Dataset Information

Genome Editing in Cowpea Vigna unguiculata Using CRISPR-Cas9.

ABSTRACT: Cowpea (Vigna unguiculata) is widely cultivated across the world. Due to its symbiotic nitrogen fixation capability and many agronomically important traits, such as tolerance to low rainfall and low fertilization requirements, as well as its high nutrition and health benefits, cowpea is an important legume crop, especially in many semi-arid countries. However, research in Vigna unguiculata is dramatically hampered by the lack of mutant resources and efficient tools for gene inactivation in vivo. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). We applied the CRISPR/Cas9-mediated genome editing technology to efficiently disrupt the representative symbiotic nitrogen fixation (SNF) gene in Vigna unguiculata. Our customized guide RNAs (gRNAs) targeting symbiosis receptor-like kinase (SYMRK) achieved ~67% mutagenic efficiency in hairy-root-transformed plants, and nodule formation was completely blocked in the mutants with both alleles disrupted. Various types of mutations were observed near the PAM region of the respective gRNA. These results demonstrate the applicability of the CRISPR/Cas9 system in Vigna unguiculata, and therefore should significantly stimulate functional genomics analyses of many important agronomical traits in this unique crop legume.

SUBMITTER: Ji J

PROVIDER: S-EPMC6566367 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Genome Editing in Cowpea Vigna unguiculata Using CRISPR-Cas9.

Ji Jie J Zhang Chunyang C Sun Zhongfeng Z Wang Longlong L Duanmu Deqiang D Fan Qiuling Q

International journal of molecular sciences 20190519 10

Cowpea (Vigna unguiculata) is widely cultivated across the world. Due to its symbiotic nitrogen fixation capability and many agronomically important traits, such as tolerance to low rainfall and low fertilization requirements, as well as its high nutrition and health benefits, cowpea is an important legume crop, especially in many semi-arid countries. However, research in Vigna unguiculata is dramatically hampered by the lack of mutant resources and efficient tools for gene inactiv ...[more]

PMID: 31109137

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Project description:Background:The legume cowpea (Vigna unguiculata L.) is extensively grown in sub-Saharan Africa. Cowpea, like many legumes has proved recalcitrant to plant transformation. A rapid transient leaf assay was developed for testing gene expression and editing constructs prior to stable cowpea transformation, to accelerate cowpea and legume crop improvement. Results:Attempts to develop a transient protoplast system for cowpea were unsuccessful. Leaflets from plants 3-4 weeks post-germination were age selected to establish a rapid Agrobacterium (Agro) infiltration-mediated transient system for efficacy testing of gene expression and CRISPR/Cas9 gene editing constructs. In planta, Agro-infiltration of leaflets with fluorescent expression constructs, resulted in necrosis. By contrast, Agro-infiltration of detached leaflets with an Arabidopsis (At) ubiquitin3 promoter:ZsGreen construct, followed by culture on solid nutrient medium resulted in fluorescence in over 48% of leaf cells. Expression efficiency was leaf age-dependent. Three cowpea meiosis genes were identified for CRISPR/Cas9 gene-editing, with the forward aim of meiosis-knock out for asexual seed induction in cowpea. Constructs were designed and tested containing candidate gene-specific guide RNAs, expressed using either the cowpea or Arabidopsis U6 promoters with Cas9 expression directed by either the Arabidopsis 40S ribosomal protein or parsley ubiquitin4-2 promoters. Leaflets were infiltrated with test gene-editing constructs and analytical methods developed to identify gene-specific mutations. A construct that produced mutations predicted to induce functional knockout of in the VuSPO11-1 meiosis gene was tested for efficacy in primary transgenic cowpea plants using a previously established stable transformation protocol. Vuspo11-1 mutants were identified, that cytologically phenocopied spo11-1 mutants previously characterized in Arabidopsis, and rice. Importantly, a biallelic male and female sterile mutant was identified in primary transgenics, exhibiting the expected defects in 100% of examined male and female meiocytes. Conclusion:The transient, detached cowpea leaf assay, and supporting analytical methods developed, provide a rapid and reproducible means for testing gene expression constructs, and constructs for inducing mutagenesis in genes involved in both vegetative and reproductive developmental programs. The method and tested editing constructs and components have potential application for a range of crop legumes.

Dataset Information

Genome Editing in Cowpea Vigna unguiculata Using CRISPR-Cas9.

Publications

Genome Editing in Cowpea <i>Vigna unguiculata</i> Using CRISPR-Cas9.

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