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Efficient Production of High Resistant Starch Rice through Targeted Mutagenesis of Starch Branching Enzyme Ⅱb By CRISPR/Cas9

Hsi-Chao Wang, Su-Ying Yeh, Yong-Pei Wu, Yu-Chia Hsu, Maurice S. B. Ku*

Rice is the staple food for half of the world’s population. Rice starch is low in Resistant Starch (RS) with a high
Glycemic Index (GI). RS has gained importance since it is beneficial in preventing various diseases. Starch Branching
Enzyme IIb (SBEIIb) plays a key role in amylopectin synthesis in the endosperm of cereals. In this study, we mutated
OsSBEIIb in the Japonica rice cultivar Tainung82 (TNG82) through CRISPR/Cas9 and investigated the molecular and
physicochemical modifications in OsSBEIIb mutant lines, e.g., gene expression, enzyme activity, Amylose Content
(AC), RS and GI. As expected, gene expression and enzyme activity of OsSBEIIb were down-regulated significantly
while AC and RS contents increased progressively from 17.4% and 0.5% in weight, respectively, to as high as
25.0% and 7.5% in heterozygous mutant lines and 36.0% and 12.0% in homozygous mutant lines. Consequently,
with increased RS and decreased rate of reducing sugar production, GI progressively decreased in heterozygous
and homozygous mutant rice endosperms by 11% and 28%, respectively. Transgene-free plants were subsequently
identified in the T1 populations. Our results demonstrate the precise and efficient generation of high RS and low
GI transgene-free rice through CRISPR/Cas9 to provide a more suitable source of starch for type II diabetes.