Multiplex CRISPR/Cas9‐mediated metabolic engineering increases soya bean isoflavone content and resistance to soya bean mosaic virus

Abstract

and (Tel 86-25-84396410; fax 86-25-84396410; email dyyu@njau.edu.cn) † These authors contribute equally to this work. SummaryIsoflavonoids, 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 T 0 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 T 0 triplemutants leaves revealed significant improvement in isoflavone content. Compared with the wild type, the T 3 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 T 2 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. 1384

Long non-coding RNAs in plants: emerging modulators of gene activity in development and stress responses

“…Large-scale functional screens of lncRNAs by CRISPR/Cas9-based mutagenesis have been established in human and flies, although only a small percentage of lncRNAs showed context-specific phenotypic changes (Liu et al 2017 ). A similar system has yet to be developed for plant lncRNAs although large-scale mutagenesis populations have been created in several plant species by transformation of sgRNA libraries targeting protein-coding genes (Jacobs et al 2017 ; Lu et al 2017 ; Meng et al 2017 ; Zhang et al 2019 ; Liu et al 2020 ; Bai et al 2020 ). Finally, we need to investigate how we can effectively utilize the knowledge on beneficial lncRNAs in breeding programs to develop novel plant germplasm and elite crop varieties.…”

t‐SNAREs bind the Rhg1 α‐SNAP and mediate soybean cyst nematode resistance

“…In soybean, one or two genes can be easily knocked out via CRISPR‐Cas9 (Cai et al ., 2015). More recently, CRISPR has been used to induce mutations in up to three genes in soybean (Zhang et al ., 2019). Here, we successfully performed multiplexed CRISPR editing of up to four genes simultaneously in roots of the Peking and Essex soybean lines.…”

CRISPR/Cas9-Based Gene Editing Using Egg Cell-Specific Promoters in Arabidopsis and Soybean

“…Nevertheless, CRISPR/Cas9 genome editing systems have been successfully utilized in soybean since the first demonstrations in 2015. It has been applied to create targeted mutations in hairy roots, somatic embryos and stable transgenic plants (Jacobs et al, 2015;Li et al, 2015Li et al, , 2019Michno et al, 2015;Sun et al, 2015;Du et al, 2016;Chilcoat et al, 2017;Cai et al, 2018Cai et al, , 2020Kanazashi et al, 2018;Al Amin et al, 2019;Bao et al, 2019;Campbell et al, 2019;Cheng et al, 2019;Do et al, 2019;Zhang et al, 2019;Michno et al, 2020;Wang et al, 2020;Wu et al, 2020). Various promoters have been deployed for expression of Cas9.…”