Targeted mutagenesis in soybean using the CRISPR-Cas9 system

Abstract

Genome editing is a valuable technique for gene function analysis and crop improvement. Over the past two years, the CRISPR-Cas9 system has emerged as a powerful tool for precisely targeted gene editing. In this study, we predicted 11 U6 genes in soybean (Glycine max L.). We then constructed two vectors (pCas9-GmU6-sgRNA and pCas9-AtU6-sgRNA) using the soybean U6-10 and Arabidopsis U6-26 promoters, respectively, to produce synthetic guide RNAs (sgRNAs) for targeted gene mutagenesis. Three genes, Glyma06g14180, Glyma08g02290 and Glyma12g37050, were selected as targets. Mutations of these three genes were detected in soybean protoplasts. The vectors were then transformed into soybean hairy roots by Agrobacterium rhizogenes infection, resulting in efficient target gene editing. Mutation efficiencies ranged from 3.2–9.7% using the pCas9-AtU6-sgRNA vector and 14.7–20.2% with the pCas9-GmU6-sgRNA vector. Biallelic mutations in Glyma06g14180 and Glyma08g02290 were detected in transgenic hairy roots. Off-target activities associated with Glyma06g14180 and Glyma12g37050 were also detected. Off-target activity would improve mutation efficiency for the construction of a saturated gene mutation library in soybean. Targeted mutagenesis using the CRISPR-Cas9 system should advance soybean functional genomic research, especially that of genes involved in the roots and nodules.

Accelerating genetic gains in legumes for the development of prosperous smallholder agriculture: integrating genomics, phenotyping, systems modelling and agronomy

“…This technology has already been optimized in three model legume crops, soybean (Sun et al, 2015;Li et al, 2015), Lotus japonicas and Medicago truncatula (Meng et al, 2017). The efficiency of genome editing using various endogenous and exogenous RNA polymerase III promoters has been compared (Sun et al, 2015;Du et al, 2016) and in soybean the Cas9-single guide RNA together with donor DNA fragments was successfully transformed into soybean using the particle bombardment method (Li et al, 2015). Medicago and Lotus, having smaller diploid genomes, are the model legume crops for conducting research in several areas including symbiotic nitrogen fixation.…”

The Amaryllidaceae alkaloids: biosynthesis and methods for enzyme discovery

“…The CRISPR/Cas system has been applied to Nicotiana benthamiana , Oryza sativa , Arabidopsis thaliana , Sorghum bicolor , Citrus sinensis var. Valencia, Solanum lycopersicum , Nicotiana tabacum , Triticum aestivum, Zea maize (in press accepted), Glycine max, Populus tomentosa, and Marchantia polymorpha showing the versatility of this technique in plants (Feng et al; Jiang et al, 2013a; Shan et al, 2013; Brooks et al, 2014; Jia and Wang, 2014; Ron et al, 2014; Sugano et al, 2014; Fan et al, 2015; Gao et al, 2015; Johnson et al, 2015; Sun et al, 2015). To apply any of these targeted gene-editing systems to the Amaryllidaceae and obtain knockouts for candidate biosynthetic genes, the ability to make stable transformants is desirable and has been demonstrated in Narcissus tazzeta (Lu et al, 2007).…”

Making complex things simpler: modern tools to edit the plant genome

“…В исследовании, проведенном на рисе, была показана большая эффективность промотора OsU6 по сравнению с OsU3, однако систематического сравнительного анализа эффективности промоторов U3 и U6 применительно к геномному редактированию растений не проводилось (Mikami et al, 2015a). Обычно для экспрессии sgRNA применяются собственные промоторы U3 и U6 растения -объекта редактирования; использование гетерологичных промоторов из других видов растений может сопрово ждаться снижением эффективности редактирования Sun et al, 2015).…”