Peipei Zhang, Hongyang Du, Jiao Wang, Yixiang Pu, Changyun Yang, Rujuan Yan, Hui Yang, Hao Cheng, Deyue Yu

Plant Biotechnol J.; 2020 Jun;18(6):1384-1395.

Abstract

Isoflavonoids, which include a variety of secondary metabolites, are derived from the phenylpropanoid pathway and are distributed predominantly in leguminous plants. These compounds play a critical role in plant-environment interactions and are beneficial to human health. Isoflavone synthase (IFS) is a key enzyme in isoflavonoid synthesis and shares a common substrate with flavanone-3-hydroxylase (F3H) and flavone synthase II (FNS II). In this study, CRISPR/Cas9-mediated multiplex gene-editing technology was employed to simultaneously target GmF3H1, GmF3H2 and GmFNSII-1 in soya bean hairy roots and plants. Various mutation types and frequencies were observed in hairy roots. Higher mutation efficiencies were found in the T₀ transgenic plants, with a triple gene mutation efficiency of 44.44%, and these results of targeted mutagenesis were stably inherited in the progeny. Metabolomic analysis of T₀ triple-mutants leaves revealed significant improvement in isoflavone content. Compared with the wild type, the T₃ generation homozygous triple mutants had approximately twice the leaf isoflavone content, and the soya bean mosaic virus (SMV) coat protein content was significantly reduced by one-third after infection with strain SC7, suggesting that increased isoflavone content enhanced the leaf resistance to SMV. The isoflavone content in the seeds of T₂ triple mutants was also significantly increased. This study provides not only materials for the improvement of soya bean isoflavone content and resistance to SMV but also a simple system to generate multiplex mutations in soya bean, which may be beneficial for further breeding and metabolic engineering.

See: https://pubmed.ncbi.nlm.nih.gov/31769589/

Figure 1: Targeted modification of GmF3H1, GmF3H2 and GmFNSII‐1 using the multiplex CRISPR/Cas9 system. (a) Schematics of the GmF3H1, GmF3H2 and GmFNSII‐1 loci. CDS regions are shown in yellow, and mRNA transcripts are depicted with black lines. The locations and sequences of the sgRNA targets are indicated, along with the protospacer adjacent motif (PAM, red underline) and GC% content. (b) Schematic of the multiplex CRISPR/Cas9 vectors used to modify GmF3H1, GmF3H2 and GmFNSII‐1. Bar, glufosinate‐resistance gene; GmUbi3pro, promoter of GmUbi3; GmU6, promoter of GmU6‐16g‐1; GmU3, promoter of GmU3‐19g‐1, CaMV‐T, terminator of Cauliflower mosaic virus (CaMV); dpCas9, dicotyledon codon‐optimized Cas9.