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Amfora is developing two categories of soybeans with increased protein content. One product is designed to increase the protein content of commodity soybeans by at least 10% with minimal impact on oil and yield. A second product is designed to deliver an ultra-high protein (ULTRAHIPRO TM) soymeal with more than 60% protein. A soybean meal with 60% protein can be used to displace soy protein concentrate in multiple applications including plant-based meats and as a high value ingredient in aquaculture feed. Amfora’s core technology is using gene editing to drive increased expression of NF-YC4, which results in a phenotype of increased protein at the expense of carbohydrates. We are using a combination of unique germplasm, modern genomics-based crop breeding and new breeding technologies to develop and commercialize valuable products for food and aquaculture applications.

The potential of genome editing to improve the agronomic performance of crops is often limited by low plant regeneration efficiencies and few transformable genotypes. Here, we show that expression of a fusion protein combining wheat GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) substantially increases the efficiency and speed of regeneration in wheat, triticale and rice and increases the number of transformable wheat genotypes. GRF4-GIF1 transgenic plants were fertile and without obvious developmental defects. Moreover, GRF4-GIF1 induced efficient wheat regeneration in the absence of exogenous cytokinins, which facilitates selection of transgenic plants without selectable markers. We also combined GRF4-GIF1 with CRISPR-Cas9 genome editing and generated 30 edited wheat plants with disruptions in the gene Q (AP2L-A5). Finally, we show that a dicot GRF-GIF chimera improves regeneration efficiency in citrus, suggesting that this strategy can be applied to dicot crops. PMID: 33046875 Funding information This work was supported by: Biotechnology and Biological Sciences Research Council, United Kingdom Grant ID: BB/P013511/1 NIGMS NIH HHS, United States Grant ID: R01 GM122968 Biotechnology and Biological Sciences Research Council, United Kingdom Grant ID: BB/L014130/1 Howard Hughes Medical Institute, United States

Rice (Oryza sativa) is one of the most important staple food crops feeding more than half of the world’s population. One of the requirements for future sustainable rice production is to develop drought tolerant varieties. We have identified a number of drought sensitive tagged lines by screening our rice activation tagging population. Two of the sensitive lines, AH13391 and AH17392, exhibit reduced drought tolerance compared to the controls, and have a single T-DNA in a region next to an ATPase-associated with diverse cellular activities (AAA)-like gene, respectively. Constitutive overexpression of either AAA-like gene (OsAAA-1 and OsAAA-2) significantly reduced the drought tolerance, whereas knocking them out by CRISPR-Cas9 significantly increased grain yield under both drought and well-watered field conditions. Comparative analysis of different OsAAA-1-edited variations shows that the core AAA ATPase domain and the C-terminal end of OsAAA-1 protein are important for its function in drought sensitivity; and OsAAA genes may regulate drought sensitivity through interacting with other drought-stress responsive partners. Our results show that OsAAA genes play an important role in drought sensitivity and demonstrate the feasibility of improving drought tolerance by CRISPR-mediated knockouts of native rice drought sensitive genes.

Plant gene editing is typically performed by delivering reagents such as Cas9 and single guide RNAs to explants in culture. Edited cells are then induced to differentiate into whole plants by exposure to various hormones. The creation of edited plants through tissue culture is often inefficient, time-consuming, works for only limited species and genotypes, and causes unintended changes to the genome and epigenome. Here we report two methods to generate gene-edited dicotyledonous plants through de novo meristem induction. Developmental regulators and gene-editing reagents are delivered to somatic cells of whole plants. This induces meristems that produce shoots with targeted DNA modifications, and gene edits are transmitted to the next generation. The de novo induction of gene-edited meristems sidesteps the need for tissue culture and promises to overcome a bottleneck in plant gene editing. PMID: 31844292 Funding information This work was supported by: NIGMS NIH HHS, United States Grant ID: T32 GM008347

5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the transfer of a carboxyvinyl group from phosphoenolpyruvate (PEP) to shikimate-3-phosphate and in plants is the target of the herbicide glyphosate. EPSPSs with high catalytic efficiency and insensitivity to glyphosate are of microbial origin, including the enzyme from Agrobacterium strain CP4, in which insensitivity is conferred by an active site alanine. In the sequence context of plant EPSPSs, alanine in place of glycine at the equivalent position interferes with the binding of both glyphosate and PEP. We show here that iterative optimization of maize EPSPS containing the G101A substitution yielded variants on par with CP4 in terms of catalytic activity in the presence of glyphosate. The improvement relative to G101A alone was entirely due to reduction in Km for PEP from 333 to 18 μm, versus 9.5 μm for native maize EPSPS. A large portion of the reduction in Km was conferred by two down-sizing substitutions (L97C and V332A) within 8 Å of glyphosate, which together reduced Km for PEP to 43 μm. Although the original optimization was conducted with maize EPSPS, contextually homologous substitutions conferred similar properties to the EPSPSs of other crops. We also discovered a variant having the known glyphosate-desensitizing substitution P106L plus three additional ones that reduced the Km for PEP from 47 μm, observed with P106L alone, to 10.3 μm. The improvements obtained with both Ala101 and Leu106 have implications regarding glyphosate-tolerant crops and weeds.