GE-SWEET rice lines were generated by CRISPR-Cas editing of the promoter region of three sugar transporter (sweet) genes in Oryza sativa line Kitaake and two mega varieties IR64 and Ciherang-Sub1.
The expression of at least one of the three host sucrose transporter genes sweet11, sweet13 and sweet14 is required for susceptibility to infection by Xanthomonas oryzae pv. Oryzae (Xoo).
CRISPR-Cas gene editing reagents were introduced into rice cells via transformation with disarmed Agrobacterium tumefaciens harboring plasmid vector IRS1132. The vector IRS1132 contained guide RNA cassettes targeting sequences specific for sweet11, sweet13, and sweet14 gene promoter regions, the gene cassette encoding CRISPR-Cas9 and the selectable hpt gene.
The identity of the introduced mutations in each of the three sweet gene promoter regions was confirmed by nucleotide sequencing of PCR amplicons.
Nine lines with different insertions, deletions and substitutions in sweet11, sweet13, and sweet14 showed resistance to Xoo-mediated bacterial blight disease.
Conventional breeding was used to generate transgene-free null-segregants. The absence of DNA insertions was confirmed by PCR amplification using ten different primer pairs corresponding to different components of the CRISPR-Cas construct.
Sources:
USDA-APHIS letter of inquiry
Oliva et al. (2019), Broad-spectrum resistance to bacterial blight in rice using genome editing. Nature Biotechnology 37:1344-1350