Progress 09/01/18 to 12/31/21
Outputs
Target Audience:The instrument developed during the Phase I award period is a core part of Nodexus' long-term plans to enable end-to-end workflows for gene edited agricultural products (see workflow diagram below, Figure 1). Figure 1: Proposed Nodexus plant cell biology platform workflow In recent years, plant biotechnology has witnessed rapid growth worldwide, and tremendous potential for optimization has been unlocked through analysis and optimization at the genetic level. It has become clear that genome editing will play a prominent role in crop improvement in the next several years, but there are still critical challenges that need to be addressed to realize the technology's full potential. In particular, there are difficulties associated with end-to-end workflows for efficient and effective gene editing that are accessible to benchtop or even field researchers in a widespread manner. In particular, current methods a) do not efficiently provide scalable protoplast selection, regeneration, and production; b) are laborious, required skilled technicians, and are expensive, making them likely nonviable for most organizations; c) require complex workflows today leading to slower time to perform trait optimization for crop production; and, d) have not solved the market need to innovate highly, as limited growth opportunities exist due to lack of new methods to improve crop quality. Successful commercialization will result in the generation of a scalable hardware platform for protoplast screening and isolation following genetic editing (using CRISPR/Cas and other methodologies) of various crop types to improve yields, soil type/stressor and drought/pest resistance, among others. Figure 2: Primer on the criticality of isolating live, viable, individual protoplasts Creating a deployable tool for isolation post-editing will provide a key platform in the arsenal for major agricultural corporations, the USDA, and other governmental entities conducting regulatory research, but also, importantly, much smaller facilities due to the accessible nature of the infrastructure in terms of cost and ease-of-use. This will help address bottlenecks in development and production while also bringing gene editing workflows a step closer to becoming more widely accessible. Figure 3: Proposed long-term protoplast regeneration workflow (both upstream and downstream of existing Nodexus platform) Current methods for single-cell isolation often suffer from numerous limitations. Single-cell analysis systems require extensive hands-on time and infrastructure, making both system and per-test cost exorbitantly expensive, and these systems are restricted to Core Facilities. Further, these methods suffer from contamination and loss of cell viability. No single-cell enrichment product meets the functional, financial, workflow, and personnel limitations of labs that do not have extensive budgets. Furthermore, none of these instruments are tailored toward protoplast selection and isolation as well as maximizing regeneration capacity. Current mechanisms fall into tools in three main categories - limiting dilution, automated cell picking, and large-particle fluorescence-activated-cell sorting. ? Table 1: Qualitative competitive landscape based upon market research from target customers Limiting dilution This option leads to very low isolation efficiency, as it is not selective. Liquid is dispensed with the hope of a single protoplast in a drop (very low success rate due to stochastic limitations). Limiting dilution does not let one select targets (e.g. fluorescence). For scalability, extensive labor or expensive robotics is involved. No information or data is provided. Automated cell picking/micro-manipulator This method can compromise cell survival due to the processing. This requires very high labor/long time for selection. For automation, these systems are very expensive. Large particle Fluorescence-Activated-Cell Sorting This does not necessarily provide access to individual particles. The shear is known to compromise cell viability. FACS requires a skilled technician for operation. Instrumentation requires very high up-front capital costs. Nodexus envisions significant usage of the proposed system (and future, pipeline products) within agricultural companies and research labs as well as governmental agencies including the USDA and other regulatory bodies for rapid and scalable enrichment and isolation of target cells for validation studies or for internal research. Ultimately, the platform will enable large and small agriculture sites to adopt/accelerate gene editing workflows for crop development. The proposed platform will offer functional, workflow, and cost benefits in the rapidly-emerging genetic engineering space while preserving sample sterility, viability, and minimizing contamination. As gene editing has immense implications for the agriculture sector, the proposed workflow improvement will ultimately increase manufacturing capability and efficiency/profitability for agricultural sites big and small while also enhancing workflows aimed at improving plant survival and resistance.For an example of market impact of the technology, the current estimated market size for sugar is roughly $100B worldwide in 2017 (source: BCC Research). Sugar beet is estimated to account for 50% of US-based sugar production, and roughly 20% of worldwide sugar production. As such, even a moderate 5% improvement in yield of the sugar content within sugar beet (leveraging the protoplast editing and regeneration from this platform) would result in a minimum of $1B worth of extra sugar produced globally every year. Deployment of this platform can replace existing single-protoplast isolation processes, leading to 10X reduction in time for processing and 10X reduction in cost compared to traditional centralized facility cell sorting equipment. Target audiences: Large and small agricultural corporations (e.g. Bayer-Monsanto) - in these organizations, the typical decision makers are business unit heads and R&D leaders. Beyond low volume instrument purchasing, widespread deployment within an organization relies on executive approval (e.g. CXOs). USDA research scientists and other domestic and international regulatory test engineers Individual academic/governmental/industrial researchers e.g. Professors, Postdoctoral Fellows, Graduate Students, and Research Scientists. To the broader community, these workflows enable tremendous potential to address crop yield, health benefits, resistance, and more. The USDA and other governmental agencies with ever-reducing budgets can utilize the low-cost, easy-to-use nature of the proposed system to enable unprecedented access to protoplasts following gene editing processes.Creating a deployable tool for isolation post-editing will provide a key platform in the arsenal for major agricultural corporations, the USDA, and other governmental entities conducting regulatory research, but also much smaller facilities due to the accessible nature of the infrastructure in terms of cost and ease-of-use. This will help address bottlenecks in development and production while also bringing gene editing workflows a step closer to becoming more widely accessible. Overall, the platform will increase the number of sites and users adopting these workflows to play a role in increasing the efficiency and overall number of gene edited crops. As an organization, we envision opportunities to license this technology in the longer term as well as further expansion by generating additional revenue streams. Changes/Problems:Through the Phase I SBIR award project period, Nodexus was able to demonstrate proof-of-concept feasibility of a hardware platform designed for the detection, sorting, and isolation of agriculturally relevant populations such as protoplasts and plant cell clusters. Although numerous challenges were faced including electrode survivability and personnel recruitment capabilities during the global COVID-19 pandemic which took place during the project period, Nodexus' hardware platform, disposable plastic cartridges, and software were all developed to the point internally that testing of model samples (e.g. polystyrene microbeads, hydrogel particles) as well as plant cell biology samples (e.g. protoplasts and cell clusters obtained from a local industry partner) was performed and promising results were obtained to achieve Technical Objective 1. As Nodexus transitions beyond the Phase I SBIR award project period, it will continue to develop the system and cartridges to support the relevant size ranges necessary for broad agtech market adoption of the proposed Nodexus platform. Further testing with protoplast samples (both internally and with the help of external partners) will take place with the goal of achieving Technical Objective 2 (regenerating a sorted and dispensed protoplast) within the company prior to seeking further USDA/NIFA support through a Phase II SBIR or beyond. What opportunities for training and professional development has the project provided?Exposure to partners in the plant cell biology sector, some of whom Nodexus intends to have ongoing relationships with. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
During the Phase I award period, a proof-of-concept hardware system was designed and fabricated, as was an injection-molded disposable cartridge. Windows®-based software control of the system was further implemented as shown in Figure 4 below. Figure 4: Proof-of-concept Nodexus hardware instrument, disposable cartridge (injection molded cyclic olefin copolymer plastic), and Windows®-based software interface Extensive work was performed by Nodexus' small in-house team in order to validate the Node-Pore Sensing (NPS) methodology on injection molded plastic cartridges, and Figure 5 below showcases the flexographic printer that was created, fabricated, and programmed in-house at Nodexus in order to "print" conductive ink electrodes onto cyclic olefin copolymer injection molded plastic cartridges. Figure 5: Proof-of-concept Nodexus disposable cartridge (injection molded cyclic olefin copolymer plastic) with conductive ink electrodes patterned using in-house developed and fabricated flexographic printer (top). After numerous person-months of effort to integrate NPS detection into the hardware system, we ultimately shelved this development in favor of proceeding with a low-cost laser and silicon photomultiplier-based detection scheme as shown below in Figure 6. Figure 6: Proof-of-concept Nodexus fluorescence and light scattering detection methodology developed for the hardware system shown above in Figure 4. Technical Objective 1 as described above was successfully achieved using the proof-of-concept system and cartridges shown in Figures 4-6. Figures 7-10 below showcase the ability of the system to use fluorescence and light scattering-based detection (which replaces the NPS methodology to discriminate particle sizing as a sorting parameter) in order to detect, sort, and dispense (1) polystyrene beads, (2) hydrogel model particles, and (3) proprietary protoplast samples obtained from a local Bay Area-based plant cell biology partner. Software was specifically written to support the Nodexus system's plate and bulk sorting, analysis, and data review capabilities. Throughout the Phase I project period, extensive validation of the system has taken place en route to commercialization.
Publications
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