Seamless Genome Editing in Rice via Gene Targeting and Precise Marker Elimination
Positive–negative selection using hygromycin phosphotransferase (hpt) and diphtheria toxin A-fragment (DT-A) as positive and negative selection markers, respectively, allows enrichment of cells harboring target genes modified via gene targeting (GT). We have developed a successful GT system employing positive–negative selection and subsequent precise marker excision via the piggyBac transposon derived from the cabbage looper moth to introduce desired modifications into target genes in the rice genome. This approach could be applied to the precision genome editing of almost all endogenous genes throughout the genome, at least in rice.
Key wordGene targeting Positive–negative selection Marker excision piggyBac transposon Homologous recombination
We acknowledge Dr. R. Terada (Meijo University) and Dr. S. Iida (Shizuoka Pref. University) for providing the DT-A gene, Dr. K. Uchino and Dr. H. Sezutsu (National Institute of Agrobiological Sciences) for providing hyPBase gene, and Dr. H. Rothnie for English editing. This research was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Genomics for Agricultural Innovation PGE1001), KAKENHI (23658012 and 23310142) and the Cross-ministerial Strategic Innovation Promotion Program (SIP).
- 12.Saika H, Onodera H, Seiichi T (2012) Visual selection in rice: a strategy for the efficient identification of transgenic calli accumulating transgene products. In: Dunwell JM, Wetten AC (eds). Transgenic plants. Humana, Totawa. Methods Mol Biol 847:67–74Google Scholar