Two GE-M28 maize lines were generated by CRISPR-Cas editing of the m28 gene promoter in Zea mays (aka zmm28).
Aim of the mutations of the m28 gene promoter was the generation of maize plants with enhanced yield potential.
GE-M28 line 1: The intended mutations were targeted base pair substitutions. The mutations were designed to enhance the expression of the native m28 gene by introducing specific changes to its promotor region.
GE-M28 line 2: The intended mutation was targeted small base pair insertion (SDN2). The mutation was designed to enhance the expression of the native m28 gene by creating an Expression Modulating Element (EME), which is a short sequence found in the maize genome.
Both maize lines were created by the particle bombardment with four plasmids containing the components of the CRISPR-Cas9 system as well as a double stranded oligonucleotide serving as the DNA repair template.
Gene editing caused in GE-M28 line 1 an almost 3-fold increase of the m28 RNA expression in preliminary assays. The preliminary assays with GE-M28 line 2 showed a several-fold increase of the m28 gene expression compared to the m28 promoter without an EME.
The targeted changes were confirmed in both maize lines by Next Generation Sequencing (NGS) analysis.
Southern-by-sequencing was used to confirm absence of unintentionally integrated DNA from the transformation plasmids in the final maize lines.
Field trials are intended to evaluate the m28 gene expression and grain yield in field conditions.
Source:
USDA APHIS letter of inquiry