Genome Editing in Cowpea Vigna unguiculata Using CRISPR-Cas9

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.

[1]  V. Orbović,et al.  CRISPR‐LbCas12a‐mediated modification of citrus , 2019, Plant biotechnology journal.

[2]  S. Gollasch,et al.  An Improved Transformation System for Cowpea (Vigna unguiculata L. Walp) via Sonication and a Kanamycin-Geneticin Selection Regime , 2019, Front. Plant Sci..

[3]  Mehmood Ali Noor,et al.  Genome editing in plants: Advancing crop transformation and overview of tools. , 2018, Plant physiology and biochemistry : PPB.

[4]  Baohong Zhang,et al.  CRISPR/Cas9: An RNA‐guided highly precise synthetic tool for plant genome editing , 2018, Journal of cellular physiology.

[5]  E. Rosa,et al.  Cowpea: a legume crop for a challenging environment. , 2017, Journal of the science of food and agriculture.

[6]  Kabin Xie,et al.  CRISPR-P 2.0: An Improved CRISPR-Cas9 Tool for Genome Editing in Plants. , 2017, Molecular plant.

[7]  Stefano Lonardi,et al.  Genome resources for climate-resilient cowpea, an essential crop for food security , 2016, bioRxiv.

[8]  T. Mockler,et al.  Precise insertion and guided editing of higher plant genomes using Cpf1 CRISPR nucleases , 2017, Scientific Reports.

[9]  Hui Zhu,et al.  Efficient Inactivation of Symbiotic Nitrogen Fixation Related Genes in Lotus japonicus Using CRISPR-Cas9 , 2016, Front. Plant Sci..

[10]  Jin-Soo Kim,et al.  Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells , 2016, Nature Biotechnology.

[11]  A. Regev,et al.  Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System , 2015, Cell.

[12]  Zhongsen Li,et al.  Cas9-Guide RNA Directed Genome Editing in Soybean[OPEN] , 2015, Plant Physiology.

[13]  Dongxue Li,et al.  Functional analysis of duplicated Symbiosis Receptor Kinase (SymRK) genes during nodulation and mycorrhizal infection in soybean (Glycine max). , 2015, Journal of plant physiology.

[14]  Kabin Xie,et al.  RNA-guided genome editing in plants using a CRISPR-Cas system. , 2013, Molecular plant.

[15]  Wen Feng Chen,et al.  High-Resolution Transcriptomic Analyses of Sinorhizobium sp. NGR234 Bacteroids in Determinate Nodules of Vigna unguiculata and Indeterminate Nodules of Leucaena leucocephala , 2013, PloS one.

[16]  M. Spalding,et al.  High-efficiency TALEN-based gene editing produces disease-resistant rice , 2012, Nature Biotechnology.

[17]  Yuriko Osakabe,et al.  Site-directed mutagenesis in Arabidopsis using custom-designed zinc finger nucleases , 2010, Proceedings of the National Academy of Sciences.

[18]  Timothy J. Close,et al.  A consensus genetic map of cowpea [Vigna unguiculata (L) Walp.] and synteny based on EST-derived SNPs , 2009, Proceedings of the National Academy of Sciences.

[19]  F. Aragão,et al.  Biolistic-mediated genetic transformation of cowpea (Vigna unguiculata) and stable Mendelian inheritance of transgenes , 2008, Plant Cell Reports.

[20]  Jiang-Ke Yang,et al.  Genetic diversity and phylogeny of indigenous rhizobia from cowpea [Vigna unguiculata (L.) Walp.] , 2007, Biology and Fertility of Soils.

[21]  Dongxue Li,et al.  Agrobacterium rhizogenes-mediated transformation of soybean to study root biology , 2007, Nature Protocols.

[22]  P. K. Jaiwal,et al.  Agrobacterium tumefaciens-mediated high frequency genetic transformation of an Indian cowpea (Vigna unguiculata L. Walp.) cultivar and transmission of transgenes into progeny , 2007 .

[23]  Dongxue Li,et al.  Agrobacterium rhizogenes transformation of the Phaseolus spp.: a tool for functional genomics. , 2006, Molecular plant-microbe interactions : MPMI.

[24]  Jeffrey C. Miller,et al.  Highly efficient endogenous human gene correction using designed zinc-finger nucleases , 2005, Nature.

[25]  A. Kereszt,et al.  A receptor kinase gene regulating symbiotic nodule development , 2002, Nature.

[26]  S. Tabata,et al.  A plant receptor-like kinase required for both bacterial and fungal symbiosis , 2002, Nature.

[27]  R. Papa,et al.  AFLP analysis of the phenetic organization and genetic diversity of Vigna unguiculata L. Walp. reveals extensive gene flow between wild and domesticated types , 2002, Theoretical and Applied Genetics.

[28]  W. Broughton,et al.  Rhizobium sp. strain NGR234 and R. fredii USDA257 share exceptionally broad, nested host ranges. , 1999, Molecular plant-microbe interactions : MPMI.

[29]  L. Segovia,et al.  Phylogenetic relationships and host range of Rhizobium spp. that nodulate Phaseolus vulgaris L , 1995, Applied and environmental microbiology.

[30]  M. Chilton,et al.  Agrobacterium rhizogenes inserts T-DNA into the genomes of the host plant root cells , 1982, Nature.

[31]  F. White,et al.  Hairy root: plasmid encodes virulence traits in Agrobacterium rhizogenes , 1980, Journal of bacteriology.

[32]  W. Broughton,et al.  Control of leghaemoglobin synthesis in snake beans. , 1971, The Biochemical journal.

[33]  H. Belshaw,et al.  The Food and Agriculture Organization of the United Nations , 1947, International Organization.

[34]  Shaun J. Curtin.  Editing the Medicago truncatula Genome: Targeted Mutagenesis Using the CRISPR-Cas9 Reagent. , 2018, Methods in molecular biology.

[35]  Longxiang Wang,et al.  Use of CRISPR/Cas9 for Symbiotic Nitrogen Fixation Research in Legumes. , 2017, Progress in molecular biology and translational science.

[36]  M. Bernards,et al.  Hairy Roots: An Ideal Platform for Transgenic Plant Production and Other Promising Applications , 2013 .

[37]  M. Savka,et al.  Induction of hairy roots on cultivated soybean genotypes and their use to propagate the soybean cyst nematode. , 1990 .

[38]  G. Zhou,et al.  Introduction of exogenous DNA into cotton embryos. , 1983, Methods in enzymology.