Should the project progress as planned, we expect to garner the following deliverables that will directly benefit soybean growers by increasing the profit of growing soybean while reducing inputs:
• A fast, cost-effective, and non-transgenic base editing protocol for accurately modifying soybean genes without disrupting their functions;
• Multiple soybean lines resistant to diverse herbicides generated with the new base editing technology;
• Multiple soybean cultivars equipped with the base editing BE3 enzyme, as well as other base-editing Cas9 with more advanced characteristics, ready to be utilized by the soybean research community for editing other soybean gene in order to improve soybean seed quality and yield.

Updated October 1, 2019:
First of all, we wish to thank NCSRP for the decision to continued support!
By now we have completed the first year of this NCSRP-funded project. We are proud to announce that we have met the milestones – with the generated soybean plants being reared for seed production.
• Specifically, we have successfully assembled a construct that contains both the BE3 base-editing Cas9 enzyme, and a guide RNA designed to guide BE3 to a specific position in the soybean ALS gene to mediate the editing of specific bases, leading to herbicide (Imazapyr) resistance. This plasmid is named as pWI-BE3-gGmALS. We also produced another construct that would express the BE3 enzyme only, designated as pWI-BE3 (milestone 2).
• Both constructs have been used to transform embryogenic tissues generated from young soybean seed, using a particle bombardment procedure. We obtained five (5) transgenic events for pWI-BE3-gGmALS, and one (1) event for pWI-BE3, from which transgenic seedlings are being induced. At least one event appears to contain the successfully base-edited ALS gene. Other events may also yield base-edited seedlings because the construct continues to carry out the editing while the tissues are being induced for seedling differentiation (milestone 1).
• Both the pWI-BE3-gGmALS and pWI-BE3 constructs have been sent to collaborators in Nebraska, Missouri, and Minnesota for producing base-edited plants in other soybean varieties (milestone 3). The transformed tissues are being selected for herbicide tolerance.

Finally, in preparation for the second year of the project, we have also been investigating the potential of adopting a new homologous recombination protocol, named as CRISPEY, in the research of generating novel herbicide tolerance traits. We are also testing a novel base-editing Cas9 that would allow us to modify a longer stretch of genes.
Again, we wish to thank NCSRP for the renewal of our project, and for the confidence you placed with us!

1. We have calibrated a streamlined protocol to quickly turn very young soybean seed into embryonic tissues in liquid cultures.
2. We have engineered the DNA constructs that once delivered into soybean cells, drive the so-called CRISPR base editing to modify specific soybean genes.
3. We used the two sets of tools outlined above to alter one of the soybean genes, known as GmALS1, at one of the amino acid positions.
4. We have succeeded in modifying the GmALS1 gene in a specific way that turned it into an enzyme that degrades the herbicide Imazapyr. This has been done in one soybean cultivar. The same set of DNA constructs are being tested in other soybean cultivars.
5. We are currently rearing the soybean seedlings in which GmALS1 has been modified in greenhouse, waiting for seed to set.