Update:
1) Testing the CRISPR/Cpf1 genome editing system in soybean. Two putative transgenic lines derived from a CRISPR/Cpf1 construct produced some seeds. The progeny plants (T1 generation) are currently being grown in the greenhouse. The genotyping of those plants for presence of Cpf1 and guide RNA and for potential edits of the two targeted FAD2 genes is in progress.

2) Improving the efficiency of CRISPR/Cas9 genome editing. The CRSPR/Cas9 system has been modified into intron Cas9 (Cas9 gene containing an intron), which is expected to produce higher expression levels of Cas9 protein in plants. The new system is currently being tested for editing in stable transgenic Arabidopsis plants with two constructs, one containing 4 guide RNAs targeting four genes of a family of 10 members and the second containing 6 guide RNAs targeting the remaining 6 genes. As controls, the original CRISPR/Cas9 was used to make two constructs with the same multiplexing guide RNAs. Transformation of Arabidopsis with the four constructs has been done and T1 plants are currently grown in the greenhouse. The preliminary genotyping results show a significant increase in the efficiency of creating edits (7 genes vs 1 gene among the 10 target genes). The results are consistent with the data from the soybean hairy root system when both the intron Cas9 was tested against the original Cas9 system. The intron Cas9 for genome editing in stable transgenic soybean plants is ongoing as plants are maturing in the greenhouse. The intron Cas9 construct targets the soybean FAD2 gene.

3) Testing CRISPR/Cas9 base editors in soybean. We have received plants from the ISU Plant Transformation Facility that have been transformed with three Cas9 base editor constructs, two of which target the genes encoding acetolactate synthase (ALS), and one that targets the gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). These plants are being grown to maturity. Successful editing of these genes will create herbicide resistant plants, which will be tested in the progeny plants.

The main goals of this project are geared to improving gene editing in soybean and expanding the genome editing tool box for soybean.

Toward this goal, we have demonstrated that expressing the Cas9 protein specifically in the egg cells of soybean results in efficient gene editing. The egg cell-specific expression of Cas9 is significant, because this enables the CRISPR/Cas9 system to create gene edits that are more likely to be inherited to the next generation than of strategies for expression. We have also demonstrated in soybean hairy roots and Arabidopsis that the addition of an intron sequence into the Cas9 gene will result in higher expression levels of the Cas9 in plant cells. The higher level of Cas9 is important, because it increases the efficiency of gene editing. Our goal now is to combine the egg cell specific expression with the intron Cas9 to maximize gene editing in soybean. The transgenic plants necessary to test this idea are now being generated.

Towards the goal of expanding the genome editing tool box for soybean, the CRISPR/Cas9 base editor constructs are being tested, and the necessary transgenic plants are being produced. The base editors enable precise DNA changes to be made, and there functions have been tested in soybean hairy roots and Arabidopsis. If the soybean base editors work as expected, we anticipate that some of the progeny from the transgenic plants that are currently growing to maturity will be resistant to sulfonyl urea or glyphosate herbicides.