GE-GBSS potato lines were generated by editing the granule-bound starch synthase (gbss) gene in tetraploid plants of Solanum tuberosum cultivar Kuras using the CRISPR/Cas9 technology.
The granule-bound starch synthase gene encodes an enzyme catalyzing production of amylose.
Aim of the modification was the generation of an altered starch quality by full knockout of GBSS function.
For targeted multiallelic mutagenesis in tetraploid potato, two target regions in gbss exon 8 (GT1, GT2) and one target region in exon 9 (GT4) were selected.
Expression of the four sgRNA-pcoCas9 constructs, pE-GT1, pE-GT2, pE-GT4 and pE-StU6GT4, as well as the combined expression of GT1-sgRNA and pcoCas9 from separate vectors was achieved through PEG-mediated protoplast transfection.
A PCR-based high-resolution fragment analysis method was used for the identification of multiple mutated alleles. Six of those lines were subjected to sequencing, where no wild-type gbss fragment was found.
Most mutations resulted in small indels of 1–10 bp, but also vector DNA inserts of 34–236 bp were found in 10 % of analysed lines.
Full knockout of GBSS enzyme activity was confirmed in four-allele mutated lines by phenotypic studies of starch. Starch in these lines lacks amylase.
Source: Andersson et al. (2017), Plant Cell Rep 36:117–128