Multiplexed CRISPR/Cas9‐mediated metabolic engineering of γ‐aminobutyric acid levels in <i>Solanum lycopersicum</i>

Lin, Rui; Li, Ran; Li, Xindi; Fu, Daqi; Zhu, Beiwei; Tian, Huiqin; Luo, Yunbo; Zhu, Hongliang

doi:10.1111/pbi.12781

Cited by 239 publications

(109 citation statements)

References 49 publications

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“…The CRISPR/Cas9 technique has several advantages in loss‐of‐function analysis: it causes knockout of the target gene with relatively low off‐target activities, it efficiently disrupts the DNA sequence and it causes heritable loss of function (Barrangou et al ., ). Hitherto, the multiplex CRISPR/Cas9 system has been successfully applied to precisely edit tomato genomic sequences and create multisite genome knockout mutations (Li et al ., ). However, there has been no report of designing lncRNAs in tomato using CRISPR/Cas9 technology.…”

Section: Introductionmentioning

confidence: 97%

CRISPR/Cas9‐mediated mutagenesis of lncRNA1459 alters tomato fruit ripening

et al. 2018

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With the development of high-throughput sequencing, many long non-coding RNAs (lncRNAs) have been found to play important roles in diverse biological processes. However, the biological functions of most plant lncRNAs are still unknown. We have previously discovered a tomato ripening-related lncRNA, lncRNA1459. Here, we cloned the full-length lncRNA1459, giving two transcript isoforms. In addition, lncRNA1459 exhibited a specific location in the nucleus. Furthermore, in order to fully identify the function of lncRNA1459 in tomato ripening, loss-of-function mutants of lncRNA1459 were developed using clustered regularly interspaced short palindromic repeats (CRISPR)/-associated protein 9 (Cas9)-induced genome editing technology. Compared with wild-type fruits, the tomato ripening process was significantly repressed in lncRNA1459 mutants. Ethylene production and lycopene accumulation were largely repressed in lncRNA1459 mutants. Additionally, genes related to ethylene and carotenoid biosynthesis were distinctly downregulated in lncRNA1459 mutants compared with wild-type fruits. Moreover, expression of numerous ripening-related genes was changed significantly when lncRNA1459 was knocked out. Expression of potential tomato ripening-related lncRNAs was also specifically changed after knocking out lncRNA1459. Taken together, these results provide insight into the role of lncRNA1459 in tomato fruit ripening.

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Section: Introductionmentioning

confidence: 97%

CRISPR/Cas9‐mediated mutagenesis of lncRNA1459 alters tomato fruit ripening

et al. 2018

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“…Another strategy is to suppress competing metabolic pathways. Using a multiplex pYLCRISPR/Cas9 system that targeted five key genes, the γ‐aminobutyric acid (GABA) shunt was converted in tomato (Li et al , ), and lycopene was enriched in tomato fruit by knocking down five genes associated with the carotenoid metabolic pathway (Li et al , ).…”

Section: Discussionmentioning

confidence: 99%

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

Zhang

Wang

et al. 2019

Plant Biotechnology Journal

212

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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

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“…In addition, the combination of natural and gene‐edited mutations of regulators of meristem maturation successfully enabled the development of weakly branched tomato hybrids with higher flower and fruit production (Soyk et al ., ). Multiplex genome editing of up to six genes with multiple gRNAs was also effective in increasing fruit nutritional value by rewiring lycopene (Li et al ., ) and GABA (Li et al ., ) metabolic pathways.…”

Section: From Trait Discovery To Trait Engineering: Gene Editingmentioning

confidence: 99%

Trait discovery and editing in tomato

2018

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Summary Tomato (Solanum lycopersicum), which is used for both processing and fresh markets, is a major crop species that is the top ranked vegetable produced over the world. Tomato is also a model species for research in genetics, fruit development and disease resistance. Genetic resources available in public repositories comprise the 12 wild related species and thousands of landraces, modern cultivars and mutants. In addition, high quality genome sequences are available for cultivated tomato and for several wild relatives, hundreds of accessions have been sequenced, and databases gathering sequence data together with genetic and phenotypic data are accessible to the tomato community. Major breeding goals are productivity, resistance to biotic and abiotic stresses, and fruit sensorial and nutritional quality. New traits, including resistance to various biotic and abiotic stresses and root architecture, are increasingly being studied. Several major mutations and quantitative trait loci (QTLs) underlying traits of interest in tomato have been uncovered to date and, thanks to new populations and advances in sequencing technologies, the pace of trait discovery has considerably accelerated. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing (GE) already proved its remarkable efficiency in tomato for engineering favorable alleles and for creating new genetic diversity by gene disruption, gene replacement, and precise base editing. Here, we provide insight into the major tomato traits and underlying causal genetic variations discovered so far and review the existing genetic resources and most recent strategies for trait discovery in tomato. Furthermore, we explore the opportunities offered by CRISPR/Cas9 and their exploitation for trait editing in tomato.

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Multiplexed CRISPR/Cas9‐mediated metabolic engineering of γ‐aminobutyric acid levels in Solanum lycopersicum

Cited by 239 publications

References 49 publications

CRISPR/Cas9‐mediated mutagenesis of lncRNA1459 alters tomato fruit ripening

CRISPR/Cas9‐mediated mutagenesis of lncRNA1459 alters tomato fruit ripening

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

Trait discovery and editing in tomato

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