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Increasing the level of resistant starch in `Presidio` rice through multiplex CRISPR–Cas9 gene editing of starch branching enzyme genes
Friday, 2022/07/01 | 16:13:17

Sudip Biswas,Oneida Ibarra,Mariam Shaphek,Marco Molina-Risco,Mayra Faion-Molina,Marcela Bellinatti-Della Gracia,Michael J. Thomson,Endang M. Septiningsih

The Plant Genome 2022:e2025; First published: 17 June 2022; https://doi.org/10.1002/tpg2.20225


Rice (Oryza sativa L.) is an excellent source of starch, which is composed of amylopectin and amylose. Resistant starch (RS) is a starch product that is not easily digestible and absorbed in the stomach or small intestine and instead is passed on directly to the large intestine. Cereals high in RS may be beneficial to improve human health and reduce the risk of diet-related chronic diseases. It has been reported through chemical mutagenesis and RNA interference studies that starch branching enzymes (SBEs) play a major role in contributing to higher levels of RS in cereal crops. In this study, we used multiplex clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR associated protein 9 (Cas9) genome editing to simultaneously target all four SBE genes in rice using the endogenous transfer RNA (tRNA)-processing system for expressing the single-guide RNAs (sgRNAs) targeting these genes. The CRISPR–Cas9 vector construct with four SBE gene sgRNAs was transformed into the U.S. rice cultivar Presidio using Agrobacterium-mediated transformation. Knockout mutations were identified at all four SBE genes across eight transgene-positive T0 plants. Transgene-free T1 lines with different combinations of disrupted SBE genes were identified, with several SBE-edited lines showing significantly increased RS content up to 15% higher than the wild-type (WT) cultivar Presidio. Although further efforts are needed to fix all of the mutant alleles as homozygous, our study demonstrated the potential of multiplex genome editing to develop high-RS lines.


See https://acsess.onlinelibrary.wiley.com/doi/10.1002/tpg2.20225


Target regions of guide RNA (gRNA) targets of fourSTARCH BRANCING ENZYME(SBE) genes, transfer TNA(tRNA)–single-guide RNA (sgRNA) construct ofSBEgenes, in vitro digestion of gRNAs and cloning of thetRNA_gRNAconstruct. (a) Schematicmap of the gRNA target sites on the genomic regions ofSBE1,SBE2,SBE3,andSBE4. (b) In vitro ribonucleotide protein digestion ofSBE1, SBE2,SBE3,andSBE4genes polymerase chain reaction amplified product. L1, L8, and L9, 1 kb+ladders; L2, uncutSBE1target region; L3,SBE1targetregion digested with Cas9 and sgRNA1 (expected bands of 466 and 246 bp); L4, uncutSBE2target region; L5,SBE2target region digested withCas9 and sgRNA2 (expected bands of 447 and 168 bp); L6, uncutSBE4target region; L7,SBE4target region digested with Cas9 and sgRNA4(expected bands of 643 and 256 bp); L10, uncutSBE3target region; L11,SBE3target region digested with Cas9 and sgRNA3 (expected bands of682 and 127 bp). (c) Schematic diagram of tRNA–gRNA construct. (d) Cloning oftRNA–gRNAconstruct into destination vector pRGEB32. L1, 1kb+ladder; L2, blank plasmid digested with specific restriction enzymes; L3–L5, positive clones ofpRGEB32_tRNA_gRNAsdigested with specificrestriction enzymes


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