ARGOS8 maize lines were generated by CRISPR/Cas9 mediated editing including homology-directed DNA repair of the argos8 gene in Zea mays inbred PH184C. The genomic sequence of the gene was edited to produce ubiquitous and elevated expression across multiple tissues at different developmental stages.
ARGOS8 is a negative regulator of ethylene responses. Transgenic plants constitutively over-expressing ARGOS8 have reduced ethylene sensitivity and improved grain yield under drought stress conditions.
The intended phenotype of ARGOS8 maize lines was an enhanced drought tolerance.
Plasmids were designed to insert the native maize gos2 promoter (P-gos2-MAIZE), which confers a moderate level of constitutive expression, into the target sequence at the argos8 locus using single guide RNA(s), the Cas9 endonuclease gene derived from Streptococcus pyogenes and the DNA repair template which carries the maize gos2 promoter.
The plasmids were delivered into immature embryos by particle bombardment. Plantlets were regenerated from embryogenic calli.
Genomic DNA modifications at the argos8 locus were verified by PCR and sequencing, two lines were identified:
All reagents used in maize DNA sequence editing as well as plasmid backbones were removed by backcrossing in the early stages of breeding.
The ARGOS8 variants were found to be stably inherited via analysis over four generations.
Both ARGOS8 lines had elevated levels of ARGOS8 transcripts relative to the native allele and these transcripts were detectable in all the tissues tested.
A field study showed that compared to the wild-type, the ARGOS8 lines increased grain yield under flowering stress conditions and had no yield loss under well-watered conditions.
Source: Shi et al. (2017), Plant Biotechnology Journal 15, 207–216