Production of High Amylose and Resistant Starch Rice Through Targeted Mutagenesis of Starch Branching Enzyme Iib by Crispr/cas9

Abstract

Rice is the staple food for half of the world’s population. Starch accounts for 80-90% of the total mass of rice seeds, and rice starch is low in resistant starch (RS) with a high glycemic index (GI). RS has gained important since it is beneficial in preventing various diseases. Starch branching enzyme IIb (SBEIIb) plays a key role in the amylopectin synthesis pathway in the endosperm of cereals. Down-regulation of SBEIIb in several important crops has led to high amylose, high RS and low GI starch. In this study, we mutated OsSBEIIb in the japonica rice cultivar TNG82 through CRISPR/Cas9 and investigated the molecular and physicochemical modifications in OsSBEIIb mutant lines, e.g., gene expression, enzyme activity, apparent amylose content (AAC), RS and GI. As expected, the levels of modification in these starch related traits in heterozygous mutant lines were about half as those of homozygous mutant lines. Gene expression and enzyme activity of OsSBEIIb were down-regulated significantly while AAC and RS contents increased progressively from 17.4% and 0.5% in WT, 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. Our results demonstrate that it has huge potential for precise and efficient generation of high RS and low GI rice through CRISPR/Cas9 to provide a more suitable source of starch for type II diabetes.

Biochemical Analysis of SBEIIb Mutant Rice Lines Generated through CRISPR/Cas-mediated Targeted Mutagenesis

“…Previous research on the indica rice cultivar (TNGS14) observed that AC increased dramatically from 15.8 % in wild type to 24.1% in the heterozygous condition and 30.8 % in the homozygous mutant lines [36]. Baysal et al [37] developed and analyzed T3 seeds from a homozygous mutant (E15 CRISPR/Cas9 edited line) with a 4 bp deletion, observed a 1.4 fold increase in amylose content in the edited line (27.4 %) compared to the wild type japonica cultivar Nipponbare (19.6 %).…”

CRISPR-Cas technology a new era in genomic engineering

“…The rice starch is low in resistant starch and has a high glycemic index. In the Japonica rice (cultivar TNG82), the CRISPR-Cas9 editing of the OsSBEIIb gene resulted in increased resistant starch production and decreased reducing sugar production, as a result, the glycemic index in homozygous and heterozygous CRISPR-Cas mutant rice endosperms was reduced by 28% and 11%, respectively [122] .…”

“…In Limosilactobacillus reuteri a two-step approach using ssDNA and the CRISPR-Cas system was used for targeted codon mutagenesis, improving the number of recoverable recombinants, as well as identification of recombinant cells in bacteria with low recombineering efficiencies. This technology allowed the selection of oligonucleotide-mediated chromosomal deletions up to 1 kb, and it excluded the need for ssDNA recombineering optimization procedures [122] .…”