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WO2025052377A1 - Cowpea plants with improved traits and methods for generating the same - Google Patents

Cowpea plants with improved traits and methods for generating the same Download PDF

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Publication number
WO2025052377A1
WO2025052377A1 PCT/IL2024/050882 IL2024050882W WO2025052377A1 WO 2025052377 A1 WO2025052377 A1 WO 2025052377A1 IL 2024050882 W IL2024050882 W IL 2024050882W WO 2025052377 A1 WO2025052377 A1 WO 2025052377A1
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Prior art keywords
plant
cowpea
seq
modified
vigna unguiculata
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French (fr)
Inventor
Ido Margalit
Tal SHERMAN
Shira COREM
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Betterseeds Ltd
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Betterseeds Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • the present disclosure relates to Cowpea plants with desirable agronomic traits. More particularly, the current invention pertains to Cowpea plants with improved plant architecture, generated by manipulating SELF PRUNING (SP) genes through gene editing techniques.
  • SP SELF PRUNING
  • Cowpea (Vigna unguiculata) is a vital legume crop, and its mechanical harvesting is hindered by its indeterminate growth habit. In such open field crops, it is essential for enabling sustainable agriculture, to be able to harvest the plants mechanically, instead of using manual labor.
  • SP SELF PRUNING
  • said determinate growth habit characteristic is selected from the group consisting of determinate or semideterminate architecture, early termination of sympodial cycling, compact growth habit, reduced height, distinct short stature, determinate ap
  • gRNA guide RNA
  • gRNA guide RNA
  • said mutation is a silencing mutation, a knockdown mutation, a knockout mutation, a loss of function mutation, downregulating, de-regulating, deactivating, reducing expression, at least partially deleting, at least partially silencing, at least partially deactivating, removing, partially removing, duplicating, inverting, missense mutation, nonsense mutation, or any combination thereof.
  • a construct comprising (a) Cas DNA and gRNA comprising a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA comprising a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3.
  • a construct comprising (a) Cas DNA and gRNA sequence comprising SEQ ID NO: 11, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence comprising SEQ ID NO: 11.
  • said gRNA sequence comprises a 3' NGG Protospacer Adjacent Motif (PAM) .
  • gRNA guide RNA
  • transforming TO Cowpea (Vigna unguiculata) plant cells with a construct comprising (a) Cas nucleotide sequence operably linked to said at least one gRNA, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and said at least one gRNA; d. sei f-pollinating said transformed TO Cowpea (Vigna unguiculata) plant and collecting transformed T1 seeds; e. screening the genome of transformed T1 Cowpea (Vigna unguiculata) cells and selecting for CRISPR/Cas negative and VuSPl and/or VuSP2 genome edited positive T1 Cowpea (Vigna unguiculata) cells; f.
  • Agrobacterium mediated transformation virus-based plasmids for delivery of the genome editing molecules or mechanical insertion such as polyethylene glycol (PEG) mediated DNA transformation, electroporation or gene gun biolistics.
  • PEG polyethylene glycol
  • step of screening the genome of said transformed plant cells for induced targeted loss of function mutation further comprises steps of obtaining a nucleic acid sample of said transformed plant and performing a nucleic acid amplification and optionally restriction enzyme digestion to detect a mutation in said VuSPl and/or VuSP2 gene .
  • VuSPl allele comprises a polynucleotide sequence selected from SEQ ID NOs:4-9 and any combination thereof
  • mutated VuSP2 allele comprises a polynucleotide sequence selected from SEQ ID NOs: 12-15 and any combination thereof.
  • Cowpea identifying at least one Cowpea (Vigna unguiculata) SELF PRUNING (SP) VuSPl and/or VuSP2 gene allele; b. synthetizing at least one guide RNA (gRNA) comprising a polynucleotide sequence complementary to the polynucleotide sequence of said at least one identified VuSPl and/or VuSP2 gene allele ; c. transforming Cowpea (Vigna unguiculata) plant cell with a construct comprising (a) Cas nucleotide sequence operably linked to said at least one gRNA, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and said at least one gRNA; d.
  • gRNA guide RNA
  • RNP ribonucleoprotein
  • gRNA guide RNA
  • VuSPl mutated allele comprises a polynucleotide sequence selected from SEQ ID NOs:4-9 and any combination thereof
  • mutated VuSP2 allele comprises a polynucleotide sequence selected from SEQ ID N0s:12-
  • It is another object of the present invention to disclose a method for identifying and/or selecting for a Cowpea (Vigna unguiculata) plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic comprises steps of: a. screening the genome of said Cowpea (Vigna unguiculata) plant for a mutated VuSPl and/or VuSP2 allele, said wherein the VuSPl mutated allele comprises a polynucleotide sequence selected from SEQ ID NOs:4-9 and any combination thereof, and the mutated VuSP2 allele comprises a polynucleotide sequence selected from SEQ ID NOs: 12-15 and any combination thereof; b. optionally, regenerating a modified Cowpea (Vigna unguiculata) plant carrying said genetic modification; and c. optionally, screening said regenerated plants for a plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic.
  • said determinate growth habit characteristic is selected from the group consisting of determinate or semi-determinate architecture, early termination of sympodial cycling, compact growth habit, reduced height, distinct short stature, determinate apical meristem growth, reduced or limited
  • FIG. 1 is presenting a wild-type (WT) genomic sequence of Cowpea (Vigna unguiculata) SELF PRUNING (SP) 1 gene (VuSPl) as set forth in SEQ ID NO: 1;
  • FIG. 2 is presenting a wild-type (WT) genomic sequence of Cowpea (Vigna unguiculata) SELF PRUNING (SP) 2 gene (VuSP2) as set forth in SEQ ID NO: 10;
  • FIGs . 3A-B are schematic representations of the location of the gRNAs on the VuSPl gene (FIG. 3A) and VuSP2 gene (FIG. 3B) ;
  • FIGs. 4A-B are photographic representations of PCR amplicons in T1 transformed Cowpea plants;
  • FIG. 4A presents amplicons of VuSPl;
  • FIG. 4B is amplicons obtained using Cas9 primers;
  • FIGs. 5A-F are presenting nucleic acid sequences of mutated alleles of VuSPl gene having the herein disclosed editing events as set forth in SEQ ID NOs:4-9, respectively;
  • FIG. 6 is presenting an alignment of nucleic acid sequences of the WT VuSPl as set forth in SEQ ID NO:1 and the mutated VuSPl alleles containing genome editing events as set forth in SEQ ID NOs:4-9;
  • FIGs. 7A-D are presenting nucleic acid sequences of mutated alleles of VuSP2 gene containing the herein disclosed editing events as set forth in SEQ ID NOs: 12-15, respectively;
  • FIG. 8 is presenting an alignment of nucleic acid sequences of WT VuSP2 as set forth in SEQ ID NO: 10 and mutated VuSPl alleles containing genome editing events as set forth in SEQ ID Nos : 12- 15;
  • FIG. 9 is a picture representing a phenotypic comparison of edited mutated (right) and wild-type (left) Cowpea plants;
  • FIGs. 10A-C are pictures representing mutated Cowpea lines with a determinate growth habit characteristics as an embodiment of the present invention
  • FIGs. 11A-C are pictures representing mutated Cowpea lines with a determinate growth habit characteristics as an embodiment of the present invention
  • FIGs. 12A-C are pictures representing mutated Cowpea lines with a determinate growth habit characteristics as an embodiment of the present invention.
  • FIGs. 13A-C are pictures representing mutated Cowpea lines with a determinate growth habit characteristics as an embodiment of the present invention.
  • FIG. 14 is a graph representing plant heigh in cm of edited mutated Cowpea lines that have undergone editing events, compared to the wild-type Cowpea.
  • Cowpea (Vigna unguiculata) is a vital legume crop, and its mechanical harvesting is hindered by its indeterminate growth habit .
  • the current invention is aimed to solve this problem by utilizing genome editing, specifically the CRISPR/Cas9 system, to edit the Cowpea homolog of SELF PRUNING (SP) and to develop determinate Cowpea lines.
  • genome editing specifically the CRISPR/Cas9 system
  • SP SELF PRUNING
  • the present invention provides a modified Cowpea plant exhibiting improved plant architecture trait conferring determinate growth phenotype .
  • the present invention provides a modified Cowpea (Vigna unguiculata) plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic.
  • the aforementioned modified plant comprises mutated SELF PRUNING (SP) VuSPl and/or VuSP2 gene comprising genome edited loss of function mutation.
  • the improved plant architecture trait of the modified Cowpea (Vigna unguiculata) plant is relative to a corresponding Cowpea (Vigna unguiculata) plant lacking the genome edited loss of function mutation.
  • the mutated VuSPl gene comprises a genomic sequence selected from SEQ ID NO: 4-9, or a functional variant thereof, or any combination thereof .
  • the mutated VuSP2 gene comprises a genomic sequence selected from SEQ ID NO: 12-15, or a functional variant thereof, or any combination thereof .
  • the modified Cowpea plant comprises at least one mutated Cowpea (Vigna unguiculata) SELF PRUNING (SP) gene selected from VuSPl and/or VuSP2 genes.
  • mutated Cowpea Vigna unguiculata
  • SP SELF PRUNING
  • the Cowpea (Vigna unguiculata) SP gene undergone targeted gene editing mutation is selected from VuSPl and/or VuSP2 comprising a nucleic acid sequence with at least 75% sequence identity to a sequence selected from SEQ ID NO:1 and/or SEQ ID NO: 10, respectively, or a functional variant thereof and any combination thereof .
  • the present invention further provides methods for producing the aforementioned modified Cowpea plant using genome editing optionally with other genome modification techniques.
  • the solution proposed by the current invention is using genome editing such as the CRISPR/Cas system in order to create cultivated Cowpea plants with determinate growth habit characteristics. Breeding using genome editing allows a precise and significantly shorter breeding process in order to achieve these goals with a much higher success rate. Thus, genome editing has the potential to generate improved lines faster and at a lower cost.
  • the new and improved Cowpea plants of the present invention are adapted for intensive and advanced agriculture.
  • the present invention provides Cowpea plants with improved plant architecture traits conferring determinate growth phenotype.
  • the current invention discloses the generation of non-transgenic Cowpea plants with determinate growth habits, using genome editing technology, e.g. , the CRISPR/Cas9 highly precise tool.
  • the generated mutations can be introduced into elite or locally adapted Cowpea lines rapidly, with relatively minimal effort and investment .
  • the edited plants exhibited a distinct short stature and bushy growth habit, which is in contrast to the unedited control indeterminate Cowpea plants. Furthermore, flowering and pod formation were observed to be synchronized and uniform across the edited plants.
  • similar denotes a correspondence or resemblance range of about ⁇ 20%, particularly ⁇ 15%, more particularly about ⁇ 10% and even more particularly about ⁇ 5%.
  • a “plant” as used herein refers to any plant at any stage of development, particularly a seed plant.
  • the term “plant” includes the whole plant or any parts or derivatives thereof, such as plant cells, seeds, pods, plant protoplasts, plant cell tissue culture from which Cowpea plants can be regenerated, plant callus or calli, meristematic cells, microspores, embryos, immature embryos, pollen, ovules, anthers, fruit, flowers, leaves, cotyledons, pistil, seeds, seed coat, pods, roots, root tips and the like.
  • plant cell refers to a structural and physiological unit of a plant, comprising a protoplast and a cell wall.
  • the plant cell may be in the form of an isolated single cell or a cultured cell.
  • plant cell culture means cultures of plant units such as, for example, protoplasts, regenerable cells, cell culture, cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development, leaves, roots, root tips, anthers, meristematic cells, microspores, flowers, cotyledons, pistil, fruit, seeds, seed coat, pods or any combination thereof.
  • plant material or “plant part” used herein refers to leaves, stems, roots, root tips, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, seed coat, pods, cuttings, cell or tissue cultures, or any other part or product of a plant or a combination thereof.
  • a "plant organ” as used herein means a distinct and visibly structured and differentiated part of a plant such as, but not limited to a root, stem, leaf, flower, flower bud, pod, seed or embryo .
  • plant tissue as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, plant organs, plant seeds, pods, tissue culture, protoplasts, meristematic cells, calli and any group of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.
  • progeny refers in a nonlimiting manner to offspring or descendant plants.
  • progeny refers to plants developed or grown or produced from the disclosed or deposited seeds as detailed inter alia.
  • the grown plants preferably have the desired traits of the disclosed or deposited seeds, i.e. loss of function mutation in at least one of Cowpea VuSPl and/or VuSP2 genes .
  • the term "Legume” refers hereinafter to a plant in the family Fabaceae (or Leguminosae) , or the fruit or seed of such a plant.
  • the legume family consists of plants that produce a pod with seeds inside.
  • the Legume is Cowpea (Vigna unguiculata) .
  • Cropea also herein refers to "Vigna unguiculata” or “Vu” is an annual herbaceous legume from the genus Vigna (peas and beans) . It is a food and animal feed crop grown in the semi-arid tropics covering Africa, Asia, Europe, the United States, and Central and South America.
  • the term “SELF- PRUNING” or “SP” in the context of the present invention refers to a gene which encodes a flowering repressor that modulates sympodial growth. It is herein shown that mutations in the SP Cowpea orthologue cause an acceleration of sympodial cycling and shoot termination.
  • SP SELF PRUNING
  • the Legumes SP genes include Cowpea VuSPl and VuSP2 genes.
  • genome editing- targeted mutation in at least one of the aforementioned Cowpea VuSPl and/or VuSP2 genes which reduces the functional expression of the gene, affect the plant sympodial growth habit which plays a key role in determining plant architecture and particularly confers determinate growth habit characteristics, highly desirable in crops and shown for the first time in Cowpea.
  • determinate growth habit refers to morphogenesis by formation of up to three to four inflorescences per shoot (IPS) , and is one of the key tools for breeding high-yielding varieties of crops.
  • IPS inflorescences per shoot
  • sympodial growth refers to a type of bifurcating branching pattern where one branch develops more strongly than the other, resulting in the stronger branches forming the primary shoot and the weaker branches appearing laterally.
  • a sympodium also referred to as a sympode or pseudaxis, is the primary shoot, comprising the stronger branches, formed during sympodial growth.
  • sympodial growth occurs when the apical meristem is terminated (as a result of the loss of function targeted mutation is VuSPl and/or VuSP2 genes) and growth is continued by one or more lateral meristems, which repeat the process.
  • the apical meristem may be consumed to make an inflorescence or other determinate structure, or it may be aborted.
  • the shoot section between two successive inflorescences is called the 'sympodium’
  • the number of leaf nodes per sympodium is referred to as the ’sympodial index’ (spi) .
  • the first termination event activates the 'sympodial cycle' .
  • the apparent main shoot consists of a reiterated array of 'sympodial units' .
  • a mutant sp gene (VuSPl and/or VuSP2 genes) accelerates the termination of sympodial units and confers determinate growth habit.
  • determinate or “determinate growth” as used herein refers to plant growth in which the main stem ends in an inflorescence or other reproductive structure (e.g. a bud) and stops continuing to elongate indefinitely with only branches from the main stem having further and similarly restricted growth. It also refers to growth characterized by sequential flowering from the central or uppermost bud to the lateral or basal buds . It further means naturally self-limited growth, resulting in a plant of a definite maximum size.
  • semi-determinate refers to plants, genotypes, lines or varieties that continue growing through the season, but generally remain smaller than indeterminate plants, with a bushier habit. According to further aspects, semideterminate genotypes are equivalent to determinate ones with extended vegetative growth, which in turn impacts shoot height, number of leaves and either stem diameter or internode length. According to one embodiment of the present invention, the term semi-determinant is included within the scope of the meaning of the term determinant or determinant growth habit. In this aspect, the modified Cowpea plants of the present invention may present determinate or semi-determinate growth habit.
  • the generated Cowpea mutated lines comprising the targeted loss of function mutations in VuSPl and/or VuSP2 genes, are determinate Cowpea plants exhibiting at least one characteristic such as compact growth habit, distinct short stature, exhausted or terminated apical meristem, bushy growth habit, lacking central growth axis and tendrils, uniform appearance of flowers, synchronized and uniform flowering, and synchronized and uniform pod formation. This is in contrast to a wild-type plant absent of the herein disclosed targeted editing events in VuSPl and/or VuSP2 genes which is characterized by indeterminate, long, viny growth habit with many tendrils.
  • the term "apical meristem” refers, without limitation, to specialized zones of growth found at the tips of plants. These are responsible for the extension of shoots and roots and are also responsible for the primary plant body. A balance between the generation of new meristematic cells, and their transition toward differentiation, permits the maintenance of the meristem and regulates its activity. In plants with determinate growth habits, the meristem is genetically programmed to stop producing new cells at a specific developmental stage. Therefore, at some point of the growth of a plant with determinate growth, the apical meristem becomes exhausted, and no new cells are produced.
  • indeterminate or indeterminate growth refers to plant growth in which the main stem continues to elongate indefinitely without being limited by a terminal inflorescence or other reproductive structure. It also refers to growth characterized by sequential flowering from the lateral or basal buds to the central or uppermost buds. Indeterminate growth habit is herein shown to be correlated with wild-type or control Cowpea plants, lacking the herein disclosed mutations in VuSPl and/or VuSP2 genes .
  • harvest index can be herein defined as the total yield per plant weight.
  • yield related traits comprise one or more of improved plant architecture, i.e. , determinate growth habit, compact growth habit, distinct short stature, exhausted apical meristem, bushy growth habit, synchronized and uniform flowering, and synchronized and uniform pod formation.
  • improved plant architecture encompasses lacking central growing axis and tendrils, with uniform appearance of flowers.
  • yield in general means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and/or weight, or the actual yield is the yield per square meter for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted square meters.
  • yield of a plant and “plant yield” are used interchangeably herein and are meant to refer to vegetative biomass such as root and/or shoot biomass, to reproductive organs, and/or to propagules such as seeds or pods of that plant.
  • Increased seed or pods yield may be defined as one or more of the following: (a) an increase in seed/pod biomass (total seed/pod weight) which may be on an individual seed/pod basis and/or per plant and/or per square meter; (b) increased number of flowers per plant; (c) increased number of seeds/pods; and (d) increased harvest index, which is expressed as a ratio of the yield of harvestable parts, such as seeds/pods, divided by the biomass of aboveground plant parts .
  • An increase in seed/pod yield may also be manifested as an increase in seed/pod size and/or seed/pod volume.
  • biomass as used herein is intended to refer to the total weight of a plant.
  • biomass a distinction may be made between the biomass of one or more parts of a plant, which may include: aboveground (harvestable) parts such as but not limited to shoot biomass, seed/pod biomass, leaf biomass, etc. and/or (harvestable) parts below ground, such as but not limited to root biomass, etc. , and/or vegetative biomass such as root biomass, shoot biomass, etc. , and/or reproductive organs, and/or propagules such as seed/pod.
  • aboveground (harvestable) parts such as but not limited to shoot biomass, seed/pod biomass, leaf biomass, etc.
  • vegetative biomass such as root biomass, shoot biomass, etc.
  • reproductive organs such as seed/pod.
  • Control plant (s) within the scope of the present invention include corresponding wild type plants or corresponding naturally occurring plants or corresponding plants lacking the edited or mutated gene of interest or the specific generated mutation.
  • the choice of suitable control plants is a routine part of an experimental setup and may include corresponding wild type plants or corresponding plants without the gene of interest.
  • the control plant is typically of the same plant species or the same genetic background or even of the same variety as the plant to be assessed.
  • the control plant of the plant to be assessed may also be plant individuals missing the transgene or modif ied/edited gene.
  • a "control plant” or a "wild type” plant as used herein refers not only to whole plants, but also to plant parts, including seeds, pods and seed or pod parts.
  • orthologue refers hereinafter to one of two or more homologous gene sequences found in different species.
  • the term "functional variant” or "functional variant of a nucleic acid or amino acid sequence” as used herein refers to a sequence or part of a sequence which retains the biological function of the full non-variant allele (e.g. Cowpea VuSPl and/or VuSP2 genes) and hence has the activity of VuSPl and/or VuSP2 expressed gene or protein.
  • a functional variant also comprises a variant of the gene of interest encoding a polypeptide which has sequence alterations that do not affect function of the resulting protein, for example, in non-conserved residues.
  • the term "functional variant” refers to a sequence which is substantially similar to another sequence, namely retains all its characteristics although having at least about 50%-99% identity with the other sequence, for example at least 75%, for example at least 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to that particular non-variant other sequence.
  • the term “variety” or “cultivar” or “line” used herein means a group of similar plants that by structural features and performance can be identified from other varieties within the same species.
  • allele used herein means any of one or more alternative or variant or mutant forms of a gene or a genetic unit at a particular locus, all of which alleles relate to one trait or characteristic at a specific locus.
  • alleles of a given gene are located at a specific location, or locus (loci plural) on a chromosome.
  • Alternative or variant forms of alleles may be the result of at least one of single nucleotide polymorphisms, indels, insertions, inversions, translocations or deletions, or the consequence of gene regulation caused by, for example, targeted genome editing, chemical or structural modification, transcription regulation or post- translational modi f ication/regulation .
  • Hemizygous is a condition or configuration where one allele is missing .
  • a wild type allele is a naturally occurring allele.
  • the term allele refers to the two identified VuSPl and VuSP2 Cowpea genes, having the genomic nucleotide (wild type) sequence as set forth in SEQ ID NO:1 and SEQ ID NO: 10, respectively.
  • the term "genetic modification” refers hereinafter to genetic manipulation or modulation, which is the direct manipulation of an organism's genes using biotechnology. It also refers to a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species, targeted mutagenesis and genome editing technologies to produce improved organisms.
  • modified Cowpea plants with improved plant architecture traits conferring determinate growth phenotype are generated using genome editing mechanism. This technique enables to achieve in planta modification of specific genes that relate to and/or control the determinate growth habits in Cowpea.
  • the modification of the genes is aimed to result in modulated expression (preferably silencing) of the targeted genes, as compared to control Cowpea plants lacking the generated modification .
  • genome editing or “genome/genetic modification” or “genome engineering” or “gene editing” generally refers hereinafter to a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike previous genetic engineering techniques that randomly insert genetic material into a host genome, genome editing targets the insertions or deletions to site specific locations (e.g. specific genomic locus or loci) within a coding or noncoding (regulatory) regions of a gene.
  • site specific locations e.g. specific genomic locus or loci
  • nucleases engineered nucleases, or "molecular scissors". These nucleases create site-specific double-strand breaks (DSBs) at desired locations in the genome. The induced double-strand breaks are repaired through nonhomologous endjoining (NHEJ) or homologous recombination (HR) , resulting in targeted mutations ( ’edits' ) .
  • NHEJ nonhomologous endjoining
  • HR homologous recombination
  • Families of engineered nucleases used by the current invention include, but are not limited to: meganucleases, zinc finger nucleases (ZFNs) , transcription activator-like effector-based nucleases (TALEN) , and the clustered regularly interspaced short palindromic repeats (CRISPR/Cas) system.
  • ZFNs zinc finger nucleases
  • TALEN transcription activator-like effector-based nucleases
  • CRISPR/Cas clustered regularly interspaced short palindromic repeats
  • the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are used for the first time for generating genome modification targeted to VuSPl and/or VuSP2 genes in Cowpea plants. It is herein acknowledged that the functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in selected bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material. These repeats were initially discovered in the 1980s in E. coll.
  • CRISPR mechanism in which invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus comprising a series of short repeats (around 20 bps) .
  • the loci are transcribed, and transcripts are then processed to generate small RNAs (crRNA, namely CRISPR RNA) , which are used to guide effector endonucleases that target invading DNA based on sequence complementarity.
  • Cas protein such as Cas9 (also known as Csnl) is required for gene silencing.
  • Cas9 participates in the processing of crRNAs and is responsible for the destruction of the target DNA.
  • Cas9's function in both of these steps relies on the presence of two nuclease domains, a RuvC-like nuclease domain located at the amino terminus, and a HNH-like nuclease domain that resides in the mid-region of the protein.
  • Cas9 is complexed with both a crRNA and a separate trans-activating crRNA (tracrRNA or trRNA) , that is partially complementary to the crRNA.
  • the tracrRNA is required for crRNA maturation from a primary transcript encoding multiple pre-crRNAs. This occurs in the presence of RNase III and Cas9.
  • the HNH and RuvC-like nuclease domains cut both DNA strands, generating double- stranded breaks (DSBs) at sites defined by a 20-nucleotide target sequence within an associated crRNA transcript.
  • the HNH domain cleaves the complementary strand
  • the RuvC domain cleaves the noncomplementary strand.
  • sgRNA single synthetic single guide RNA
  • Cas9 nuclease variants include wild-type Cas9, Cas9D10A and nuclease-deficient Cas9 (dCas9) .
  • RNA-guided genome editing in plants using a CRISPR-Cas system "Molecular plant 6.6 (2013) : 1975-1983, incorporated herein by reference.
  • the Cas9 endonuclease forms a complex with a chimeric RNA (called guide RNA or gRNA) , replacing the crRNA- trans crRNA heteroduplex, and the gRNA could be programmed to target specific sites.
  • guide RNA or gRNA chimeric RNA
  • the gRNA-Cas9 should comprise at least 15-base-pairing (gRNA seed region) without mismatch between the 5 '-end of engineered gRNA and targeted genomic site, and an NGG motif (called protospacer-adjacent motif or PAM) that follows the base-pairing region in the complementary strand of the targeted DNA.
  • NGG motif protospacer-adjacent motif or PAM
  • meganucleases refers hereinafter to endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs) ; as a result this site generally occurs only once in any given genome. Meganucleases are therefore considered to be the most specific naturally occurring restriction enzymes.
  • PAM protospacsr adjacent motif
  • next-generation sequencing or “NGS” as used herein refers hereinafter to massively, parallel, high- throughput or deep sequencing technology platforms that perform sequencing of millions of small fragments of DNA in parallel. Bioinformatics analyses are used to piece together these fragments by mapping the individual reads to the reference genome.
  • gene knockdown refers hereinafter to an experimental technique by which the expression of one or more of an organism's genes is reduced and/or it's function is significantly reduced or eliminated.
  • the reduction can occur through genetic modification, i.e. targeted genome editing or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.
  • the reduced expression can be at the level of RNA and/or at the level of protein.
  • gene knockdown also refers to a loss of function mutation, gene knockout or silencing mutation in which an organism's gene is made inoperative or nonfunctional.
  • gene silencing refers hereinafter to the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation. In certain aspects of the invention, gene silencing is considered to have a similar meaning as gene knockdown. When genes are silenced, their expression is reduced. In contrast, when genes are knocked out, they are completely not expressed. Gene silencing may be considered a gene knockdown mechanism since the methods used to silence genes, such as RNAi, CRISPR, or siRNA, generally reduce the expression of a gene by at least 70% but do not completely eliminate it.
  • loss of function mutation refers to a type of mutation in which the altered gene product lacks the function of the wild-type gene.
  • a synonym of the term included within the scope of the present invention is null mutation.
  • in planta means in the context of the present invention within the plant or plant cells. More specifically, it means introducing CRISPR/Cas complex into plant material comprising a tissue culture of several cells, a whole plant, or into a single plant cell, without introducing a foreign gene or a mutated gene into its genome. It is also used to describe conditions present in a non-laboratory environment (e.g. in vivo) .
  • locus means a specific place (s) or region (s) or a site (s) on a chromosome where for example a gene or genetic marker element or factor is found. In specific embodiments, such a genetic element is contributing to a trait .
  • homozygous refers to a genetic condition or configuration existing when two identical or like alleles reside at a specific locus but are positioned individually on corresponding pairs of homologous chromosomes in the cell of a diploid organism.
  • Cowpea plants of the present invention comprise homozygous configuration of at least one of the mutated Cowpea VuSPl and/or VuSP2 genes.
  • heterozygous means a genetic condition or configuration existing when two different or unlike alleles reside at a specific locus but are positioned individually on corresponding pairs of homologous chromosomes in the cell of a diploid organism.
  • hemizygous refers to a genetic condition or configuration existing in a diploid cell in which there is only one copy of a gene instead of the typical two copies (i.e. , the gene has no counterpart on a homologous chromosome) .
  • a heterologous transgene can be present in a hemizygous state.
  • Cowpea plants of the present invention comprise hemizygous configuration of at least one of the mutated Cowpea SP genes.
  • the phrase "genetic marker” or “molecular marker” or “biomarker” refers to a feature in an individual's genome e.g. , a nucleotide or a polynucleotide sequence that is associated with one or more loci or trait of interest.
  • a genetic marker is polymorphic in a population of interest, or the locus occupied by the polymorphism, depending on context.
  • Genetic markers or molecular markers include, for example, single nucleotide polymorphisms (SNPs) , indels (i.e.
  • DNA sequence per se can, for example, be used to locate genetic loci containing alleles on a chromosome that contribute to variability of phenotypic traits.
  • genetic marker or “molecular marker” or “biomarker” can also refer to a polynucleotide sequence complementary or corresponding to a genomic sequence, such as a sequence of a nucleic acid used as a probe or primer.
  • genomic sequence such as a sequence of a nucleic acid used as a probe or primer.
  • genetic marker refers to the totality of the genotypes of a population or other group of individuals (e.g. , a species) .
  • the term “germplasm” can also refer to plant material; e.g. , a group of plants that act as a repository for various alleles.
  • Such germplasm genotypes or populations include plant materials of proven genetic superiority; e.g. , for a given environment or geographical area, and plant materials of unknown or unproven genetic value; that are not part of an established breeding population and that do not have a known relationship to a member of the established breeding population.
  • hybrid refers to an individual produced from genetically different parents (e.g. , a genetically heterozygous, hemizygous or mostly heterozygous individual) .
  • the inventors have generated mutant Cowpea lines with mutations inactivating the Cowpea VuSPl and/or VuSP2 gene homologous alleles which confer determinate growth habit characteristics. In this way, at least one of the VuSPl and/or VuSP2 proteins is not functional. Thus, the invention relates to these mutant Cowpea lines and related methods.
  • VuSPl and/or VuSP2 allele enables the mechanical harvest of the plant, due to the determinant growth habit of the plant.
  • loss of VuSPl and/or VuSP2 function results in compact Cowpea plants with determinate growth phenotype when compared with corresponding WT Cowpea plants. More specifically, the generated plants with determinate growth habit characteristics present reduced height, reduced number of sympodial units, compact growth habit, distinct short stature, exhausted apical meristem, bushy growth habit, synchronized and uniform flowering, and synchronized and uniform pod formation.
  • modifying Cowpea shoot architecture by selection for mutations in VuSPl and/or VuSP2 genes allowed major improvements in plant architecture and yield.
  • a mutation in the VuSPl and/or VuSP2 genes provided compact 'determinate' growth that translated to a burst of flowers, thereby enabling largescale field production .
  • CRISPR/Cas9 can be used to create heritable mutations in Cowpea homologs of the florigen pathway family members that result in desirable phenotypic effects.
  • Cowpea VuSPl and VuSP2 genes having genomic nucleotide sequence as set forth in SEQ ID NO:1 and SEQ ID NO: 10, respectively, are silenced by genome editing. These two mutated alleles have been identified. Notably, the plants with these two genome-edited mutated sp alleles presented determinate growth habits and, consequently, were more compact than the wild type plants lacking these mutated alleles and showing indeterminate growth.
  • the loss of function mutation may be a deletion or insertion ("indels") with reference to the wild-type CowpeaVuSPl and/or VuSP2 gene allele sequences (genomic nucleotide sequence as set forth in SEQ ID NO:1 and SEQ ID NO: 10, respectively) .
  • the deletion may comprise 1-20 or more nucleotides (e.g. up to 160 bp) , for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18 or 20 nucleotides or more in one or more strand.
  • the insertion may comprise 1-20 or more nucleotides, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18 or 20 or more nucleotides in one or more strand.
  • the plant of the invention includes plants wherein the plant is heterozygous for each of the mutations.
  • the plant is homozygous for each of the mutations.
  • Progeny that is also homozygous can be generated from these plants according to methods known in the art.
  • the plant is hemizygous for each of the mutations.
  • variants of Cowpea VuSPl and/or VuSP2 genes nucleotide or amino acid encoded sequence according to the various aspects of the invention will have at least about 50%- 99%, for example at least 75%, for example at least 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to that particular non-variant Cowpea VuSPl and/or VuSP2 gene nucleotide sequence.
  • the various aspects of the invention encompass not only Cowpea VuSPl and/or VuSP2 gene nucleic acid sequence or amino acid sequence, but also fragments thereof.
  • fragment it is intended to mean a portion of the nucleotide sequence or a portion of the amino acid sequence and hence of the protein encoded thereby. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein, in this case improved plant architecture trait conferring determinate growth phenotype .
  • Cowpea SP1 and/or SP2 genes have been targeted using the CRISPR/Cas system.
  • DNA introduction into the plant cells can be done by Agrobacterium infiltration, virus-based plasmids for delivery of the genome editing molecules and mechanical insertion of DNA (PEG mediated DNA transformation, biolistics, etc. ) .
  • the Cas9 protein is directly inserted together with a gRNA (ribonucleoprotein- RNP's) in order to bypass the need for in vivo transcription and translation of the Cas9+gRNA plasmid in planta to achieve gene editing.
  • gRNA ribonucleoprotein- RNP's
  • CRISPR/Cas system for the generation of Cowpea plants with determinate growth habit phenotype, allows the modification of predetermined specific DNA sequences without introducing foreign DNA into the genome by GMO techniques.
  • this is achieved by combining the Cas nuclease (e.g. Cas9, Cpfl and the like) with a predefined guide RNA molecule (gRNA) .
  • the gRNA is complementary to a specific DNA sequence targeted for editing in the plant genome and which guides the Cas nuclease to a specific nucleotide sequence.
  • the predefined gene specific gRNA' s are cloned into the same plasmid as the Cas gene and this plasmid is inserted into plant cells. Insertion of the aforementioned plasmid DNA can be done, but not limited to, using different delivery systems, biological and/or mechanical, e.g. Agrobacterium infiltration, virus based plasmids for delivery of the genome editing molecules and mechanical insertion of DNA (PEG mediated DNA transformation, biolistics, etc. ) .
  • the Cas9 nuclease upon reaching the specific predetermined DNA sequence, cleaves both DNA strands to create double stranded breaks leaving blunt ends. This cleavage site is then repaired by the cellular non homologous end joining DNA repair mechanism resulting in insertions or deletions which eventually create a mutation at the cleavage site.
  • a deletion form of the mutation consists of at least 1 base pair deletion. As a result of this base pair deletion the gene coding sequence is disrupted, and the translation of the encoded protein is compromised either by a premature stop codon or disruption of a functional or structural property of the protein.
  • DNA is cut by the Cas9 protein and re-assembled by the cell's DNA repair mechanism.
  • improved architecture traits conferring determinate growth phenotype in Cowpea plants is herein produced by generating gRNA with homology to a specific site of VuSPl and/or VuSP2 genes in the Cowpea genome (in the coding and/or regulatory non-coding regions within VuSPl and/or VuSP2 genomic sequence) , sub cloning this gRNA into a plasmid containing the Cas9 gene, and insertion of the plasmid into the Cowpea plant cells.
  • site specific mutations in the VuSPl and/or VuSP2 genes are generated thus effectively creating non-active VuSPl and/or VuSP2 molecules, resulting in determinate growth habit of the genome edited plant.
  • the present invention to provides a modified Cowpea (Vigna unguiculata) plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic, wherein the plant comprises mutated SELF PRUNING (SP) VuSPl and/or VuSP2 gene comprising genome edited loss of function mutation.
  • SP SELF PRUNING
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined above, wherein the improved plant architecture trait is relative to a corresponding Cowpea (Vigna unguiculata) plant lacking the genome edited loss of function mutation.
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutated VuSPl gene comprises a genomic sequence selected from SEQ ID NO: 4-9, or a functional variant thereof, or any combination thereof .
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutated VuSP2 gene comprises a genomic sequence selected from SEQ ID NO: 12-15, or a functional variant thereof, or any combination thereof .
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the functional variant comprises at least 90% sequence identity to the corresponding mutated VuSPl or mutated VuSP2 sequence.
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the determinate growth habit characteristic is selected from the group consisting of determinate or semideterminate architecture, early termination of sympodial cycling, compact growth habit, reduced height, distinct short stature, determinate apical meristem growth, reduced or limited apical meristem growth, interrupted or exhausted apical meristem growth, bushy growth habit, synchronized flowering, uniform flowering, synchronized pod formation, uniform pod formation, reduced number of sympodial units, suppressed sympodial shoot termination, lack of central growing axis and tendrils, uniform appearance of flowers, adaptation to mechanical harvest, higher harvest index and any combination thereof.
  • the determinate growth habit characteristic is selected from the group consisting of determinate or semideterminate architecture, early termination of sympodial cycling, compact growth habit, reduced height, distinct short stature, determinate apical meristem
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the height of the modified plant is in the range of 60cm to 100cm, such as in the range of 65cm to 90cm.
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the modified plant height is about 30%-50% lower than a control Cowpea plant lacking the genome edited loss of function mutation in the VuSPl and/or VuSP2 genes.
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the modified plant has pod number similar or at least 10% higher than a control Cowpea plant lacking the genome edited loss of function mutation in the VuSPl and/or VuSP2 genes.
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutation in the VuSPl gene is generated using a guide RNA (gRNA) comprising a polynucleotide sequence selected from SEQ ID NO: 2 and SEQ ID NO: 3 or a combination thereof, and the mutation in the VuSP2 gene is generated using a guide RNA (gRNA) sequence comprising a polynucleotide sequence comprising SEQ ID NO: 11.
  • gRNA guide RNA
  • gRNA guide RNA
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutated VuSPl and/or VuSP2 gene is a CRISPR/Cas- induced heritable mutated allele.
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the Cas gene is selected from the group consisting of Cas9, Casl2, Casl3, Casl4, CasX, CasY, Csnl, Cpfl and any combination thereof.
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutation is an insertion, deletion, indel, inversion, substitution, duplication or any combination thereof .
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutation is a silencing mutation, a knockdown mutation, a knockout mutation, a loss of function mutation, downregulating, de-regulating, deactivating, reducing expression, at least partially deleting, at least partially silencing, at least partially deactivating, removing, partially removing, duplicating, inverting, missense mutation, nonsense mutation, or any combination thereof.
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutation is in the coding sequence or in a regulatory sequence such as a promoter, terminator sequence, of the VuSPl and/or VuSP2 gene.
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the plant is homozygous, heterozygous or hemizygous for the genome edited loss of function mutation in the VuSPl and/or VuSP2 gene.
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutation in the VuSPl gene is generated in planta via introduction of a construct comprising (a) Cas DNA and gRNA comprising a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA comprising a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3.
  • a construct comprising (a) Cas DNA and gRNA comprising a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA comprising a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3.
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutation in the VuSP2 gene is generated in planta via introduction of a construct comprising (a) Cas DNA and gRNA sequence comprising SEQ ID NO: 11, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence comprising SEQ ID NO: 11.
  • a construct comprising (a) Cas DNA and gRNA sequence comprising SEQ ID NO: 11, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence comprising SEQ ID NO: 11.
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the gRNA sequence comprises a 3' NGG Protospacer Adjacent Motif (PAM) .
  • the gRNA sequence comprises a 3' NGG Protospacer Adjacent Motif (PAM) .
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutation confers determinate growth habit phenotype .
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the modified plant flowers earlier than a corresponding control Cowpea plant lacking the mutated VuSPl and/or VuSP2 gene.
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the improved plant architecture trait is selected from the group consisting of reduced flowering time, earliness, synchronous flowering, reduced day-length sensitivity, determinant growth habit, early termination of sympodial cycling, earlier axillary shoot flowering, compact growth habit, reduced height, reduced number of sympodial units, adaptation to mechanical harvest, higher harvest index and any combination thereof .
  • the improved plant architecture trait is selected from the group consisting of reduced flowering time, earliness, synchronous flowering, reduced day-length sensitivity, determinant growth habit, early termination of sympodial cycling, earlier axillary shoot flowering, compact growth habit, reduced height, reduced number of sympodial units, adaptation to mechanical harvest, higher harvest index and any combination thereof .
  • the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the genome editing events located in VuSPl are selected from the group consisting of SEQ ID NOs:4-9 and any combination thereof, and the genome editing events located in VuSP2 are selected from the group consisting of SEQ ID NOs: 12-15 and any combination thereof.
  • the present invention provides a modified Cowpea plant, plant part, plant pot or plant cell as defined in any of the above, wherein the plant does not comprise a transgene .
  • the present invention provides a plant part, plant cell, plant progeny, plant pod or plant seed of a modified Cowpea (Vigna unguiculata) plant as defined in any of the above.
  • the present invention provides a tissue culture of regenerable cells, protoplasts or callus obtained from the modified Cowpea (Vigna unguiculata) plant as defined in any of the above.
  • the present invention provides harvestable parts of a modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the harvestable parts are preferably shoot biomass and/or pods or seeds.
  • a modified Cowpea Vehicle unguiculata
  • the present invention provides the method as defined above, wherein the method comprises steps of : a. identifying in Cowpea (Vigna unguiculata) plant cells, VuSPl and/or VuSP2 genes comprising a nucleic acid sequence with at least 75% sequence identity to a sequence selected from SEQ ID NO:1 and/or SEQ ID NO: 10, respectively; b. synthetizing at least one guide RNA (gRNA) complementary to the VuSPl and/or VuSP2 genes, comprising a nucleotide sequence selected from SEQ ID NO: 2-3 and/or SEQ ID NO: 11, respectively; c.
  • gRNA guide RNA
  • transforming TO Cowpea (Vigna unguiculata) plant cells with a construct comprising (a) Cas nucleotide sequence operably linked to the at least one gRNA, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and the at least one gRNA; d. sei f-pollinating the transformed TO Cowpea (Vigna unguiculata) plant and collecting transformed T1 seeds; e. screening the genome of transformed T1 Cowpea (Vigna unguiculata) cells and selecting for CRISPR/Cas negative and VuSPl and/or VuSP2 genome edited positive T1 Cowpea (Vigna unguiculata) cells; f. screening the genome of the selected T1 Cowpea (Vigna unguiculata) cells for induced targeted loss of function mutation in the VuSPl and/or VuSP2 genes; g. regenerating Cowpea plants from the selected transformed T1
  • Cowpea Vigna unguiculata cells for phenotyping and selecting plants exhibiting improved plant architecture trait conferring determinate growth habit characteristic.
  • the present invention provides the method as defined in any of the above, wherein the construct is introduced into the plant cells via Agrobacterium mediated transformation, virus-based plasmids for delivery of the genome editing molecules or mechanical insertion such as polyethylene glycol (PEG) mediated DNA transformation, electroporation or gene gun biolistics.
  • Agrobacterium mediated transformation virus-based plasmids for delivery of the genome editing molecules or mechanical insertion such as polyethylene glycol (PEG) mediated DNA transformation, electroporation or gene gun biolistics.
  • PEG polyethylene glycol
  • the present invention provides the method as defined in any of the above, wherein the edited plants were screened for loss of function mutations using molecular techniques such as PCR and DNA sequencing.
  • the present invention provides the method as defined in any of the above, wherein the phenotyping analysis comprises evaluating plant height, flowering synchronization, and pod formation parameters.
  • the present invention provides the method as defined in any of the above, wherein the step of screening the genome of the transformed plant cells for induced targeted loss of function mutation further comprises steps of obtaining a nucleic acid sample of the transformed plant and performing a nucleic acid amplification and optionally restriction enzyme digestion to detect a mutation in the VuSPl and/or VuSP2 gene .
  • the present invention provides the method as defined in any of the above, wherein the VuSPl allele comprises a polynucleotide sequence selected from SEQ ID NOs:4-9 and any combination thereof, and mutated VuSP2 allele comprises a polynucleotide sequence selected from SEQ ID NOs: 12-15 and any combination thereof.
  • the present invention provides a modified Cowpea (Vigna unguiculata) plant, plant part, plant seed or pod or plant cell produced by the method as defined in any of the above, wherein the modified plant does not comprise a CRIPPR/Cas related transgene.
  • the present invention provides a tissue culture of regenerable cells, protoplasts or callus obtained from the modified Cowpea (Vigna unguiculata) plant produced by the method as defined in any of the above.
  • the present invention provides a method of improving at least one plant architecture trait conferring determinate growth habit characteristic in a Cowpea (Vigna unguiculata) plant, comprising steps of producing using genome editing, a modified Cowpea (Vigna unguiculata) plant as defined in any of the above, seed, pod or plant part thereof, preferably in a method as defined in any of the above, and enabling growth of the modified Cowpea (Vigna unguiculata) plant, seed or plant part thereof.
  • the present invention provides the method as defined in any of the above, wherein the method comprises steps of: a. identifying at least one Cowpea (Vigna unguiculata) SELF PRUNING (SP) VuSPl and/or VuSP2 gene allele; b. synthetizing at least one guide RNA (gRNA) comprising a polynucleotide sequence complementary to the polynucleotide sequence of the at least one identified VuSPl and/or VuSP2 gene allele ; c.
  • gRNA guide RNA
  • transforming Cowpea (Vigna unguiculata) plant cell with a construct comprising (a) Cas nucleotide sequence operably linked to the at least one gRNA, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and the at least one gRNA; d. screening the genome of the transformed plant cell for induced targeted loss of function mutation in the VuSPl and/or VuSP2 gene to generate a mutated VuSPl and/or VuSP2 allele; e. regenerating Cowpea (Vigna unguiculata) plant carrying the mutated VuSPl and/or VuSP2allele; and f. screening the regenerated plants for a Cowpea (Vigna unguiculata) plant with improved plant architecture trait conferring determinate growth habit characteristic.
  • a construct comprising (a) Cas nucleotide sequence operably linked to the at least one gRNA, or (b) a ribonucleo
  • the present invention provides the method as defined in any of the above, wherein the guide RNA (gRNA) complementary to the VuSPl and/or VuSP2 genes, comprising a nucleotide sequence selected from SEQ ID NO: 2-3 and/or SEQ ID NO: 11, respectively.
  • gRNA guide RNA
  • the present invention provides the method as defined in any of the above, wherein the VuSPl mutated allele comprises a polynucleotide sequence selected from SEQ ID NOs:4-9 and any combination thereof, and the mutated VuSP2 allele comprises a polynucleotide sequence selected from SEQ ID NOs: 12-15 and any combination thereof.
  • the present invention provides a method for identifying and/or selecting for a Cowpea (Vigna unguiculata) plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic, the method comprises steps of: a. screening the genome of the Cowpea (Vigna unguiculata) plant for a mutated VuSPl and/or VuSP2 allele, the wherein the VuSPl mutated allele comprises a polynucleotide sequence selected from SEQ ID NOs:4-9 and any combination thereof, and the mutated VuSP2 allele comprises a polynucleotide sequence selected from SEQ ID NOs: 12-15 and any combination thereof; b. optionally, regenerating a modified Cowpea (Vigna unguiculata) plant carrying the genetic modification; and c. optionally, screening the regenerated plants for a plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic.
  • the present invention provides the method as defined in any of the above, wherein the genomic modification is a loss of function mutation.
  • the present invention provides the method as defined in any of the above, wherein the determinate growth habit characteristic is selected from the group consisting of determinate or semi-determinate architecture, early termination of sympodial cycling, compact growth habit, reduced height, distinct short stature, determinate apical meristem growth, reduced or limited apical meristem growth, interrupted or exhausted apical meristem growth, bushy growth habit, synchronized flowering, uniform flowering, synchronized pod formation, uniform pod formation, reduced number of sympodial units, suppressed sympodial shoot termination, lack of central growing axis and tendrils, uniform appearance of flowers, adaptation to mechanical harvest, higher harvest index and any combination thereof .
  • the determinate growth habit characteristic is selected from the group consisting of determinate or semi-determinate architecture, early termination of sympodial cycling, compact growth habit, reduced height, distinct short stature, determinate apical meristem growth, reduced or limited apical mer
  • the present invention provides a method of determining the presence of a mutated VuSPl and/or VuSP2 allele in a Cowpea (Vigna unguiculata) plant, conferring determinate growth habit characteristic, wherein the method comprising assaying the genome of the Cowpea plant for the presence of a polynucleotide sequence selected from SEQ ID NOs:4-9 and any combination thereof, and SEQ ID NOs: 12-15 and any combination thereof, respectively.
  • the present invention provides an isolated polynucleotide sequence having at least 80% sequence similarity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 4-9 and any combination thereof, and SEQ ID NOs: 12-15 and any combination thereof .
  • the present invention provides use of (a) a nucleic acid sequence as set forth in SEQ ID NO: 1 and/or SEQ ID NO: 10 and/or (b) a nucleotide sequence as set forth in SEQ ID NOs: 2-3 and/or SEQ ID NO: 11 and any combination thereof for targeted genome modification of Cowpea (Vigna unguiculata) , for generating and/or producing a modified Cowpea (Vigna unguiculata) plant with at least one genetically modified VuSPl and/or VuSP2 genes involved in determinate growth habits.
  • the present invention provides use of a nucleotide sequence having at least 80% sequence similarity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 4-9 and any combination thereof, and SEQ ID NOs: 12-15 and any combination thereof, or a complementary sequence thereof, for generating, identifying and/or screening for a modified Cowpea (Vigna unguiculata) plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic .
  • the present invention provides the use as defined in any of the above, wherein the presence of at least one polynucleotide sequence selected from SEQ ID NOs: 4-9 and any combination thereof, or a complementary sequence thereof indicates that the Cowpea plant comprises a VuSPl allele comprising a loss of function mutation, and the presence of at least one polynucleotide sequence selected from at least one polynucleotide sequence selected from SEQ ID NOs: 12-15 and any combination thereof, or a complementary sequence thereof indicates that the Cowpea plant comprises a VuSP2 allele comprising a loss of function mutation .
  • the present invention provides a detection kit for determining the presence or absence of a mutated VuSPl and/or VuSP2 gene conferring determinate growth habit characteristic in a Cowpea (Vigna unguiculata) plant, comprising a polynucleotide fragment comprising a polynucleotide sequence having at least 80% similarity to a sequence selected from SEQ ID NOs: 4-9, any combination thereof, or a complementary sequence thereof, and /or at least 80% similarity to a sequence selected from SEQ ID NOs: 12-15 any combination thereof, or a complementary sequence thereof, respectively.
  • Cowpea lines with mutated VuSPl and/or VuSP2 genes was achieved by the following breeding/cultivation method. It should be noted that other breeding/cultivation methods known in the art can be executed.
  • Cowpea Vigna unguiculata homologs of SELF PRUNING (SP) , namely VuSPl and/or VuSP2.
  • Constructs containing the CRISPR/Cas9 machinery and specific guide RNA (gRNA) targeting the Cowpea SP homologs were designed and introduced into Cowpea tissue using Agrobacterium-mediated transformation.
  • the edited plants were screened for desired mutations using molecular techniques known in the art, such as PCR and DNA sequencing. Phenotypic analysis was performed to evaluate plant height, flowering synchronization, and pod formation.
  • Table 1 presenting the wild-type Cowpea's genomic sequences related to VuSPl and VuSP2.
  • the wild-type genomic sequence for VuSPl (SEQ ID NO:1) is represented on Fig. 1
  • wild-type genomic sequence for VuSP2 (SEQ ID NO: 10) is represented on Fig. 2.
  • Figs. 3A-B schematically presenting the locations of the gRNA molecules on the respective VuSPl (Fig.3A) and VuSP2 (Fig. 3B) genes.
  • the scale bar represents 100 bp
  • the white box represents UTR (untranslated region)
  • the black box represents an exon
  • the black line represents an intron.
  • the chromosome locations and the gRNA target sequences are shown in Table 2 below.
  • the PAM sequences (NGG) are presented in bold and underlined.
  • the sequences of gRNAl (SEQ ID NO: 2) and gRNA2 (SEQ ID NO: 3) are underlined in Fig. 1, and the sequence of gRNA3 (SEQ ID NO: 11) is underlined in Fig. 2.
  • VuSPl SEQ ID NO:1
  • VuSP2 SEQ ID NO: 10
  • the corresponding gRNA molecules were designed and synthesized as shown in Table 2 above. These gRNA molecules were then cloned into suitable vectors, and their sequences were verified. Optionally, the efficiency of the designed gRNA molecules was validated by transiently transforming Cowpea tissue culture as described hereinafter .
  • Cowpea seedlings were transformed with a binary vector containing the CRISPR machinery (Cas9 and gRNA) alongside an eYGFPuv reporter and an antibiotic resistance gene.
  • CRISPR machinery Cas9 and gRNA
  • T2 individuals underwent phenotyping in the greenhouse, revealing a consistent and reproducible determinate phenotype among all plants, unaffected by the specific genome edits they harbored.
  • Figs. 4A-B DNA was extracted from T1 seeds and a PCR using VuSPl as well as a PCR using Cas9 primers was performed.
  • Fig. 4A it is shown that amplicons (442 bp) of the VuSPl PCR are present in all samples.
  • This PCR serves as a positive control indicating genomic DNA was extracted successfully and samples are usable for the downstream applications.
  • Fig. 4B a PCR using Cas9 primers was performed. It can be seen that Cas9 DNA was detected in samples 1, 2, 5, 6, 8, 10, 14, 15 & 16. These plants were not used in the greenhouse experiment. In the remaining plants, the SP1 and SP2 genes were sequenced, and only edited plants have been selected for phenotypic analysis in the greenhouse .
  • T1 edits have been determined by sequencing (prior to planting) .
  • T1 non transgenic, but edited plants have been grown and compared to the WT non edited control. Plant architecture, flowering time, flowering synchronization, and yield properties have been compared.
  • Table 3 summarizing genomic sequences of mutated VuSPl fragments, from the first generation of mutated Cowpea plants (Tl) , containing genome editing events obtained using the first guide RNA (gRNAl) having nucleic acid sequence as set forth in SEQ ID NO: 2 and the second guide RNA ( gRNA2 ) having nucleic acid sequence as set forth in SEQ ID NO: 3.
  • Figs. 5A-F presenting the nucleic acid sequences of VuSPl gene editing events as set forth in SEQ ID NOs:4-9, respectively.
  • Fig. 6 presents the alignment of nucleic acid sequences from the WT VuSPl (SEQ ID NO:1) and the mutated VuSPl fragments containing genome editing events (SEQ ID NOs:4-9) , shown on Table 3. The alignment was performed with CLUSTAL multiple sequence alignment by Kalign.
  • Fig. 8 presents the alignment of nucleic acid sequences from the WT VuSP12 (SEQ ID NO: 10) and the mutated VuSPl fragments containing genome editing events (SEQ ID NOs: 12-15) , shown on Table 4. The alignment was performed with CLUSTAL multiple sequence alignment by Kalign.
  • SP2_pl editing event on VuSP2 has an insertion ( + 1) on position 41 of SEQ ID NO: 10, and an inversion from TO to AA.
  • This insertion (A) and inversion (TO to AA) on SP2_pl appears in italic in Fig. 8.
  • VuSP2 constructs containing editing events of SEQ ID NO: 12-15 were associated with the determinate growth habit phenotype as described herein (as shown in Figs 9- 14) .
  • the edited plants exhibited a distinct short stature and bushy growth habit, which contrasts with the unedited control indeterminate Cowpea plants.
  • flowering and pod formation were observed to be synchronized and uniform across the edited plants.
  • Table 5 summarizing mutated nucleic acid sequences of the second generation (T2) Cowpea plants comprising a combination of mutated VuSPl allele (Table 3 and Figs. 5A-F) and mutated VuSP2 allele (Table 4 and Figs.7A-D) from the first generation (Tl) .
  • Fig. 9 photographically presenting a phenotypic comparison of exemplified edited (doubled-edited in VuSPl & VuSP2 or edited in VuSP2 gene) versus wild-type Cowpea plants.
  • the mutated edited plant (on the right) has a determinate growth habit phenotype, lacks central growing axis and tendrils, with uniform appearance of flowers.
  • the mutated edited plant displays a shorter stature and bushy growth habit.
  • the wild-type control plant (left) has an indeterminate phenotype, characterized by long and viny plant architecture, with many tendrils .
  • Figs. 10-13 The determinant growth habit phenotype resulting from the herein disclosed editing events combinations in VuSPl & VuSP2 as exemplified in Table 5, is further confirmed and demonstrated in Figs. 10-13, as follows: Reference is now made to Figs. 10A-C, Figs. 11A-C, Figs. 12A-C, and Figs. 13A-C, presenting Cowpea plants that undergone through edit events 3, 8, 12, and 14 (see Table 5) , respectively, having determinate growth habit characteristics.
  • Fig. 14 graphically presenting plant height in cm of edited Cowpea lines (edit events 3, 12, 14 and 8, see Table 5) , measured during day 49 (dark grey) , day 53 (light grey) and day 67 (grey) , compared to the wild-type Cowpea. It can be seen that the edited Cowpea plants presented a general height range from approximately 62 to 98 cm, while the wild-type Cowpea presented a height range from approximately 120 cm to 150 cm. More specifically, the height range varied from approximately 65 cm to 98 cm after 67 days, which is significantly lower than the wildtype Cowpea height after the same period of time.
  • RNA-guided genome editing in plants using a CRISPR-Cas system Molecular plant 6.6 (2013) : 1975-1983.

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Abstract

A modified Cowpea (Vigna unguiculata") plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic. The plant comprises mutated SELF PRUNING (SP) VuSPl and/or VuSP2 gene comprising genome edited loss of function mutation.

Description

COWPEA PLANTS WITH IMPROVED TRAITS AND METHODS FOR GENERATING THE SAME
FIELD OF THE INVENTION
The present disclosure relates to Cowpea plants with desirable agronomic traits. More particularly, the current invention pertains to Cowpea plants with improved plant architecture, generated by manipulating SELF PRUNING (SP) genes through gene editing techniques.
BACKGROUND OF THE INVENTION
Labor scarcity in agriculture poses significant challenges to crop production. For many crops, artificial selection for modified shoot architectures provided critical steps towards improving yield, followed by improvements enabling large-scale field production .
For example, in the industrial tomato industry, the natural mutation causing determinate growth, SELF PRUNING (SP) , has revolutionized cultivation.
Cowpea (Vigna unguiculata) is a vital legume crop, and its mechanical harvesting is hindered by its indeterminate growth habit. In such open field crops, it is essential for enabling sustainable agriculture, to be able to harvest the plants mechanically, instead of using manual labor.
In view of the above, there is still a long felt and unmet need to manipulate Cowpea plant architecture in a rapid and efficient way to increase yield and reduce production costs.
SUMMARY OF THE INVENTION
It is one object of the present invention to disclose a modified Cowpea (Vigna unguiculata) plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic, wherein said plant comprises mutated SELF PRUNING (SP) VuSPl and/or VuSP2 gene comprising genome edited loss of function mutation.
It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined above, wherein said improved plant architecture trait is relative to a corresponding Cowpea (Vigna unguiculata) plant lacking the genome edited loss of function mutation.
It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein said mutated VuSPl gene comprises a genomic sequence selected from SEQ ID NO: 4-9, or a functional variant thereof, or any combination thereof .
It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein said mutated VuSP2 gene comprises a genomic sequence selected from SEQ ID NO: 12-15, or a functional variant thereof, or any combination thereof .
It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein said functional variant comprises at least 90% sequence identity to the corresponding mutated VuSPl or mutated VuSP2 sequence.
It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein said determinate growth habit characteristic is selected from the group consisting of determinate or semideterminate architecture, early termination of sympodial cycling, compact growth habit, reduced height, distinct short stature, determinate apical meristem growth, reduced or limited apical meristem growth, interrupted or exhausted apical meristem growth, bushy growth habit, synchronized flowering, uniform flowering, synchronized pod formation, uniform pod formation, reduced number of sympodial units, suppressed sympodial shoot termination, lack of central growing axis and tendrils, uniform appearance of flowers, adaptation to mechanical harvest, higher harvest index and any combination thereof.
It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the height of said modified plant is in the range of 60cm to 100cm, such as in the range of 65cm to 90cm.
It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein said modified plant height is about 30%-50% lower than a control Cowpea plant lacking the genome edited loss of function mutation in said VuSPl and/or VuSP2 genes.
It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein said modified plant has pod number similar or at least 10% higher than a control Cowpea plant lacking the genome edited loss of function mutation in said VuSPl and/or VuSP2 genes.
It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein said mutation in said VuSPl gene is generated using a guide RNA (gRNA) comprising a polynucleotide sequence selected from SEQ ID NO: 2 and SEQ ID NO: 3 or a combination thereof, and said mutation in said VuSP2 gene is generated using a guide RNA (gRNA) sequence comprising a polynucleotide sequence comprising SEQ ID NO: 11. It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutated VuSPl and/or VuSP2 gene is a CRISPR/Cas- induced heritable mutated allele.
It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein said Cas gene is selected from the group consisting of Cas9, Casl2, Casl3, Casl4, CasX, CasY, Csnl, Cpfl and any combination thereof.
It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein said mutation is an insertion, deletion, indel, inversion, substitution, duplication or any combination thereof .
It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein said mutation is a silencing mutation, a knockdown mutation, a knockout mutation, a loss of function mutation, downregulating, de-regulating, deactivating, reducing expression, at least partially deleting, at least partially silencing, at least partially deactivating, removing, partially removing, duplicating, inverting, missense mutation, nonsense mutation, or any combination thereof.
It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein said mutation is in the coding sequence or in a regulatory sequence such as a promoter, terminator sequence, of the VuSPl and/or VuSP2 gene.
It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein said plant is homozygous, heterozygous or hemizygous for said genome edited loss of function mutation in said VuSPl and/or VuSP2 gene.
It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein said mutation in said VuSPl gene is generated in planta via introduction of a construct comprising (a) Cas DNA and gRNA comprising a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA comprising a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3.
It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein said mutation in said VuSP2 gene is generated in planta via introduction of a construct comprising (a) Cas DNA and gRNA sequence comprising SEQ ID NO: 11, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence comprising SEQ ID NO: 11.
It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein said gRNA sequence comprises a 3' NGG Protospacer Adjacent Motif (PAM) .
It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein said mutation confers determinate growth habit phenotype .
It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein said modified plant flowers earlier than a corresponding control Cowpea plant lacking said mutated VuSPl and/or VuSP2 gene. It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein said improved plant architecture trait is selected from the group consisting of reduced flowering time, earliness, synchronous flowering, reduced day-length sensitivity, determinant growth habit, early termination of sympodial cycling, earlier axillary shoot flowering, compact growth habit, reduced height, reduced number of sympodial units, adaptation to mechanical harvest, higher harvest index and any combination thereof.
It is another object of the present invention to disclose the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the genome editing events located in VuSPl are selected from the group consisting of SEQ ID NOs:4-9 and any combination thereof, and the genome editing events located in VuSP2 are selected from the group consisting of SEQ ID NOs: 12-15 and any combination thereof.
It is another object of the present invention to disclose a modified Cowpea plant, plant part, plant pot or plant cell as defined in any of the above, wherein said plant does not comprise a transgene .
It is another object of the present invention to disclose a plant part, plant cell, plant progeny, plant pod or plant seed of a modified Cowpea (Vigna unguiculata) plant as defined in any of the above .
It is another object of the present invention to disclose a tissue culture of regenerable cells, protoplasts or callus obtained from the modified Cowpea (Vigna unguiculata) plant as defined in any of the above .
It is another object of the present invention to disclose harvestable parts of a modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein said harvestable parts are preferably shoot biomass and/or pods or seeds.
It is another object of the present invention to disclose products derived from the modified Cowpea (Vigna unguiculata) plant as defined in any of the above and/or from harvestable parts of a modified Cowpea (Vigna unguiculata) plant as defined in any of the above .
It is another object of the present invention to disclose a method for producing a modified Cowpea (Vigna unguiculata) plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic as defined in any of the above, wherein said method comprises steps of mutating a Vigna unguiculata SELF PRUNING (SP) VuSPl and/or VuSP2 gene by introducing using targeted genome editing a loss of function mutation .
It is another object of the present invention to disclose the method as defined above, wherein said method comprises steps of: a. identifying in Cowpea (Vigna unguiculata) plant cells, VuSPl and/or VuSP2 genes comprising a nucleic acid sequence with at least 75% sequence identity to a sequence selected from SEQ ID NO:1 and/or SEQ ID NO: 10, respectively; b. synthetizing at least one guide RNA (gRNA) complementary to said VuSPl and/or VuSP2 genes, comprising a nucleotide sequence selected from SEQ ID NO: 2-3 and/or SEQ ID NO: 11, respectively; c. transforming TO Cowpea (Vigna unguiculata) plant cells with a construct comprising (a) Cas nucleotide sequence operably linked to said at least one gRNA, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and said at least one gRNA; d. sei f-pollinating said transformed TO Cowpea (Vigna unguiculata) plant and collecting transformed T1 seeds; e. screening the genome of transformed T1 Cowpea (Vigna unguiculata) cells and selecting for CRISPR/Cas negative and VuSPl and/or VuSP2 genome edited positive T1 Cowpea (Vigna unguiculata) cells; f. screening the genome of said selected T1 Cowpea (Vigna unguiculata) cells for induced targeted loss of function mutation in said VuSPl and/or VuSP2 genes; g. regenerating Cowpea plants from said selected transformed T1 Cowpea (Vigna unguiculata) cells for phenotyping and selecting plants exhibiting improved plant architecture trait conferring determinate growth habit characteristic.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said construct is introduced into the plant cells via Agrobacterium mediated transformation, virus-based plasmids for delivery of the genome editing molecules or mechanical insertion such as polyethylene glycol (PEG) mediated DNA transformation, electroporation or gene gun biolistics.
It is another object of the present invention to disclose the method as defined in any of the above, wherein the edited plants were screened for loss of function mutations using molecular techniques such as PCR and DNA sequencing.
It is another object of the present invention to disclose the method as defined in any of the above, wherein the phenotyping analysis comprises evaluating plant height, flowering synchronization, and pod formation parameters.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said step of screening the genome of said transformed plant cells for induced targeted loss of function mutation further comprises steps of obtaining a nucleic acid sample of said transformed plant and performing a nucleic acid amplification and optionally restriction enzyme digestion to detect a mutation in said VuSPl and/or VuSP2 gene .
It is another object of the present invention to disclose the method as defined in any of the above, wherein the VuSPl allele comprises a polynucleotide sequence selected from SEQ ID NOs:4-9 and any combination thereof, and mutated VuSP2 allele comprises a polynucleotide sequence selected from SEQ ID NOs: 12-15 and any combination thereof.
It is another object of the present invention to disclose a modified Cowpea (Vigna unguiculata) plant, plant part, plant seed or pod or plant cell produced by the method as defined in any of the above, wherein said modified plant does not comprise a CRIPPR/Cas related transgene.
It is another object of the present invention to disclose a tissue culture of regenerable cells, protoplasts or callus obtained from the modified Cowpea (Vigna unguiculata) plant produced by the method as defined in any of the above.
It is another object of the present invention to disclose a method of improving at least one plant architecture trait conferring determinate growth habit characteristic in a Cowpea (Vigna unguiculata) plant, comprising steps of producing using genome editing, a modified Cowpea (Vigna unguiculata) plant as defined in any of the above, seed, pod or plant part thereof, preferably in a method as defined in any of the above, and enabling growth of said modified Cowpea (Vigna unguiculata) plant, seed or plant part thereof . It is another object of the present invention to disclose the method as defined in any of the above, wherein said method comprises steps of: a. identifying at least one Cowpea (Vigna unguiculata) SELF PRUNING (SP) VuSPl and/or VuSP2 gene allele; b. synthetizing at least one guide RNA (gRNA) comprising a polynucleotide sequence complementary to the polynucleotide sequence of said at least one identified VuSPl and/or VuSP2 gene allele ; c. transforming Cowpea (Vigna unguiculata) plant cell with a construct comprising (a) Cas nucleotide sequence operably linked to said at least one gRNA, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and said at least one gRNA; d. screening the genome of said transformed plant cell for induced targeted loss of function mutation in said VuSPl and/or VuSP2 gene to generate a mutated VuSPl and/or VuSP2 allele; e. regenerating Cowpea (Vigna unguiculata) plant carrying said mutated VuSPl and/or VuSP2allele; and f. screening said regenerated plants for a Cowpea (Vigna unguiculata) plant with improved plant architecture trait conferring determinate growth habit characteristic.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said guide RNA (gRNA) complementary to said VuSPl and/or VuSP2 genes, comprising a nucleotide sequence selected from SEQ ID NO: 2-3 and/or SEQ ID NO: 11, respectively.
It is another object of the present invention to disclose the method as defined in any of the above, wherein the VuSPl mutated allele comprises a polynucleotide sequence selected from SEQ ID NOs:4-9 and any combination thereof, and the mutated VuSP2 allele comprises a polynucleotide sequence selected from SEQ ID N0s:12-
15 and any combination thereof.
It is another object of the present invention to disclose a method for identifying and/or selecting for a Cowpea (Vigna unguiculata) plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic, said method comprises steps of: a. screening the genome of said Cowpea (Vigna unguiculata) plant for a mutated VuSPl and/or VuSP2 allele, said wherein the VuSPl mutated allele comprises a polynucleotide sequence selected from SEQ ID NOs:4-9 and any combination thereof, and the mutated VuSP2 allele comprises a polynucleotide sequence selected from SEQ ID NOs: 12-15 and any combination thereof; b. optionally, regenerating a modified Cowpea (Vigna unguiculata) plant carrying said genetic modification; and c. optionally, screening said regenerated plants for a plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said genomic modification is a loss of function mutation.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said determinate growth habit characteristic is selected from the group consisting of determinate or semi-determinate architecture, early termination of sympodial cycling, compact growth habit, reduced height, distinct short stature, determinate apical meristem growth, reduced or limited apical meristem growth, interrupted or exhausted apical meristem growth, bushy growth habit, synchronized flowering, uniform flowering, synchronized pod formation, uniform pod formation, reduced number of sympodial units, suppressed sympodial shoot termination, lack of central growing axis and tendrils, uniform appearance of flowers, adaptation to mechanical harvest, higher harvest index and any combination thereof .
It is another object of the present invention to disclose a method of determining the presence of a mutated VuSPl and/or VuSP2 allele in a Cowpea (Vigna unguiculata) plant, conferring determinate growth habit characteristic, wherein the method comprising assaying the genome of said Cowpea plant for the presence of a polynucleotide sequence selected from SEQ ID NOs:4-9 and any combination thereof, and SEQ ID NOs: 12-15 and any combination thereof, respectively.
It is another object of the present invention to disclose an isolated polynucleotide sequence having at least 80% sequence similarity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 4-9 and any combination thereof, and SEQ ID NOs: 12-15 and any combination thereof .
It is another object of the present invention to disclose use of (a) a nucleic acid sequence as set forth in SEQ ID NO:1 and/or SEQ ID NO: 10 and/or (b) a nucleotide sequence as set forth in SEQ ID NOs: 2-3 and/or SEQ ID NO: 11 and any combination thereof for targeted genome modification of Cowpea (Vigna unguiculata) , for generating and/or producing a modified Cowpea (Vigna unguiculata) plant with at least one genetically modified VuSPl and/or VuSP2 genes involved in determinate growth habits.
It is another object of the present invention to disclose use of a nucleotide sequence having at least 80% sequence similarity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 4-9 and any combination thereof, and SEQ ID NOs: 12-15 and any combination thereof, or a complementary sequence thereof, for generating, identifying and/or screening for a modified Cowpea (Vigna unguiculata) plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic.
It is another object of the present invention to disclose the use as defined in any of the above, wherein the presence of at least one polynucleotide sequence selected from SEQ ID NOs:4-9 and any combination thereof, or a complementary sequence thereof indicates that the Cowpea plant comprises a VuSPl allele with a loss of function mutation, and the presence of at least one polynucleotide sequence selected from at least one polynucleotide sequence selected from SEQ ID NOs: 12-15 and any combination thereof, or a complementary sequence thereof indicates that the Cowpea plant comprises a VuSP2 allele with a loss of function mutation.
It is another object of the present invention to disclose a detection kit for determining the presence or absence of a mutated VuSPl and/or VuSP2 gene conferring determinate growth habit characteristic in a Cowpea (Vigna unguiculata) plant, comprising a polynucleotide fragment comprising a polynucleotide sequence having at least 80% similarity to a sequence selected from SEQ ID NOs: 4-9, any combination thereof, or a complementary sequence thereof, and /or at least 80% similarity to a sequence selected from SEQ ID NOs: 12-15 any combination thereof, or a complementary sequence thereof, respectively.
BRIEF DESCRIPTION OF THE DRAWINGS
The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention is best understood in view of the accompanying drawings in which: FIG. 1 is presenting a wild-type (WT) genomic sequence of Cowpea (Vigna unguiculata) SELF PRUNING (SP) 1 gene (VuSPl) as set forth in SEQ ID NO: 1;
FIG. 2 is presenting a wild-type (WT) genomic sequence of Cowpea (Vigna unguiculata) SELF PRUNING (SP) 2 gene (VuSP2) as set forth in SEQ ID NO: 10;
FIGs . 3A-B are schematic representations of the location of the gRNAs on the VuSPl gene (FIG. 3A) and VuSP2 gene (FIG. 3B) ;
FIGs. 4A-B are photographic representations of PCR amplicons in T1 transformed Cowpea plants; FIG. 4A presents amplicons of VuSPl; FIG. 4B is amplicons obtained using Cas9 primers;
FIGs. 5A-F are presenting nucleic acid sequences of mutated alleles of VuSPl gene having the herein disclosed editing events as set forth in SEQ ID NOs:4-9, respectively;
FIG. 6 is presenting an alignment of nucleic acid sequences of the WT VuSPl as set forth in SEQ ID NO:1 and the mutated VuSPl alleles containing genome editing events as set forth in SEQ ID NOs:4-9;
FIGs. 7A-D are presenting nucleic acid sequences of mutated alleles of VuSP2 gene containing the herein disclosed editing events as set forth in SEQ ID NOs: 12-15, respectively;
FIG. 8 is presenting an alignment of nucleic acid sequences of WT VuSP2 as set forth in SEQ ID NO: 10 and mutated VuSPl alleles containing genome editing events as set forth in SEQ ID Nos : 12- 15;
FIG. 9 is a picture representing a phenotypic comparison of edited mutated (right) and wild-type (left) Cowpea plants;
FIGs. 10A-C are pictures representing mutated Cowpea lines with a determinate growth habit characteristics as an embodiment of the present invention; FIGs . 11A-C are pictures representing mutated Cowpea lines with a determinate growth habit characteristics as an embodiment of the present invention;
FIGs. 12A-C are pictures representing mutated Cowpea lines with a determinate growth habit characteristics as an embodiment of the present invention;
FIGs. 13A-C are pictures representing mutated Cowpea lines with a determinate growth habit characteristics as an embodiment of the present invention; and
FIG. 14 is a graph representing plant heigh in cm of edited mutated Cowpea lines that have undergone editing events, compared to the wild-type Cowpea.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
In the following detailed description of the preferred embodiments, reference is made to the accompanying drawings that form a part hereof, and in which are shown by way of illustration specific embodiments in which the invention may be practiced. It is understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention. The present invention may be practiced according to the claims without some or all of these specific details. For the purpose of clarity, technical material that is known in the technical fields related to the invention has not been described in detail so that the present invention is not unnecessarily obscured .
Cowpea (Vigna unguiculata) is a vital legume crop, and its mechanical harvesting is hindered by its indeterminate growth habit .
The current invention is aimed to solve this problem by utilizing genome editing, specifically the CRISPR/Cas9 system, to edit the Cowpea homolog of SELF PRUNING (SP) and to develop determinate Cowpea lines.
The present invention provides a modified Cowpea plant exhibiting improved plant architecture trait conferring determinate growth phenotype .
According to one embodiment, the present invention provides a modified Cowpea (Vigna unguiculata) plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic. The aforementioned modified plant comprises mutated SELF PRUNING (SP) VuSPl and/or VuSP2 gene comprising genome edited loss of function mutation.
According to a further embodiment, the improved plant architecture trait of the modified Cowpea (Vigna unguiculata) plant is relative to a corresponding Cowpea (Vigna unguiculata) plant lacking the genome edited loss of function mutation.
It is further within the scope of the present invention that the mutated VuSPl gene comprises a genomic sequence selected from SEQ ID NO: 4-9, or a functional variant thereof, or any combination thereof .
It is further within the scope of the present invention that the mutated VuSP2 gene comprises a genomic sequence selected from SEQ ID NO: 12-15, or a functional variant thereof, or any combination thereof .
According to a further embodiment, the modified Cowpea plant comprises at least one mutated Cowpea (Vigna unguiculata) SELF PRUNING (SP) gene selected from VuSPl and/or VuSP2 genes.
According to a further embodiment of the present invention, the Cowpea (Vigna unguiculata) SP gene undergone targeted gene editing mutation is selected from VuSPl and/or VuSP2 comprising a nucleic acid sequence with at least 75% sequence identity to a sequence selected from SEQ ID NO:1 and/or SEQ ID NO: 10, respectively, or a functional variant thereof and any combination thereof .
The present invention further provides methods for producing the aforementioned modified Cowpea plant using genome editing optionally with other genome modification techniques.
The solution proposed by the current invention is using genome editing such as the CRISPR/Cas system in order to create cultivated Cowpea plants with determinate growth habit characteristics. Breeding using genome editing allows a precise and significantly shorter breeding process in order to achieve these goals with a much higher success rate. Thus, genome editing has the potential to generate improved lines faster and at a lower cost.
It is further noted that the resulted edited mutated plants may desirably and advantageously be considered as non-GMO, as no foreign DNA is integrated into the plants (T1 plants were selected to be CRISPR/Cas9 negative) .
Thus, the new and improved Cowpea plants of the present invention are adapted for intensive and advanced agriculture.
The present invention provides Cowpea plants with improved plant architecture traits conferring determinate growth phenotype. The current invention discloses the generation of non-transgenic Cowpea plants with determinate growth habits, using genome editing technology, e.g. , the CRISPR/Cas9 highly precise tool. The generated mutations can be introduced into elite or locally adapted Cowpea lines rapidly, with relatively minimal effort and investment .
It is within the scope that the edited plants exhibited a distinct short stature and bushy growth habit, which is in contrast to the unedited control indeterminate Cowpea plants. Furthermore, flowering and pod formation were observed to be synchronized and uniform across the edited plants.
Genome editing is an efficient and useful tool for increasing crop productivity, and there is particular interest in advancing manipulation of domestication genes in Cowpea wild species, which often have undesirable characteristics.
Genome-editing technologies, such as the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -CRISPR-associated protein-9 nuclease (Cas9) (CRISPR-Cas9) provide opportunities to address undesirable characteristics of Cowpea plants, with the aims of increasing quality and yield, improve adaptation and expand geographical ranges of cultivation.
A major obstacle for CRISPR-Cas9 plant genome editing is lack of efficient tissue culture and transformation methodologies. The present invention achieves these aims and provides transformed and regenerated Cowpea plants with modified desirable plant architecture genes.
To that end, guide RNAs (gRNAs) were designed for each of the target genes identified in Cowpea to induce loss of function mutations in VuSPl and/or VuSP2 genes through genome editing.
As used herein the term "about" denotes ± 25% of the defined amount or measure or value.
As used herein the term "similar" denotes a correspondence or resemblance range of about ± 20%, particularly ± 15%, more particularly about ± 10% and even more particularly about ± 5%.
As used herein the term "corresponding" generally means similar, analogous, like, alike, akin, parallel, identical, resembling or comparable. In further aspects it means having or participating in the same relationship (such as type or species, kind, degree, position, correspondence, or function) . It further means related or accompanying. In some embodiments of the present invention refers to plants of the same Cowpea species or strain or variety or to sibling plant, or one or more individuals having one or both parents in common.
A "plant" as used herein refers to any plant at any stage of development, particularly a seed plant. The term "plant" includes the whole plant or any parts or derivatives thereof, such as plant cells, seeds, pods, plant protoplasts, plant cell tissue culture from which Cowpea plants can be regenerated, plant callus or calli, meristematic cells, microspores, embryos, immature embryos, pollen, ovules, anthers, fruit, flowers, leaves, cotyledons, pistil, seeds, seed coat, pods, roots, root tips and the like.
The term "plant cell" used herein refers to a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in the form of an isolated single cell or a cultured cell.
The term "plant cell culture" as used herein means cultures of plant units such as, for example, protoplasts, regenerable cells, cell culture, cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development, leaves, roots, root tips, anthers, meristematic cells, microspores, flowers, cotyledons, pistil, fruit, seeds, seed coat, pods or any combination thereof.
The term "plant material" or "plant part" used herein refers to leaves, stems, roots, root tips, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, seed coat, pods, cuttings, cell or tissue cultures, or any other part or product of a plant or a combination thereof.
A "plant organ" as used herein means a distinct and visibly structured and differentiated part of a plant such as, but not limited to a root, stem, leaf, flower, flower bud, pod, seed or embryo .
The term "plant tissue" as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, plant organs, plant seeds, pods, tissue culture, protoplasts, meristematic cells, calli and any group of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.
As used herein, the term "progeny" or "progenies" refers in a nonlimiting manner to offspring or descendant plants. According to certain embodiments, the term "progeny" or "progenies" refers to plants developed or grown or produced from the disclosed or deposited seeds as detailed inter alia. The grown plants preferably have the desired traits of the disclosed or deposited seeds, i.e. loss of function mutation in at least one of Cowpea VuSPl and/or VuSP2 genes .
The term "Legume" refers hereinafter to a plant in the family Fabaceae (or Leguminosae) , or the fruit or seed of such a plant. The legume family consists of plants that produce a pod with seeds inside. According to the present invention, the Legume is Cowpea (Vigna unguiculata) .
The term "Cowpea" also herein refers to "Vigna unguiculata" or "Vu" is an annual herbaceous legume from the genus Vigna (peas and beans) . It is a food and animal feed crop grown in the semi-arid tropics covering Africa, Asia, Europe, the United States, and Central and South America. The term "SELF- PRUNING" or "SP" in the context of the present invention refers to a gene which encodes a flowering repressor that modulates sympodial growth. It is herein shown that mutations in the SP Cowpea orthologue cause an acceleration of sympodial cycling and shoot termination. It is further acknowledged that the SELF PRUNING (SP) gene controls the regularity of the vegetative- reproductive switch along the compound shoot and thus conditions the 'determinate' (sp/sp) and 'indeterminate' (SP) growth habits of the plant. SP is a developmental regulator which is considered as similar to CENTRORADIAL IS (CEN) from Antirrhinum and TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT) from Arabidopsis.
According to certain aspects of the present invention, the Legumes SP genes include Cowpea VuSPl and VuSP2 genes.
According to main aspects of the present invention, genome editing- targeted mutation in at least one of the aforementioned Cowpea VuSPl and/or VuSP2 genes, which reduces the functional expression of the gene, affect the plant sympodial growth habit which plays a key role in determining plant architecture and particularly confers determinate growth habit characteristics, highly desirable in crops and shown for the first time in Cowpea.
In some aspects of the present invention, determinate growth habit, refers to morphogenesis by formation of up to three to four inflorescences per shoot (IPS) , and is one of the key tools for breeding high-yielding varieties of crops.
The term "sympodial growth" as used herein refers to a type of bifurcating branching pattern where one branch develops more strongly than the other, resulting in the stronger branches forming the primary shoot and the weaker branches appearing laterally. A sympodium, also referred to as a sympode or pseudaxis, is the primary shoot, comprising the stronger branches, formed during sympodial growth. In some aspects of the present invention, sympodial growth occurs when the apical meristem is terminated (as a result of the loss of function targeted mutation is VuSPl and/or VuSP2 genes) and growth is continued by one or more lateral meristems, which repeat the process. The apical meristem may be consumed to make an inflorescence or other determinate structure, or it may be aborted.
It is further within the scope of the current invention that the shoot section between two successive inflorescences is called the 'sympodium’ , and the number of leaf nodes per sympodium is referred to as the ’sympodial index’ (spi) . The first termination event activates the 'sympodial cycle' . In sympodial plants, the apparent main shoot consists of a reiterated array of 'sympodial units' . It is herein shown in Cowpea that a mutant sp gene (VuSPl and/or VuSP2 genes) accelerates the termination of sympodial units and confers determinate growth habit.
The term "determinate" or "determinate growth" as used herein refers to plant growth in which the main stem ends in an inflorescence or other reproductive structure (e.g. a bud) and stops continuing to elongate indefinitely with only branches from the main stem having further and similarly restricted growth. It also refers to growth characterized by sequential flowering from the central or uppermost bud to the lateral or basal buds . It further means naturally self-limited growth, resulting in a plant of a definite maximum size.
The term "semi-determinate" as used herein refers to plants, genotypes, lines or varieties that continue growing through the season, but generally remain smaller than indeterminate plants, with a bushier habit. According to further aspects, semideterminate genotypes are equivalent to determinate ones with extended vegetative growth, which in turn impacts shoot height, number of leaves and either stem diameter or internode length. According to one embodiment of the present invention, the term semi-determinant is included within the scope of the meaning of the term determinant or determinant growth habit. In this aspect, the modified Cowpea plants of the present invention may present determinate or semi-determinate growth habit.
According to some embodiments of the present invention, the generated Cowpea mutated lines, comprising the targeted loss of function mutations in VuSPl and/or VuSP2 genes, are determinate Cowpea plants exhibiting at least one characteristic such as compact growth habit, distinct short stature, exhausted or terminated apical meristem, bushy growth habit, lacking central growth axis and tendrils, uniform appearance of flowers, synchronized and uniform flowering, and synchronized and uniform pod formation. This is in contrast to a wild-type plant absent of the herein disclosed targeted editing events in VuSPl and/or VuSP2 genes which is characterized by indeterminate, long, viny growth habit with many tendrils.
The term "apical meristem" refers, without limitation, to specialized zones of growth found at the tips of plants. These are responsible for the extension of shoots and roots and are also responsible for the primary plant body. A balance between the generation of new meristematic cells, and their transition toward differentiation, permits the maintenance of the meristem and regulates its activity. In plants with determinate growth habits, the meristem is genetically programmed to stop producing new cells at a specific developmental stage. Therefore, at some point of the growth of a plant with determinate growth, the apical meristem becomes exhausted, and no new cells are produced.
The term "indeterminate" or "indeterminate growth" as used herein refers to plant growth in which the main stem continues to elongate indefinitely without being limited by a terminal inflorescence or other reproductive structure. It also refers to growth characterized by sequential flowering from the lateral or basal buds to the central or uppermost buds. Indeterminate growth habit is herein shown to be correlated with wild-type or control Cowpea plants, lacking the herein disclosed mutations in VuSPl and/or VuSP2 genes .
The term "harvest index" can be herein defined as the total yield per plant weight.
It is within the scope of the present invention that ’yield related traits’ comprise one or more of improved plant architecture, i.e. , determinate growth habit, compact growth habit, distinct short stature, exhausted apical meristem, bushy growth habit, synchronized and uniform flowering, and synchronized and uniform pod formation. According to some embodiments, improved plant architecture encompasses lacking central growing axis and tendrils, with uniform appearance of flowers.
The term "yield" in general means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and/or weight, or the actual yield is the yield per square meter for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted square meters. The terms "yield" of a plant and "plant yield" are used interchangeably herein and are meant to refer to vegetative biomass such as root and/or shoot biomass, to reproductive organs, and/or to propagules such as seeds or pods of that plant.
The terms "increase", "improve" or "enhance" are interchangeable and shall mean in the sense of the application at least a 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35% or 40% more yield, growth or any other agronomic trait such as height, domestication trait or plant architecture, in comparison to control plants as defined herein.
Increased seed or pods yield may be defined as one or more of the following: (a) an increase in seed/pod biomass (total seed/pod weight) which may be on an individual seed/pod basis and/or per plant and/or per square meter; (b) increased number of flowers per plant; (c) increased number of seeds/pods; and (d) increased harvest index, which is expressed as a ratio of the yield of harvestable parts, such as seeds/pods, divided by the biomass of aboveground plant parts .
An increase in seed/pod yield may also be manifested as an increase in seed/pod size and/or seed/pod volume.
The term "biomass" as used herein is intended to refer to the total weight of a plant. Within the definition of biomass, a distinction may be made between the biomass of one or more parts of a plant, which may include: aboveground (harvestable) parts such as but not limited to shoot biomass, seed/pod biomass, leaf biomass, etc. and/or (harvestable) parts below ground, such as but not limited to root biomass, etc. , and/or vegetative biomass such as root biomass, shoot biomass, etc. , and/or reproductive organs, and/or propagules such as seed/pod.
Control plant (s) within the scope of the present invention include corresponding wild type plants or corresponding naturally occurring plants or corresponding plants lacking the edited or mutated gene of interest or the specific generated mutation. The choice of suitable control plants is a routine part of an experimental setup and may include corresponding wild type plants or corresponding plants without the gene of interest. The control plant is typically of the same plant species or the same genetic background or even of the same variety as the plant to be assessed. The control plant of the plant to be assessed may also be plant individuals missing the transgene or modif ied/edited gene. A "control plant" or a "wild type" plant as used herein refers not only to whole plants, but also to plant parts, including seeds, pods and seed or pod parts.
The term "orthologue" as used herein refers hereinafter to one of two or more homologous gene sequences found in different species.
The term "functional variant" or "functional variant of a nucleic acid or amino acid sequence" as used herein refers to a sequence or part of a sequence which retains the biological function of the full non-variant allele (e.g. Cowpea VuSPl and/or VuSP2 genes) and hence has the activity of VuSPl and/or VuSP2 expressed gene or protein. A functional variant also comprises a variant of the gene of interest encoding a polypeptide which has sequence alterations that do not affect function of the resulting protein, for example, in non-conserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example, in non-conserved residues, to the wild type nucleic acid or amino acid sequences of the alleles as shown herein, and is biologically active.
It is also within the scope that the term "functional variant" refers to a sequence which is substantially similar to another sequence, namely retains all its characteristics although having at least about 50%-99% identity with the other sequence, for example at least 75%, for example at least 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to that particular non-variant other sequence. The term "variety" or "cultivar" or "line" used herein means a group of similar plants that by structural features and performance can be identified from other varieties within the same species.
The term "allele" used herein means any of one or more alternative or variant or mutant forms of a gene or a genetic unit at a particular locus, all of which alleles relate to one trait or characteristic at a specific locus. In a diploid cell of an organism, alleles of a given gene are located at a specific location, or locus (loci plural) on a chromosome. Alternative or variant forms of alleles may be the result of at least one of single nucleotide polymorphisms, indels, insertions, inversions, translocations or deletions, or the consequence of gene regulation caused by, for example, targeted genome editing, chemical or structural modification, transcription regulation or post- translational modi f ication/regulation . An allele associated with a qualitative trait may comprise alternative or variant forms of various genetic units including those that are identical or associated with a single gene or multiple genes or their products or even a gene disrupting or controlled by a genetic factor contributing to the phenotype represented by the locus. According to further embodiments, the term "allele" designates any of one or more alternative forms of a gene at a particular locus. Heterozygous alleles are two different alleles at the same locus. Homozygous alleles are two identical alleles at a particular locus.
Hemizygous is a condition or configuration where one allele is missing .
Nullizygous is a condition where both alleles are missing.
A wild type allele is a naturally occurring allele. In the context of the current invention, the term allele refers to the two identified VuSPl and VuSP2 Cowpea genes, having the genomic nucleotide (wild type) sequence as set forth in SEQ ID NO:1 and SEQ ID NO: 10, respectively.
As used herein the term "genetic modification" refers hereinafter to genetic manipulation or modulation, which is the direct manipulation of an organism's genes using biotechnology. It also refers to a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species, targeted mutagenesis and genome editing technologies to produce improved organisms. According to main embodiments of the present invention, modified Cowpea plants with improved plant architecture traits conferring determinate growth phenotype are generated using genome editing mechanism. This technique enables to achieve in planta modification of specific genes that relate to and/or control the determinate growth habits in Cowpea. The modification of the genes is aimed to result in modulated expression (preferably silencing) of the targeted genes, as compared to control Cowpea plants lacking the generated modification .
The term "genome editing", or "genome/genetic modification" or "genome engineering" or "gene editing" generally refers hereinafter to a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike previous genetic engineering techniques that randomly insert genetic material into a host genome, genome editing targets the insertions or deletions to site specific locations (e.g. specific genomic locus or loci) within a coding or noncoding (regulatory) regions of a gene.
It is within the scope of the present invention that the common methods for such editing use engineered nucleases, or "molecular scissors". These nucleases create site-specific double-strand breaks (DSBs) at desired locations in the genome. The induced double-strand breaks are repaired through nonhomologous endjoining (NHEJ) or homologous recombination (HR) , resulting in targeted mutations ( ’edits' ) . Families of engineered nucleases used by the current invention include, but are not limited to: meganucleases, zinc finger nucleases (ZFNs) , transcription activator-like effector-based nucleases (TALEN) , and the clustered regularly interspaced short palindromic repeats (CRISPR/Cas) system.
Reference is now made to exemplary genome editing terms used by the current disclosure:
Figure imgf000031_0001
According to specific aspects of the present invention, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are used for the first time for generating genome modification targeted to VuSPl and/or VuSP2 genes in Cowpea plants. It is herein acknowledged that the functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in selected bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material. These repeats were initially discovered in the 1980s in E. coll. Without wishing to be bound by theory, reference is now made to a type of CRISPR mechanism, in which invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus comprising a series of short repeats (around 20 bps) . The loci are transcribed, and transcripts are then processed to generate small RNAs (crRNA, namely CRISPR RNA) , which are used to guide effector endonucleases that target invading DNA based on sequence complementarity.
According to further aspects of the invention, Cas protein, such as Cas9 (also known as Csnl) is required for gene silencing. Cas9 participates in the processing of crRNAs and is responsible for the destruction of the target DNA. Cas9's function in both of these steps relies on the presence of two nuclease domains, a RuvC-like nuclease domain located at the amino terminus, and a HNH-like nuclease domain that resides in the mid-region of the protein. To achieve site-specific DNA recognition and cleavage, Cas9 is complexed with both a crRNA and a separate trans-activating crRNA (tracrRNA or trRNA) , that is partially complementary to the crRNA. The tracrRNA is required for crRNA maturation from a primary transcript encoding multiple pre-crRNAs. This occurs in the presence of RNase III and Cas9.
Without wishing to be bound by theory, it is herein acknowledged that during the destruction of target DNA, the HNH and RuvC-like nuclease domains cut both DNA strands, generating double- stranded breaks (DSBs) at sites defined by a 20-nucleotide target sequence within an associated crRNA transcript. The HNH domain cleaves the complementary strand, while the RuvC domain cleaves the noncomplementary strand.
It is further noted that the double-stranded endonuclease activity of Cas9 also requires that a short-conserved sequence, (2-5 nts) known as protospacer-associated motif (PAM) , follows immediately 3'- of the crRNA complementary sequence. According to further aspects of the invention, a two-component system may be used by the current invention, combining trRNA and crRNA into a single synthetic single guide RNA (sgRNA) for guiding targeted gene alterations.
It is further within the scope that Cas9 nuclease variants include wild-type Cas9, Cas9D10A and nuclease-deficient Cas9 (dCas9) .
Reference is now made to an example of CRISPR/Cas9 mechanism of action as depicted by Xie, Kabin, and Yinong Yang. "RNA-guided genome editing in plants using a CRISPR-Cas system. " Molecular plant 6.6 (2013) : 1975-1983, incorporated herein by reference. As shown in this scientific publication, the Cas9 endonuclease forms a complex with a chimeric RNA (called guide RNA or gRNA) , replacing the crRNA- trans crRNA heteroduplex, and the gRNA could be programmed to target specific sites. The gRNA-Cas9 should comprise at least 15-base-pairing (gRNA seed region) without mismatch between the 5 '-end of engineered gRNA and targeted genomic site, and an NGG motif (called protospacer-adjacent motif or PAM) that follows the base-pairing region in the complementary strand of the targeted DNA.
The term "meganucleases" as used herein refers hereinafter to endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs) ; as a result this site generally occurs only once in any given genome. Meganucleases are therefore considered to be the most specific naturally occurring restriction enzymes.
The term "protospacsr adjacent motif" or "PAM” as used herein refers hereinafter to a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Gas 9 nuclease in the CRISPR bacterial adaptive immune system. PAM is a component of the invading virus or plasmid but is not a component of the bacterial CRISPR locus. PAM is an essential targeting component which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by nuclease .
The term "Next-generation sequencing" or "NGS" as used herein refers hereinafter to massively, parallel, high- throughput or deep sequencing technology platforms that perform sequencing of millions of small fragments of DNA in parallel. Bioinformatics analyses are used to piece together these fragments by mapping the individual reads to the reference genome.
The term "gene knockdown" as used herein refers hereinafter to an experimental technique by which the expression of one or more of an organism's genes is reduced and/or it's function is significantly reduced or eliminated. The reduction can occur through genetic modification, i.e. targeted genome editing or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript. The reduced expression can be at the level of RNA and/or at the level of protein. It is within the scope of the present invention that the term gene knockdown also refers to a loss of function mutation, gene knockout or silencing mutation in which an organism's gene is made inoperative or nonfunctional.
The term "gene silencing" as used herein refers hereinafter to the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation. In certain aspects of the invention, gene silencing is considered to have a similar meaning as gene knockdown. When genes are silenced, their expression is reduced. In contrast, when genes are knocked out, they are completely not expressed. Gene silencing may be considered a gene knockdown mechanism since the methods used to silence genes, such as RNAi, CRISPR, or siRNA, generally reduce the expression of a gene by at least 70% but do not completely eliminate it.
The term "loss of function mutation" as used herein refers to a type of mutation in which the altered gene product lacks the function of the wild-type gene. A synonym of the term included within the scope of the present invention is null mutation.
The term "in planta" means in the context of the present invention within the plant or plant cells. More specifically, it means introducing CRISPR/Cas complex into plant material comprising a tissue culture of several cells, a whole plant, or into a single plant cell, without introducing a foreign gene or a mutated gene into its genome. It is also used to describe conditions present in a non-laboratory environment (e.g. in vivo) .
As used herein, the term "locus" (loci plural) means a specific place (s) or region (s) or a site (s) on a chromosome where for example a gene or genetic marker element or factor is found. In specific embodiments, such a genetic element is contributing to a trait .
As used herein, the term "homozygous" refers to a genetic condition or configuration existing when two identical or like alleles reside at a specific locus but are positioned individually on corresponding pairs of homologous chromosomes in the cell of a diploid organism.
In specific embodiments, the Cowpea plants of the present invention comprise homozygous configuration of at least one of the mutated Cowpea VuSPl and/or VuSP2 genes.
Conversely, as used herein, the term "heterozygous" means a genetic condition or configuration existing when two different or unlike alleles reside at a specific locus but are positioned individually on corresponding pairs of homologous chromosomes in the cell of a diploid organism.
As used herein, the term "hemizygous" refers to a genetic condition or configuration existing in a diploid cell in which there is only one copy of a gene instead of the typical two copies (i.e. , the gene has no counterpart on a homologous chromosome) . As an illustration, a heterologous transgene can be present in a hemizygous state.
In specific embodiments, the Cowpea plants of the present invention comprise hemizygous configuration of at least one of the mutated Cowpea SP genes.
As used herein, the phrase "genetic marker" or "molecular marker" or "biomarker" refers to a feature in an individual's genome e.g. , a nucleotide or a polynucleotide sequence that is associated with one or more loci or trait of interest. In some embodiments, a genetic marker is polymorphic in a population of interest, or the locus occupied by the polymorphism, depending on context. Genetic markers or molecular markers include, for example, single nucleotide polymorphisms (SNPs) , indels (i.e. insertions deletions) , simple sequence repeats (SSRs) , restriction fragment length polymorphisms (RFLPs) , random amplified polymorphic DNAs (RAFDs) , cleaved amplified polymorphic sequence (CAPS) markers, Diversity Arrays Technology (DArT) markers, and amplified fragment length polymorphisms (AFLPs) or combinations thereof, among many other examples such as the DNA sequence per se. Genetic markers can, for example, be used to locate genetic loci containing alleles on a chromosome that contribute to variability of phenotypic traits. The phrase "genetic marker" or "molecular marker" or "biomarker" can also refer to a polynucleotide sequence complementary or corresponding to a genomic sequence, such as a sequence of a nucleic acid used as a probe or primer. As used herein, the term "germplasm" refers to the totality of the genotypes of a population or other group of individuals (e.g. , a species) . The term "germplasm" can also refer to plant material; e.g. , a group of plants that act as a repository for various alleles. Such germplasm genotypes or populations include plant materials of proven genetic superiority; e.g. , for a given environment or geographical area, and plant materials of unknown or unproven genetic value; that are not part of an established breeding population and that do not have a known relationship to a member of the established breeding population.
The terms "hybrid", "hybrid plant" and "hybrid progeny" used herein refers to an individual produced from genetically different parents (e.g. , a genetically heterozygous, hemizygous or mostly heterozygous individual) .
As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. , charge or hydrophobicity) and therefore do not change the functional properties of the molecule. The term further refers hereinafter to the amount of characters which match exactly between two different sequences. Hereby, gaps are not counted, and the measurement is relational to the shorter of the two sequences.
It is further within the scope that the terms "similarity" and "identity" additionally refer to local homology, identifying domains that are homologous or similar (in nucleotide and/or amino acid sequence) . It is acknowledged that bioinformatics tools such as BLAST, SSEARCH, FASTA, and HMMER calculate local sequence alignments which identify the most similar region between two sequences. For domains that are found in different sequence contexts in different proteins, the alignment should be limited to the homologous domain, since the domain homology provides the sequence similarity captured in the score. According to some aspects the term similarity or identity further includes a sequence motif, which is a nucleotide or amino-acid sequence pattern that is widespread and has, or is conjectured to have, a biological significance. Proteins may have a sequence motif and/or a structural motif, a motif formed by the three- dimensional arrangement of amino acids which may not be adjacent.
As used herein, the terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g. , cDNA or genomic DNA) , RNA molecules (e.g. , mRNA) , natural occurring, mutated, synthetic DNA or RNA molecules, and analogs of the DNA or RNA generated using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term "gene", "allele" or "gene sequence" is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence or may comprise only a coding sequence as in cDNAs, in their gene expressed form, and/or may include cDNAs in combination with regulatory sequences. Thus, according to the various aspects of the invention, genomic DNA, cDNA or coding DNA may be used. In one embodiment, the nucleic acid is cDNA or coding DNA. The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds .
According to other aspects of the invention, a "modified" or a "mutant" or "mutated" plant is a plant that has been altered compared to the naturally occurring wild type (WT) or control plant. Specifically, the endogenous nucleic acid sequences of the two VuSPl and VuSP2 homologs in Cowpea (nucleic acid sequences comprising at least 75% sequence identity to SEQ ID NO:1 and SEQ ID NO: 10, respectively) have been altered compared to wild type sequences using targeted genome editing methods as described herein. This causes inactivation of the endogenous VuSPl and/or VuSP2 genes and thus disables VuSPl and/or VuSP2 function and/or expression. Such Cowpea plants have an altered phenotype and show improved plant architecture traits conferring determinate growth habit characteristics compared to corresponding wild type plants or control plants lacking the VuSPl and/or VuSP2 modification and showing indeterminate growth. Therefore, the determinate growth habit phenotype is conferred by the presence of at least one mutated (loss of function) endogenous Cowpea VuSPl and/or VuSP2 gene in the Cowpea plant genome which has been specifically targeted using genome editing technique.
According to further aspects of the present invention, the at least one improved plant architecture trait conferring determinate growth phenotype is not conferred by the presence of transgenes expressed in Cowpea plants.
It is further within the scope of the current invention that VuSPl and/or VuSP2 mutations that down-regulate or disrupt functional expression of the wild-type VuSPl and/or VuSP2 gene sequences, may be recessive, such that they are complemented by expression of a wild-type sequence. It is further noted that according to certain aspects of the present invention, a wild-type Cowpea plant is a plant that does not have any mutant sp allele.
Main aspects of the invention involve targeted mutagenesis methods, specifically genome editing, and exclude embodiments that are solely based on generating plants by traditional breeding methods. In a further embodiment of the current invention, as explained herein, the determinate growth habit phenotype is not due to the presence of a transgene.
The inventors have generated mutant Cowpea lines with mutations inactivating the Cowpea VuSPl and/or VuSP2 gene homologous alleles which confer determinate growth habit characteristics. In this way, at least one of the VuSPl and/or VuSP2 proteins is not functional. Thus, the invention relates to these mutant Cowpea lines and related methods.
It is further within the scope of the present invention that breeding Cowpea lines with mutated (loss of function) VuSPl and/or VuSP2 allele enables the mechanical harvest of the plant, due to the determinant growth habit of the plant. According to a further aspect of the present invention, loss of VuSPl and/or VuSP2 function results in compact Cowpea plants with determinate growth phenotype when compared with corresponding WT Cowpea plants. More specifically, the generated plants with determinate growth habit characteristics present reduced height, reduced number of sympodial units, compact growth habit, distinct short stature, exhausted apical meristem, bushy growth habit, synchronized and uniform flowering, and synchronized and uniform pod formation.
According to a main aspect of the present invention, modifying Cowpea shoot architecture by selection for mutations in VuSPl and/or VuSP2 genes allowed major improvements in plant architecture and yield. In particular, a mutation in the VuSPl and/or VuSP2 genes provided compact 'determinate' growth that translated to a burst of flowers, thereby enabling largescale field production .
The work inter alia described has important implications. The results have shown that CRISPR/Cas9 can be used to create heritable mutations in Cowpea homologs of the florigen pathway family members that result in desirable phenotypic effects.
It is further within the scope of the current invention that Cowpea VuSPl and VuSP2 genes having genomic nucleotide sequence as set forth in SEQ ID NO:1 and SEQ ID NO: 10, respectively, are silenced by genome editing. These two mutated alleles have been identified. Notably, the plants with these two genome-edited mutated sp alleles presented determinate growth habits and, consequently, were more compact than the wild type plants lacking these mutated alleles and showing indeterminate growth.
The loss of function mutation may be a deletion or insertion ("indels") with reference to the wild-type CowpeaVuSPl and/or VuSP2 gene allele sequences (genomic nucleotide sequence as set forth in SEQ ID NO:1 and SEQ ID NO: 10, respectively) . The deletion may comprise 1-20 or more nucleotides (e.g. up to 160 bp) , for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18 or 20 nucleotides or more in one or more strand. The insertion may comprise 1-20 or more nucleotides, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18 or 20 or more nucleotides in one or more strand.
The plant of the invention includes plants wherein the plant is heterozygous for each of the mutations. In a preferred embodiment however, the plant is homozygous for each of the mutations. Progeny that is also homozygous can be generated from these plants according to methods known in the art. In another embodiment however, the plant is hemizygous for each of the mutations.
It is further within the scope that variants of Cowpea VuSPl and/or VuSP2 genes nucleotide or amino acid encoded sequence according to the various aspects of the invention will have at least about 50%- 99%, for example at least 75%, for example at least 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to that particular non-variant Cowpea VuSPl and/or VuSP2 gene nucleotide sequence.
Also, the various aspects of the invention encompass not only Cowpea VuSPl and/or VuSP2 gene nucleic acid sequence or amino acid sequence, but also fragments thereof. By "fragment" it is intended to mean a portion of the nucleotide sequence or a portion of the amino acid sequence and hence of the protein encoded thereby. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein, in this case improved plant architecture trait conferring determinate growth phenotype .
According to a further embodiment of the invention, the herein identified Cowpea SP1 and/or SP2 genes have been targeted using the CRISPR/Cas system.
According to further embodiments of the present invention, DNA introduction into the plant cells can be done by Agrobacterium infiltration, virus-based plasmids for delivery of the genome editing molecules and mechanical insertion of DNA (PEG mediated DNA transformation, biolistics, etc. ) .
In addition, it is within the scope of the present invention that the Cas9 protein is directly inserted together with a gRNA (ribonucleoprotein- RNP's) in order to bypass the need for in vivo transcription and translation of the Cas9+gRNA plasmid in planta to achieve gene editing.
It is within the scope of the present invention that the usage of CRISPR/Cas system for the generation of Cowpea plants with determinate growth habit phenotype, allows the modification of predetermined specific DNA sequences without introducing foreign DNA into the genome by GMO techniques. According to one embodiment of the present invention, this is achieved by combining the Cas nuclease (e.g. Cas9, Cpfl and the like) with a predefined guide RNA molecule (gRNA) . The gRNA is complementary to a specific DNA sequence targeted for editing in the plant genome and which guides the Cas nuclease to a specific nucleotide sequence. The predefined gene specific gRNA' s are cloned into the same plasmid as the Cas gene and this plasmid is inserted into plant cells. Insertion of the aforementioned plasmid DNA can be done, but not limited to, using different delivery systems, biological and/or mechanical, e.g. Agrobacterium infiltration, virus based plasmids for delivery of the genome editing molecules and mechanical insertion of DNA (PEG mediated DNA transformation, biolistics, etc. ) .
It is further within the scope of the present invention that upon reaching the specific predetermined DNA sequence, the Cas9 nuclease cleaves both DNA strands to create double stranded breaks leaving blunt ends. This cleavage site is then repaired by the cellular non homologous end joining DNA repair mechanism resulting in insertions or deletions which eventually create a mutation at the cleavage site. For example, it is acknowledged that a deletion form of the mutation consists of at least 1 base pair deletion. As a result of this base pair deletion the gene coding sequence is disrupted, and the translation of the encoded protein is compromised either by a premature stop codon or disruption of a functional or structural property of the protein. Thus, DNA is cut by the Cas9 protein and re-assembled by the cell's DNA repair mechanism.
It is further within the scope that improved architecture traits conferring determinate growth phenotype in Cowpea plants is herein produced by generating gRNA with homology to a specific site of VuSPl and/or VuSP2 genes in the Cowpea genome (in the coding and/or regulatory non-coding regions within VuSPl and/or VuSP2 genomic sequence) , sub cloning this gRNA into a plasmid containing the Cas9 gene, and insertion of the plasmid into the Cowpea plant cells. In this way, site specific mutations in the VuSPl and/or VuSP2 genes are generated thus effectively creating non-active VuSPl and/or VuSP2 molecules, resulting in determinate growth habit of the genome edited plant.
According to one embodiment, the present invention to provides a modified Cowpea (Vigna unguiculata) plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic, wherein the plant comprises mutated SELF PRUNING (SP) VuSPl and/or VuSP2 gene comprising genome edited loss of function mutation.
According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined above, wherein the improved plant architecture trait is relative to a corresponding Cowpea (Vigna unguiculata) plant lacking the genome edited loss of function mutation.
According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutated VuSPl gene comprises a genomic sequence selected from SEQ ID NO: 4-9, or a functional variant thereof, or any combination thereof . According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutated VuSP2 gene comprises a genomic sequence selected from SEQ ID NO: 12-15, or a functional variant thereof, or any combination thereof .
According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the functional variant comprises at least 90% sequence identity to the corresponding mutated VuSPl or mutated VuSP2 sequence.
According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the determinate growth habit characteristic is selected from the group consisting of determinate or semideterminate architecture, early termination of sympodial cycling, compact growth habit, reduced height, distinct short stature, determinate apical meristem growth, reduced or limited apical meristem growth, interrupted or exhausted apical meristem growth, bushy growth habit, synchronized flowering, uniform flowering, synchronized pod formation, uniform pod formation, reduced number of sympodial units, suppressed sympodial shoot termination, lack of central growing axis and tendrils, uniform appearance of flowers, adaptation to mechanical harvest, higher harvest index and any combination thereof.
According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the height of the modified plant is in the range of 60cm to 100cm, such as in the range of 65cm to 90cm.
According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the modified plant height is about 30%-50% lower than a control Cowpea plant lacking the genome edited loss of function mutation in the VuSPl and/or VuSP2 genes.
According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the modified plant has pod number similar or at least 10% higher than a control Cowpea plant lacking the genome edited loss of function mutation in the VuSPl and/or VuSP2 genes.
According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutation in the VuSPl gene is generated using a guide RNA (gRNA) comprising a polynucleotide sequence selected from SEQ ID NO: 2 and SEQ ID NO: 3 or a combination thereof, and the mutation in the VuSP2 gene is generated using a guide RNA (gRNA) sequence comprising a polynucleotide sequence comprising SEQ ID NO: 11.
According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutated VuSPl and/or VuSP2 gene is a CRISPR/Cas- induced heritable mutated allele.
According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the Cas gene is selected from the group consisting of Cas9, Casl2, Casl3, Casl4, CasX, CasY, Csnl, Cpfl and any combination thereof.
According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutation is an insertion, deletion, indel, inversion, substitution, duplication or any combination thereof . According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutation is a silencing mutation, a knockdown mutation, a knockout mutation, a loss of function mutation, downregulating, de-regulating, deactivating, reducing expression, at least partially deleting, at least partially silencing, at least partially deactivating, removing, partially removing, duplicating, inverting, missense mutation, nonsense mutation, or any combination thereof.
According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutation is in the coding sequence or in a regulatory sequence such as a promoter, terminator sequence, of the VuSPl and/or VuSP2 gene.
According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the plant is homozygous, heterozygous or hemizygous for the genome edited loss of function mutation in the VuSPl and/or VuSP2 gene.
According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutation in the VuSPl gene is generated in planta via introduction of a construct comprising (a) Cas DNA and gRNA comprising a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA comprising a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3.
According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutation in the VuSP2 gene is generated in planta via introduction of a construct comprising (a) Cas DNA and gRNA sequence comprising SEQ ID NO: 11, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence comprising SEQ ID NO: 11.
According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the gRNA sequence comprises a 3' NGG Protospacer Adjacent Motif (PAM) .
According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the mutation confers determinate growth habit phenotype .
According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the modified plant flowers earlier than a corresponding control Cowpea plant lacking the mutated VuSPl and/or VuSP2 gene.
According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the improved plant architecture trait is selected from the group consisting of reduced flowering time, earliness, synchronous flowering, reduced day-length sensitivity, determinant growth habit, early termination of sympodial cycling, earlier axillary shoot flowering, compact growth habit, reduced height, reduced number of sympodial units, adaptation to mechanical harvest, higher harvest index and any combination thereof .
According to a further embodiment, the present invention provides the modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the genome editing events located in VuSPl are selected from the group consisting of SEQ ID NOs:4-9 and any combination thereof, and the genome editing events located in VuSP2 are selected from the group consisting of SEQ ID NOs: 12-15 and any combination thereof.
According to a further embodiment, the present invention provides a modified Cowpea plant, plant part, plant pot or plant cell as defined in any of the above, wherein the plant does not comprise a transgene .
According to a further embodiment, the present invention provides a plant part, plant cell, plant progeny, plant pod or plant seed of a modified Cowpea (Vigna unguiculata) plant as defined in any of the above.
According to a further embodiment, the present invention provides a tissue culture of regenerable cells, protoplasts or callus obtained from the modified Cowpea (Vigna unguiculata) plant as defined in any of the above.
According to a further embodiment, the present invention provides harvestable parts of a modified Cowpea (Vigna unguiculata) plant as defined in any of the above, wherein the harvestable parts are preferably shoot biomass and/or pods or seeds.
According to a further embodiment, the present invention provides products derived from the modified Cowpea (Vigna unguiculata) plant as defined in any of the above and/or from harvestable parts of a modified Cowpea (Vigna unguiculata) plant as defined in any of the above.
According to a further embodiment, the present invention provides a method for producing a modified Cowpea (Vigna unguiculata) plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic as defined in any of the above, wherein the method comprises steps of mutating a Vigna unguiculata SELF PRUNING (SP) VuSPl and/or VuSP2 gene by introducing using targeted genome editing a loss of function mutation .
According to a further embodiment, the present invention provides the method as defined above, wherein the method comprises steps of : a. identifying in Cowpea (Vigna unguiculata) plant cells, VuSPl and/or VuSP2 genes comprising a nucleic acid sequence with at least 75% sequence identity to a sequence selected from SEQ ID NO:1 and/or SEQ ID NO: 10, respectively; b. synthetizing at least one guide RNA (gRNA) complementary to the VuSPl and/or VuSP2 genes, comprising a nucleotide sequence selected from SEQ ID NO: 2-3 and/or SEQ ID NO: 11, respectively; c. transforming TO Cowpea (Vigna unguiculata) plant cells with a construct comprising (a) Cas nucleotide sequence operably linked to the at least one gRNA, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and the at least one gRNA; d. sei f-pollinating the transformed TO Cowpea (Vigna unguiculata) plant and collecting transformed T1 seeds; e. screening the genome of transformed T1 Cowpea (Vigna unguiculata) cells and selecting for CRISPR/Cas negative and VuSPl and/or VuSP2 genome edited positive T1 Cowpea (Vigna unguiculata) cells; f. screening the genome of the selected T1 Cowpea (Vigna unguiculata) cells for induced targeted loss of function mutation in the VuSPl and/or VuSP2 genes; g. regenerating Cowpea plants from the selected transformed T1
Cowpea (Vigna unguiculata) cells for phenotyping and selecting plants exhibiting improved plant architecture trait conferring determinate growth habit characteristic.
According to a further embodiment, the present invention provides the method as defined in any of the above, wherein the construct is introduced into the plant cells via Agrobacterium mediated transformation, virus-based plasmids for delivery of the genome editing molecules or mechanical insertion such as polyethylene glycol (PEG) mediated DNA transformation, electroporation or gene gun biolistics.
According to a further embodiment, the present invention provides the method as defined in any of the above, wherein the edited plants were screened for loss of function mutations using molecular techniques such as PCR and DNA sequencing.
According to a further embodiment, the present invention provides the method as defined in any of the above, wherein the phenotyping analysis comprises evaluating plant height, flowering synchronization, and pod formation parameters.
According to a further embodiment, the present invention provides the method as defined in any of the above, wherein the step of screening the genome of the transformed plant cells for induced targeted loss of function mutation further comprises steps of obtaining a nucleic acid sample of the transformed plant and performing a nucleic acid amplification and optionally restriction enzyme digestion to detect a mutation in the VuSPl and/or VuSP2 gene .
According to a further embodiment, the present invention provides the method as defined in any of the above, wherein the VuSPl allele comprises a polynucleotide sequence selected from SEQ ID NOs:4-9 and any combination thereof, and mutated VuSP2 allele comprises a polynucleotide sequence selected from SEQ ID NOs: 12-15 and any combination thereof.
According to a further embodiment, the present invention provides a modified Cowpea (Vigna unguiculata) plant, plant part, plant seed or pod or plant cell produced by the method as defined in any of the above, wherein the modified plant does not comprise a CRIPPR/Cas related transgene.
According to a further embodiment, the present invention provides a tissue culture of regenerable cells, protoplasts or callus obtained from the modified Cowpea (Vigna unguiculata) plant produced by the method as defined in any of the above.
According to a further embodiment, the present invention provides a method of improving at least one plant architecture trait conferring determinate growth habit characteristic in a Cowpea (Vigna unguiculata) plant, comprising steps of producing using genome editing, a modified Cowpea (Vigna unguiculata) plant as defined in any of the above, seed, pod or plant part thereof, preferably in a method as defined in any of the above, and enabling growth of the modified Cowpea (Vigna unguiculata) plant, seed or plant part thereof.
According to a further embodiment, the present invention provides the method as defined in any of the above, wherein the method comprises steps of: a. identifying at least one Cowpea (Vigna unguiculata) SELF PRUNING (SP) VuSPl and/or VuSP2 gene allele; b. synthetizing at least one guide RNA (gRNA) comprising a polynucleotide sequence complementary to the polynucleotide sequence of the at least one identified VuSPl and/or VuSP2 gene allele ; c. transforming Cowpea (Vigna unguiculata) plant cell with a construct comprising (a) Cas nucleotide sequence operably linked to the at least one gRNA, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and the at least one gRNA; d. screening the genome of the transformed plant cell for induced targeted loss of function mutation in the VuSPl and/or VuSP2 gene to generate a mutated VuSPl and/or VuSP2 allele; e. regenerating Cowpea (Vigna unguiculata) plant carrying the mutated VuSPl and/or VuSP2allele; and f. screening the regenerated plants for a Cowpea (Vigna unguiculata) plant with improved plant architecture trait conferring determinate growth habit characteristic.
According to a further embodiment, the present invention provides the method as defined in any of the above, wherein the guide RNA (gRNA) complementary to the VuSPl and/or VuSP2 genes, comprising a nucleotide sequence selected from SEQ ID NO: 2-3 and/or SEQ ID NO: 11, respectively.
According to a further embodiment, the present invention provides the method as defined in any of the above, wherein the VuSPl mutated allele comprises a polynucleotide sequence selected from SEQ ID NOs:4-9 and any combination thereof, and the mutated VuSP2 allele comprises a polynucleotide sequence selected from SEQ ID NOs: 12-15 and any combination thereof.
According to a further embodiment, the present invention provides a method for identifying and/or selecting for a Cowpea (Vigna unguiculata) plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic, the method comprises steps of: a. screening the genome of the Cowpea (Vigna unguiculata) plant for a mutated VuSPl and/or VuSP2 allele, the wherein the VuSPl mutated allele comprises a polynucleotide sequence selected from SEQ ID NOs:4-9 and any combination thereof, and the mutated VuSP2 allele comprises a polynucleotide sequence selected from SEQ ID NOs: 12-15 and any combination thereof; b. optionally, regenerating a modified Cowpea (Vigna unguiculata) plant carrying the genetic modification; and c. optionally, screening the regenerated plants for a plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic.
According to a further embodiment, the present invention provides the method as defined in any of the above, wherein the genomic modification is a loss of function mutation.
According to a further embodiment, the present invention provides the method as defined in any of the above, wherein the determinate growth habit characteristic is selected from the group consisting of determinate or semi-determinate architecture, early termination of sympodial cycling, compact growth habit, reduced height, distinct short stature, determinate apical meristem growth, reduced or limited apical meristem growth, interrupted or exhausted apical meristem growth, bushy growth habit, synchronized flowering, uniform flowering, synchronized pod formation, uniform pod formation, reduced number of sympodial units, suppressed sympodial shoot termination, lack of central growing axis and tendrils, uniform appearance of flowers, adaptation to mechanical harvest, higher harvest index and any combination thereof .
According to a further embodiment, the present invention provides a method of determining the presence of a mutated VuSPl and/or VuSP2 allele in a Cowpea (Vigna unguiculata) plant, conferring determinate growth habit characteristic, wherein the method comprising assaying the genome of the Cowpea plant for the presence of a polynucleotide sequence selected from SEQ ID NOs:4-9 and any combination thereof, and SEQ ID NOs: 12-15 and any combination thereof, respectively.
According to a further embodiment, the present invention provides an isolated polynucleotide sequence having at least 80% sequence similarity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 4-9 and any combination thereof, and SEQ ID NOs: 12-15 and any combination thereof .
According to a further embodiment, the present invention provides use of (a) a nucleic acid sequence as set forth in SEQ ID NO: 1 and/or SEQ ID NO: 10 and/or (b) a nucleotide sequence as set forth in SEQ ID NOs: 2-3 and/or SEQ ID NO: 11 and any combination thereof for targeted genome modification of Cowpea (Vigna unguiculata) , for generating and/or producing a modified Cowpea (Vigna unguiculata) plant with at least one genetically modified VuSPl and/or VuSP2 genes involved in determinate growth habits.
According to a further embodiment, the present invention provides use of a nucleotide sequence having at least 80% sequence similarity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 4-9 and any combination thereof, and SEQ ID NOs: 12-15 and any combination thereof, or a complementary sequence thereof, for generating, identifying and/or screening for a modified Cowpea (Vigna unguiculata) plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic .
According to a further embodiment, the present invention provides the use as defined in any of the above, wherein the presence of at least one polynucleotide sequence selected from SEQ ID NOs: 4-9 and any combination thereof, or a complementary sequence thereof indicates that the Cowpea plant comprises a VuSPl allele comprising a loss of function mutation, and the presence of at least one polynucleotide sequence selected from at least one polynucleotide sequence selected from SEQ ID NOs: 12-15 and any combination thereof, or a complementary sequence thereof indicates that the Cowpea plant comprises a VuSP2 allele comprising a loss of function mutation .
According to a further embodiment, the present invention provides a detection kit for determining the presence or absence of a mutated VuSPl and/or VuSP2 gene conferring determinate growth habit characteristic in a Cowpea (Vigna unguiculata) plant, comprising a polynucleotide fragment comprising a polynucleotide sequence having at least 80% similarity to a sequence selected from SEQ ID NOs: 4-9, any combination thereof, or a complementary sequence thereof, and /or at least 80% similarity to a sequence selected from SEQ ID NOs: 12-15 any combination thereof, or a complementary sequence thereof, respectively.
In order to understand the invention and to see how it may be implemented in practice, a plurality of preferred embodiments will now be described, by way of non-limiting example only, with reference to the following examples.
EXAMPLE 1
Using genome editing to generate determinate Cowpea plants
The production of Cowpea lines with mutated VuSPl and/or VuSP2 genes was achieved by the following breeding/cultivation method. It should be noted that other breeding/cultivation methods known in the art can be executed.
In this study, the CRISPR/Cas9 system was employed to target and modify Cowpea (Vigna unguiculata) homologs of SELF PRUNING (SP) , namely VuSPl and/or VuSP2. Constructs containing the CRISPR/Cas9 machinery and specific guide RNA (gRNA) targeting the Cowpea SP homologs were designed and introduced into Cowpea tissue using Agrobacterium-mediated transformation. The edited plants were screened for desired mutations using molecular techniques known in the art, such as PCR and DNA sequencing. Phenotypic analysis was performed to evaluate plant height, flowering synchronization, and pod formation.
As presented below, successful genome editing of the two homologs of Cowpea SP genes was demonstrated, resulting in the development of determinate Cowpea varieties/lines . Several editing events were identified, resulting in small deletions/insertions of a few base pairs and/or up to 160 base pairs deletions. The examined editing events led to the same determinate phenotype. The edited plants exhibited a distinct short stature and bushy growth habit, which is in contrast to the unedited control indeterminate Cowpea plants. Furthermore, flowering and pod formation were observed to be synchronized and uniform across the edited plants.
Reference is now made to Table 1, presenting the wild-type Cowpea's genomic sequences related to VuSPl and VuSP2. The wild-type genomic sequence for VuSPl (SEQ ID NO:1) is represented on Fig. 1, and wild-type genomic sequence for VuSP2 (SEQ ID NO: 10) is represented on Fig. 2.
Table 1: Cowpea (Vigna unguiculata) VuSPl and VuSP2 genomic sequences .
Figure imgf000057_0001
To perform the editing events, two gRNA molecules (sense and antisense) were applied for VuSPl gene, and one gRNA molecule (sense) for the VuSP2 gene (see Table 2) . Reference is now made to Figs. 3A-B schematically presenting the locations of the gRNA molecules on the respective VuSPl (Fig.3A) and VuSP2 (Fig. 3B) genes. In these figures the scale bar represents 100 bp, the white box represents UTR (untranslated region) , the black box represents an exon, and the black line represents an intron. The chromosome locations and the gRNA target sequences are shown in Table 2 below. In this table, the PAM sequences (NGG) are presented in bold and underlined. The sequences of gRNAl (SEQ ID NO: 2) and gRNA2 (SEQ ID NO: 3) are underlined in Fig. 1, and the sequence of gRNA3 (SEQ ID NO: 11) is underlined in Fig. 2.
Table 2. gRNAs for VuSPl and VuSP2.
Figure imgf000058_0001
After identifying, sequencing, and mapping the two SP orthologues in Cowpea, VuSPl (SEQ ID NO:1) and VuSP2 (SEQ ID NO: 10) , the corresponding gRNA molecules were designed and synthesized as shown in Table 2 above. These gRNA molecules were then cloned into suitable vectors, and their sequences were verified. Optionally, the efficiency of the designed gRNA molecules was validated by transiently transforming Cowpea tissue culture as described hereinafter .
Reference is now made to non-limiting steps that have been used for the production of the mutated SP Cowpea plants by genome editing : Cowpea seedlings were transformed with a binary vector containing the CRISPR machinery (Cas9 and gRNA) alongside an eYGFPuv reporter and an antibiotic resistance gene.
Successfully regenerated plants exhibiting GFP fluorescence that were able to grow and survive on a selective media containing the antibiotic were sampled. DNA was extracted followed by amplification of the gRNA flanking region through PCR. PCR products were then sequenced using NGS technology, and editing events were characterized. Plants that were successfully edited were transferred to soil and grown to maturity.
In the next steps, seeds were collected from the TO plants. The genotyping procedure was then uniformly applied to all T1 plants to confirm the transmission of the edits to the succeeding generation .
T2 individuals underwent phenotyping in the greenhouse, revealing a consistent and reproducible determinate phenotype among all plants, unaffected by the specific genome edits they harbored.
Results
As described above, plants (TO) were transformed with the CRISPR/Cs9 system. TO plants were sei f -pollinated and set seeds. All T1 plants were selected to be CRISPR/Cas9 negative, and genome edited positive. It is emphasized that only non-transgenic, edited plants were further used in the experiment. This is exemplified in Figs. 4A-B. In this exemplified experiment, DNA was extracted from T1 seeds and a PCR using VuSPl as well as a PCR using Cas9 primers was performed. In Fig. 4A, it is shown that amplicons (442 bp) of the VuSPl PCR are present in all samples. This PCR serves as a positive control indicating genomic DNA was extracted successfully and samples are usable for the downstream applications. In Fig. 4B a PCR using Cas9 primers was performed. It can be seen that Cas9 DNA was detected in samples 1, 2, 5, 6, 8, 10, 14, 15 & 16. These plants were not used in the greenhouse experiment. In the remaining plants, the SP1 and SP2 genes were sequenced, and only edited plants have been selected for phenotypic analysis in the greenhouse .
Thus, all plants used in the phenotypic experiment were T1 transgene-null segregants. No foreign DNA is integrated into the plants of this experiment.
In the next step, T1 edits have been determined by sequencing (prior to planting) . T1 non transgenic, but edited plants have been grown and compared to the WT non edited control. Plant architecture, flowering time, flowering synchronization, and yield properties have been compared.
Reference is now made to Table 3 summarizing genomic sequences of mutated VuSPl fragments, from the first generation of mutated Cowpea plants (Tl) , containing genome editing events obtained using the first guide RNA (gRNAl) having nucleic acid sequence as set forth in SEQ ID NO: 2 and the second guide RNA ( gRNA2 ) having nucleic acid sequence as set forth in SEQ ID NO: 3.
Table 3. VuSPl gene editing events obtained using gRNA sequences having a nucleic acid sequence as set forth in SEQ ID NO: 2 and SEQ
ID NO: 3.
Figure imgf000060_0001
Figure imgf000061_0001
Reference is now made to Figs. 5A-F, presenting the nucleic acid sequences of VuSPl gene editing events as set forth in SEQ ID NOs:4-9, respectively.
Fig. 6 presents the alignment of nucleic acid sequences from the WT VuSPl (SEQ ID NO:1) and the mutated VuSPl fragments containing genome editing events (SEQ ID NOs:4-9) , shown on Table 3. The alignment was performed with CLUSTAL multiple sequence alignment by Kalign.
Reference is now made to Table 4 summarizing genomic sequences of mutated VuSP2 fragments, containing genome editing events obtained using the third guide RNA (gRNA3) having nucleic acid sequence as set forth in SEQ ID NO: 11.
Table 4. VuSP2 gene editing events obtained using gRNA having a nucleic acid sequence as set forth in SEQ ID NO: 11.
Figure imgf000061_0002
Reference is now made to Figs. 7A-F, presenting the nucleic acid sequences of VuSP2 gene editing events as set forth in SEQ ID NOs:12-15, respectively.
Fig. 8 presents the alignment of nucleic acid sequences from the WT VuSP12 (SEQ ID NO: 10) and the mutated VuSPl fragments containing genome editing events (SEQ ID NOs: 12-15) , shown on Table 4. The alignment was performed with CLUSTAL multiple sequence alignment by Kalign.
It should be noted that the SP2_pl editing event on VuSP2, has an insertion ( + 1) on position 41 of SEQ ID NO: 10, and an inversion from TO to AA. This insertion (A) and inversion (TO to AA) on SP2_pl appears in italic in Fig. 8.
It was found that all the VuSP2 constructs containing editing events of SEQ ID NO: 12-15 were associated with the determinate growth habit phenotype as described herein (as shown in Figs 9- 14) . The edited plants exhibited a distinct short stature and bushy growth habit, which contrasts with the unedited control indeterminate Cowpea plants. Furthermore, flowering and pod formation were observed to be synchronized and uniform across the edited plants.
Reference is now made to Table 5 summarizing mutated nucleic acid sequences of the second generation (T2) Cowpea plants comprising a combination of mutated VuSPl allele (Table 3 and Figs. 5A-F) and mutated VuSP2 allele (Table 4 and Figs.7A-D) from the first generation (Tl) .
Table 5. Cowpea mutants containing combinations of VuSPl editing events and VuSP2 editing events.
Figure imgf000062_0001
Figure imgf000063_0001
As shown above, several editing events were identified in VuSPl and in VuSP2 genes. It was found that all editing events led to the same determinate growth habit phenotype. The edited plants exhibited a distinct short stature and bushy growth habit, which contrasts with the unedited control indeterminate Cowpea plants. Furthermore, flowering and pod formation were observed to be synchronized and uniform across the edited plants.
Reference is now made to Fig. 9, photographically presenting a phenotypic comparison of exemplified edited (doubled-edited in VuSPl & VuSP2 or edited in VuSP2 gene) versus wild-type Cowpea plants. The mutated edited plant (on the right) has a determinate growth habit phenotype, lacks central growing axis and tendrils, with uniform appearance of flowers. The mutated edited plant displays a shorter stature and bushy growth habit. In contrast, the wild-type control plant (left) has an indeterminate phenotype, characterized by long and viny plant architecture, with many tendrils .
The determinant growth habit phenotype resulting from the herein disclosed editing events combinations in VuSPl & VuSP2 as exemplified in Table 5, is further confirmed and demonstrated in Figs. 10-13, as follows: Reference is now made to Figs. 10A-C, Figs. 11A-C, Figs. 12A-C, and Figs. 13A-C, presenting Cowpea plants that undergone through edit events 3, 8, 12, and 14 (see Table 5) , respectively, having determinate growth habit characteristics.
The effect of editing events in VuSPl & VuSP2 as exemplified in Tables 3- 5 on plant height is demonstrated in Fig. 14.
Reference is now made to Fig. 14, graphically presenting plant height in cm of edited Cowpea lines (edit events 3, 12, 14 and 8, see Table 5) , measured during day 49 (dark grey) , day 53 (light grey) and day 67 (grey) , compared to the wild-type Cowpea. It can be seen that the edited Cowpea plants presented a general height range from approximately 62 to 98 cm, while the wild-type Cowpea presented a height range from approximately 120 cm to 150 cm. More specifically, the height range varied from approximately 65 cm to 98 cm after 67 days, which is significantly lower than the wildtype Cowpea height after the same period of time.
It is noted that the same determinant growth-habit phenotype characteristics disclosed herein (Figs 9-13) , including reduced plant height (Fig. 14) , were shown to be associated with any of the editing events constructs of VuSP2 described in Table 4, as well. Thus, each of the editing events of VuSP2 gene (presented in Table 4) , and/or any of the combination of editing events presented in Table 5, were associated with the same determinate growth habit phenotype as described herein.
The results of this study clearly demonstrated successful genome editing of two homologs of Cowpea SP (VuSPl, VuSP2) , resulting in the development of determinate Cowpea lines. The effect on development of determinate Cowpea lines may be a synergistic effect resulting from editing events (to produce loss of function mutation) within the two homologs of Cowpea SP (VuSPl, VuSP2) genes. This is in contrast to the unedited control Cowpea plants showing indeterminate growth. The edited plants exhibited a distinct short stature and bushy growth habit. Furthermore, flowering and pod formation were observed to be synchronized and uniform across the edited plants.
Conclusions
It can be concluded that genome editing, specifically utilizing the CRISPR/Cas9 system, enabled successful modification of the Cowpea homolog of SELF PRUNING (SP) , resulting in the development of determinate Cowpea plants. The edited plants exhibited a compact growth habit, which facilitates mechanical harvesting, overcoming the labor limitations faced in traditional indeterminate Cowpea cultivation. Additionally, the synchronized and uniform flowering and pod formation in the edited plants offer advantages in terms of crop management and yield prediction. The findings of this study demonstrate the potential of genome editing technologies to enhance crop productivity and to address challenges associated with labor scarcity in agriculture, and specifically in Cowpea.
References :
Xie Kabin, and Yinong Yang. "RNA-guided genome editing in plants using a CRISPR-Cas system" Molecular plant 6.6 (2013) : 1975-1983.
Eshed, Yuval, and Zachary B. Lippman. "Revolutions in agriculture chart a course for targeted breeding of old and new crops . " Science 366.6466 eaax0025 (2019) : 1-7.
Che, Ping, et al. "Developing a rapid and highly efficient cowpea regeneration, transformation and genome editing system using embryonic axis explants." The Plant Journal 106.3 (2021) : 817-830.

Claims

1. A modified Cowpea (Vigna unguiculata) plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic, wherein said plant comprises mutated SELF PRUNING (SP) VuSPl and/or VuSP2 gene comprising genome edited loss of function mutation.
2. The modified Cowpea (Vigna unguiculata) plant according to claim 1, wherein said improved plant architecture trait is relative to a corresponding Cowpea (Vigna unguiculata) plant lacking the genome edited loss of function mutation.
3. The modified Cowpea (Vigna unguiculata) plant according to any one of claims 1 and 2, wherein said mutated VuSPl gene comprises a genomic sequence selected from SEQ ID NO: 4-9, or a functional variant thereof, or any combination thereof.
4. The modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-3, wherein said mutated VuSP2 gene comprises a genomic sequence selected from SEQ ID NO: 12-15, or a functional variant thereof, or any combination thereof .
5. The modified Cowpea (Vigna unguiculata) plant according to any one of claims 3-4, wherein said functional variant comprises at least 90% sequence identity to the corresponding mutated VuSPl or mutated VuSP2 sequence.
6. The modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-5, wherein said determinate growth habit characteristic is selected from the group consisting of determinate or semi-determinate architecture, early termination of sympodial cycling, compact growth habit, reduced height, distinct short stature, determinate apical meristem growth, reduced or limited apical meristem growth, interrupted or exhausted apical meristem growth, bushy growth habit, synchronized flowering, uniform flowering, synchronized pod formation, uniform pod formation, reduced number of sympodial units, suppressed sympodial shoot termination, lack of central growing axis and tendrils, uniform appearance of flowers, adaptation to mechanical harvest, higher harvest index and any combination thereof.
7. The modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-6, wherein the height of said modified plant is in the range of 60cm to 100cm, such as in the range of 65cm to 90cm.
8. The modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-7, wherein said modified plant height is about 30%-50% lower than a control Cowpea plant lacking the genome edited loss of function mutation in said VuSPl and/or VuSP2 genes .
9. The modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-8, wherein said modified plant has pod number similar or at least 10% higher than a control Cowpea plant lacking the genome edited loss of function mutation in said VuSPl and/or VuSP2 genes.
10. The modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-9, wherein said mutation in said VuSPl gene is generated using a guide RNA (gRNA) comprising a polynucleotide sequence selected from SEQ ID NO: 2 and SEQ ID NO: 3 or a combination thereof, and said mutation in said VuSP2 gene is generated using a guide RNA (gRNA) sequence comprising a polynucleotide sequence comprising SEQ ID NO: 11.
11. The modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-10, wherein the mutated VuSPl and/or VuSP2 gene is a CRISPR/Cas- induced heritable mutated allele.
12. The modified Cowpea (Vigna unguiculata) plant according to claim 11, wherein said Cas gene is selected from the group consisting of Cas9, Casl2, Casl3, Casl4, CasX, CasY, Csnl, Cpfl and any combination thereof.
13. The modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-12, wherein said mutation is an insertion, deletion, indel, inversion, substitution, duplication or any combination thereof.
14. The modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-13, wherein said mutation is a silencing mutation, a knockdown mutation, a knockout mutation, a loss of function mutation, downregulating, de-regulating, deactivating, reducing expression, at least partially deleting, at least partially silencing, at least partially deactivating, removing, partially removing, duplicating, inverting, missense mutation, nonsense mutation, or any combination thereof.
15. The modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-14, wherein said mutation is in the coding sequence or in a regulatory sequence such as a promoter, terminator sequence, of the VuSPl and/or VuSP2 gene.
16. The modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-15, wherein said plant is homozygous, heterozygous or hemizygous for said genome edited loss of function mutation in said VuSPl and/or VuSP2 gene.
17. The modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-16, wherein said mutation in said VuSPl gene is generated in planta via introduction of a construct comprising (a) Cas DNA and gRNA comprising a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA comprising a sequence selected from the group consisting of SEQ ID NO : 2 and SEQ ID NO:3.
18. The modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-17, wherein said mutation in said VuSP2 gene is generated in planta via introduction of a construct comprising (a) Cas DNA and gRNA sequence comprising SEQ ID NO: 11, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence comprising SEQ ID NO: 11.
19. The modified Cowpea (Vigna unguiculata) plant according to any one of claims 10, 17 and 18, wherein said gRNA sequence comprises a 3' NGG Protospacer Adjacent Motif (PAM) .
20. The modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-19, wherein said mutation confers determinate growth habit phenotype.
21. The modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-20, wherein said modified plant flowers earlier than a corresponding control Cowpea plant lacking said mutated VuSPl and/or VuSP2 gene.
22. The modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-21, wherein said improved plant architecture trait is selected from the group consisting of reduced flowering time, earliness, synchronous flowering, reduced day-length sensitivity, determinant growth habit, early termination of sympodial cycling, earlier axillary shoot flowering, compact growth habit, reduced height, reduced number of sympodial units, adaptation to mechanical harvest, higher harvest index and any combination thereof.
23. The modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-22, wherein the genome editing events located in VuSPl are selected from the group consisting of SEQ ID NOs:4-9 and any combination thereof, and the genome editing events located in VuSP2 are selected from the group consisting of SEQ ID NOs: 12-15 and any combination thereof.
24. A modified Cowpea plant, plant part, plant pot or plant cell according to any one of claims 1-23, wherein said plant does not comprise a transgene.
25. A plant part, plant cell, plant progeny, plant pod or plant seed of a modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-24.
26. A tissue culture of regenerable cells, protoplasts or callus obtained from the modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-24.
27. Harvestable parts of a modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-24, wherein said harvestable parts are preferably shoot biomass and/or pods or seeds .
28. Products derived from the modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-24 and/or from harvestable parts of a modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-24.
29. A method for producing a modified Cowpea (Vigna unguiculata) plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic according to any one of claims 1-24, wherein said method comprises steps of mutating a Vigna unguiculata SELF PRUNING (SP) VuSPl and/or VuSP2 gene by introducing using targeted genome editing a loss of function mutation.
30. The method according to claim 29, wherein said method comprises steps of : a. identifying in Cowpea (Vigna unguiculata) plant cells, VuSPl and/or VuSP2 genes comprising a nucleic acid sequence with at least 75% sequence identity to a sequence selected from SEQ ID NO:1 and/or SEQ ID NO: 10, respectively; b. synthetizing at least one guide RNA (gRNA) complementary to said VuSPl and/or VuSP2 genes, comprising a nucleotide sequence selected from SEQ ID NO:2-3 and/or SEQ ID NO: 11, respectively; c. transforming TO Cowpea (Vigna unguiculata) plant cells with a construct comprising (a) Cas nucleotide sequence operably linked to said at least one gRNA, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and said at least one gRNA; d. sei f-pollinating said transformed TO Cowpea ( Vigna unguiculata) plant and collecting transformed T1 seeds; e. screening the genome of transformed T1 Cowpea (Vigna unguiculata) cells and selecting for CRISPR/Cas negative and VuSPl and/or VuSP2 genome edited positive T1 Cowpea (Vigna unguiculata) cells; f. screening the genome of said selected T1 Cowpea (Vigna unguiculata) cells for induced targeted loss of function mutation in said VuSPl and/or VuSP2 genes; g. regenerating Cowpea plants from said selected transformed T1 Cowpea (Vigna unguiculata) cells for phenotyping and selecting plants exhibiting improved plant architecture trait conferring determinate growth habit characteristic.
31. The method according to any one of claims 29-30, wherein said construct is introduced into the plant cells via Agrobacterium mediated transformation, virus-based plasmids for delivery of the genome editing molecules or mechanical insertion such as polyethylene glycol (PEG) mediated DNA transformation, electroporation or gene gun biolistics.
32. The method according to any one of claims 29-31, wherein the edited plants were screened for loss of function mutations using molecular techniques such as PCR and DNA sequencing.
33. The method according to any one of claims 29-32, wherein the phenotyping analysis comprises evaluating plant height, flowering synchronization, and pod formation parameters.
34. The method according to any one of claims 29-33, wherein said step of screening the genome of said transformed plant cells for induced targeted loss of function mutation further comprises steps of obtaining a nucleic acid sample of said transformed plant and performing a nucleic acid amplification and optionally restriction enzyme digestion to detect a mutation in said VuSPl and/or VuSP2 gene.
35. The method according to any one of claims 29-34, wherein the VuSPl allele comprises a polynucleotide sequence selected from SEQ ID NOs:4-9 and any combination thereof, and mutated VuSP2 allele comprises a polynucleotide sequence selected from SEQ ID NOs: 12-15 and any combination thereof.
36. A modified Cowpea (Vigna unguiculata) plant, plant part, plant seed or pod or plant cell produced by the method according to any one of claims 29-35, wherein said modified plant does not comprise a CRIPPR/Cas related transgene.
37. A tissue culture of regenerable cells, protoplasts or callus obtained from the modified Cowpea (Vigna unguiculata) plant produced by the method according to any one of claims 29-35.
38. A method of improving at least one plant architecture trait conferring determinate growth habit characteristic in a Cowpea (Vigna unguiculata) plant, comprising steps of producing using genome editing, a modified Cowpea (Vigna unguiculata) plant according to any one of claims 1-24, seed, pod or plant part thereof, preferably in a method according to any one of claims 29-35, and enabling growth of said modified Cowpea (Vigna unguiculata) plant, seed or plant part thereof.
39. The method according to claim 38, wherein said method comprises steps of : a. identifying at least one Cowpea (Vigna unguiculata) SELF PRUNING (SP) VuSPl and/or VuSP2 gene allele; b. synthetizing at least one guide RNA (gRNA) comprising a polynucleotide sequence complementary to the polynucleotide sequence of said at least one identified VuSPl and/or VuSP2 gene allele; c. transforming Cowpea (Vigna unguiculata) plant cell with a construct comprising (a) Cas nucleotide sequence operably linked to said at least one gRNA, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and said at least one gRNA; d. screening the genome of said transformed plant cell for induced targeted loss of function mutation in said VuSPl and/or VuSP2 gene to generate a mutated VuSPl and/or VuSP2 allele; e. regenerating Cowpea (Vigna unguiculata) plant carrying said mutated VuSPl and/or VuSP2allele; and f . screening said regenerated plants for a Cowpea (Vigna unguiculata) plant with improved plant architecture trait conferring determinate growth habit characteristic .
40. The method according to any one of claims 38 and 39, wherein said guide RNA (gRNA) complementary to said VuSPl and/or VuSP2 genes, comprising a nucleotide sequence selected from SEQ ID NO:2-3 and/or SEQ ID NO: 11, respectively.
41. The method according to any one of claims 38-40, wherein the VuSPl mutated allele comprises a polynucleotide sequence selected from SEQ ID NOs:4-9 and any combination thereof, and the mutated VuSP2 allele comprises a polynucleotide sequence selected from SEQ ID NOs: 12-15 and any combination thereof .
42. A method for identifying and/or selecting for a Cowpea (Vigna unguiculata) plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic, said method comprises steps of: a. screening the genome of said Cowpea (Vigna unguiculata) plant for a mutated VuSPl and/or VuSP2 allele, wherein the VuSPl mutated allele comprises a polynucleotide sequence selected from SEQ ID NOs:4-9 and any combination thereof, and the mutated VuSP2 allele comprises a polynucleotide sequence selected from SEQ ID NOs: 12-15 and any combination thereof; b. optionally, regenerating a modified Cowpea (Vigna unguiculata) plant carrying said genetic modification; and c. optionally, screening said regenerated plants for a plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic.
43. The method according to claim 42, wherein said genomic modification is a loss of function mutation.
44. The method according to any one of claims 42-43, wherein said determinate growth habit characteristic is selected from the group consisting of determinate or semi-determinate architecture, early termination of sympodial cycling, compact growth habit, reduced height, distinct short stature, determinate apical meristem growth, reduced or limited apical meristem growth, interrupted or exhausted apical meristem growth, bushy growth habit, synchronized flowering, uniform flowering, synchronized pod formation, uniform pod formation, reduced number of sympodial units, suppressed sympodial shoot termination, lack of central growing axis and tendrils, uniform appearance of flowers, adaptation to mechanical harvest, higher harvest index and any combination thereof.
45. A method of determining the presence of a mutated VuSPl and/or
VuSP2 allele in a Cowpea (Vigna unguiculata) plant conferring determinate growth habit characteristic, wherein the method comprising assaying the genome of said Cowpea plant for the presence of a polynucleotide sequence selected from SEQ ID NOs:4-9 and any combination thereof, and SEQ ID NOs: 12-15 and any combination thereof, respectively.
46. An isolated polynucleotide sequence having at least 80% sequence similarity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 4-9 and any combination thereof, and SEQ ID NOs: 12-15 and any combination thereof.
47. Use of (a) a nucleic acid sequence as set forth in SEQ ID NO:1 and/or SEQ ID NO: 10 and/or (b) a nucleotide sequence as set forth in SEQ ID NOs: 2-3 and/or SEQ ID NO: 11 and any combination thereof for targeted genome modification of Cowpea (Vigna unguiculata) , for generating and/or producing a modified Cowpea (Vigna unguiculata) plant with at least one genetically modified VuSPl and/or VuSP2 genes involved in determinate growth habits.
48. Use of a nucleotide sequence having at least 80% sequence similarity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 4-9 and any combination thereof, and SEQ ID NOs: 12-15 and any combination thereof, or a complementary sequence thereof, for generating, identifying and/or screening for a modified Cowpea (Vigna unguiculata) plant exhibiting improved plant architecture trait conferring determinate growth habit characteristic.
49. The use according to claim 48, wherein the presence of at least one polynucleotide sequence selected from SEQ ID NOs: 4- 9 and any combination thereof, or a complementary sequence thereof indicates that the Cowpea plant comprises a VuSPl allele with a loss of function mutation, and the presence of at least one polynucleotide sequence selected from at least one polynucleotide sequence selected from SEQ ID NOs: 12-15 and any combination thereof, or a complementary sequence thereof indicates that the Cowpea plant comprises a VuSP2 allele with a loss of function mutation.
50. A detection kit for determining the presence or absence of a mutated VuSPl and/or VuSP2 gene conferring determinate growth habit characteristic in a Cowpea (Vigna unguiculata) plant, comprising a polynucleotide fragment comprising a polynucleotide sequence having at least 80% similarity to a sequence selected from SEQ ID NOs:4-9, any combination thereof, or a complementary sequence thereof, and /or at least 80% similarity to a sequence selected from SEQ ID NOs: 12-15, any combination thereof, or a complementary sequence thereof, respectively .
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