Application of ZFN for Site Directed Mutagenesis of Rice SSIVa Gene

Journal Article / 2018

Jung, Yu-Jin; Nogoy, Franz Marielle; Lee, Sang-Kyu; Cho, Yong-Gu; Kang, Kwon-Kyoo

Many successful studies on genome editing in plants have been reported and one of the popular genome editing technology used in plants is Zinc Finger Nucleases (ZFN), which are chimeric proteins composed of synthetic zinc finger-based DNA binding domain and a DNA cleavage domain. The objective of this research was to utilize ZFNs to induce a double-stranded break in SSIVa, a soluble starch synthase involved in starch biosynthesis pathway, leading to the regulation of the SSIVa expression. The isoform SSIVa is not yet well studied, thus, by modifying the endogenous loci in SSIVa, we can explore on the specific roles of this gene in starch biosynthesis and other possible functions it might play. In this study, we used ZFN-mediated targeted gene disruption in the coding sequence of the SSIVa rice gene in an effort to elucidate the functional role of the gene. Generation of transgenic plants carrying premature stop codons and substitution events, revealed no SSIVa mRNA expression, low starch contents and dwarf phenotypes. Remarkably, based on our analysis SSIVa gene disruption had no effect on other starch synthesis related genes as their expression remained at wild type levels. Therefore, the engineered ZFNs can efficiently cleave and stimulate mutations at SSIVa locus in rice to affect plant height, grain filling and starch content.