HO-0 soybean lines were generated by targeted knock-out of the fatty acid desaturase 2 (FAD2) gene in Glycine max L. cultivar Kwangan.
FAD2 is known as a regulator of the unsaturated fatty acid content in soybean.
The intended phenotype was a soybean cultivar with high oleic acid content in the seeds.
To edit the fad2 gene by using CRISPR/Cas9 system, the CRISPR-Cas9 vector pPZP-FAD2-7 with the selectable bar gene and a fad2 targeting guide RNA was delivered into the plant cells by a disarmed Agrobacterium-mediated transformation system.
The edited soybeans were selected by next-generation sequencing, and the transgene-free soybeans were obtained by self-crossing.
T1 transformants showed small deletions or insertions at the target site in the fad2 gene in chromosome 10 and chromosome 20. Line FAD2-7-30 #21-2 was chosen for further study.
T1 seeds of the HO-0 soybean lines had oleic acid levels of approximately 80-85%.
Source:
USDA APHIS letter of inquiry