthrough yield gains will help protect our environment by reducing the need for increasing farmland by deforestation, which is a major contributor to climate change. Our first objective was to identify sequences useful for maintaining exogenous DNA in plant mitochondria. We identified in the literature several good candidates for functional replication elements. We tested one of these elements in the Phase I Project. Constructs having this element showed mitochondrial gene expression of our selectable marker gene and enabled gene editing in mitochondria. Our second objective was to identify sequences that convey mitochondrial-specific gene expression. Based on the literature, we designed plasmid vectors having mitochondrial-specific expression elements. After biolistic transformation, vectors containing mitochondrial-specific expression elements produced rice calli with similar growth behavior, showing that these elements are functional and efficacious, i.e., the plasmids were transformed into mitochondria. Our third objective was to develop a screenable marker that is only expressed in mitochondria. Our first experiments involved the use of green fluorescent protein as a screenable marker. We encountered a number of experimental difficulties with this approach, as described in the later section on "Changes/Problems". Consequently, we switched to the approach of using a selectable marker instead of a screenable marker. In our first proof-of-concept experiments, we wanted to show that a nuclear-encoded selectable marker protein could be targeted to the mitochondria and used as a selectable marker in both yeast (Saccharomyces cerevisiae) and rice (Oryza sativa). For these experiments, we designed a selectable marker gene encoding a protein fused to a mitochondrial targeting sequence (MTS) to allow import of the selectable marker protein into the mitochondria. In both yeast and rice, nuclear transformants showed the ability to grow on the medium containing the selective agent. Encouraged by these nuclear gene transformation results, we made constructs to transform yeast and rice mitochondria with plasmid DNAs carrying the selectable marker gene. In yeast, we optimized the gene for yeast mitochondrial expression. After transforming the plasmid into wild-type yeast cells, cells were selected on a medium containing the selective agent. We obtained multiple transformants. Additionally, the transformants selected on the selective medium also had the Donor DNA integrated at the site designed for gene editing (our fourth objective). For experiments in rice, we designed three mitochondrial expression cassettes to have varying gene expression levels. Plasmid DNA for mitochondrial transformation was co-bombarded with another DNA that allowed selection of nuclear transformation using the hygromycin resistant gene (HPT). As we expect the frequency of mitochondrial transformation to be significantly less than that of nuclear transformation, we tried to enrich for mitochondrial transformants by selecting them among cells that also received the nuclear selection marker. The double selection was performed by using media containing both hygromycin and our mitochondrial selective agent. We observed that several independent rice calli grew on the medium with the double selection. No growth was observed among negative control samples. PCR analysis of several positive events showed the presence of not only the selectable marker gene but also mitochondrial plasmid DNA. Our fourth objective was to use CRISPR technology to gene edit wheat mitochondrial DNA. We changed our initial experiments from wheat to rice for reasons described in the later section on "Changes/Problems". For rice mitochondrial gene editing, we chose the region downstream of the mitochondrial ATP6 gene, where one of the CMS genes, orf79, is localized in existing rice lines. We chose the MAD7 site-specific nuclease, which belongs to the Cas12 class, as the CRISPR enzyme due to its availability for industrial use without a license. We chose two pairs of guide RNAs (total four cleavage sites) that were unique to rice mitochondrial DNA of the Nipponbare cultivar. Donor DNAs were designed to contain the regions of homology for insertion into the target sites. The target sequence of the gRNAs in the Donor DNAs were modified so that they would not be targets of CRISPR, i.e., gene edited mitochondrial DNA would be stable in the presence of MAD7 and gRNAs. Rice callus tissue was transformed with plasmids using the biolistic method and grown on selective media over two months. Gene editing events were analyzed by PCR reactions that amplify the junction regions of the Donor DNA integration. We observed the integration of Donor DNA multiple times among more than 200 independent events created by the combination of 12 different constructs we transformed. The integration of Donor DNA was further confirmed by sequencing of the PCR fragments of multiple events. In all cases, the junction fragments contained the sequences as predicted from the precise integration of Donor DNA near the cleavage sites induced by MAD7 site-specific nuclease. The ultimate goal of producing cytoplasmic male-sterile lines directly in elite wheat backgrounds is the key to enable global hybrid wheat seed production. To accomplish this objective requires the ability to transform plant mitochondrial DNA. The main reason for the prior inability to transform plant mitochondrial DNA has been the lack of a selectable marker gene. We have developed an approach that enables mitochondrial transformation in plants for the first time by using our selectable marker gene. The selectable marker system for plant mitochondrial transformation is the subject of a US Provisional Patent Application, No. 63/111,543, filed on 11/9/2020. By using the selectable marker gene and our existing technology of organelle gene editing, Edit Plasmids, which we have developed prior to this project (PCT International patent applications PCT/US2018/047566 and PCT/US2020/040730; US patent applications US 16/109,523 and US 16/641,073), we could demonstrate the gene editing of rice mitochondrial DNA through the integration of new DNA compositions for the first time in plant science. Our distinctive progress in plant mitochondrial gene editing should facilitate the creation of male-sterile crop plants by CRISPR technology.
Publications
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