Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
  • A01H5/02—Flowers
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
  • A01H6/28—Cannabaceae, e.g. cannabis
• • 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

Definitions

• the present disclosure relates to conferring desirable agronomic traits in Cannabis plants. More particularly, the current invention pertains to producing Cannabis plants with improved yield traits by manipulating genes controlling day-length sensitivity and plant architecture.
• Cannabis is one of the oldest domesticated plants with evidence of being used by a vast array of ancient cultures. It is thought to have originated from central Asia from which it was spread by humans to China, Europe, the Middle East and the Americas. Thus, Cannabis has been bred by many different cultures for various uses such as food, fiber and medicine since the dawn of agricultural societies. In the last few decades, Cannabis breeding has stopped as it became illegal and non-economic to do so. With the recent legislation converting Cannabis back to legality, there is a growing need for the implementation of new and advanced breeding techniques in future Cannabis breeding programs. This will allow speeding up the long process of classical breeding and accelerate reaching new and genetically improved Cannabis varieties for fiber, food and medicine products. Developing and implementing molecular biology tools to support the breeders, will allow creating new traits and tracking the movement of such desired traits across breeders germplasm.
• Cannabis plants are mostly done by small Cannabis growers. There is very limited if any molecular tools supporting or leading the breeding process.
• Traditional Cannabis breeding is done by mixing breeding material with hope to find the desired traits and phenotypes by random crosses. These methods have allowed the construction of the leading Cannabis varieties on the market today.
• plant architecture One of the most important determinants of crop productivity is plant architecture.
• artificial selection for modified shoot architectures provided critical steps towards improving yield, followed by innovations enabling large- scale field production.
• a prominent example is tomato, in which the discovery of a mutation in the antiflorigen-encoding self-pruning gene (sp), led to determinate plants that provided a burst of flowering and synchronized fruit ripening, permitting mechanical harvesting.
• sp antiflorigen-encoding self-pruning gene
• day-length sensitivity in crops limits their geographical range of cultivation, and thus modification of the photoperiod response was critical for their domestication.
• PCT application WO2017180474 discloses a tomato plant that is a sp5g sp double mutant and that flowers earlier than the corresponding sp [sibling] tomato plant, as measured with reference to the number of leaves produced prior to appearance of first inflorescence.
• Cannabis architecture or earliness traits were not targeted during domestication.
• SP mutated Cannabis SELF PRUNING
• SP5G mutated Cannabis SELF PRUNING 5G
• SP SELF PRUNING
• SELF PRUNING 5G (SP5G) Cannabis gene is selected from the group consisting of CsSP5G-l having a genomic nucleotide sequence as set forth in SEQ ID NO: 10 or a functional variant thereof, CsSP5G-2 having a genomic nucleotide sequence as set forth in SEQ ID NO: 13 or a functional variant thereof, CsSP5G-3 having a genomic nucleotide sequence as set forth in SEQ ID NO: 16 or a functional variant thereof, CsSP5G-4 having a genomic nucleotide sequence as set forth in SEQ ID NO: 19 or a functional variant thereof and any combination thereof.
• SP5G SELF PRUNING 5G
• siRNA small interfering RNA
• miRNA microRNA
• amiRNA artificial miRNA
• CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
• Cas CRISPR/Cas
• TALEN Transcription activator-like effector nuclease
• ZFN Zinc Finger Nuclease
• Cas gene is selected from the group consisting of Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, CastlOd, Casl2, Casl3, Casl4, CasX, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csnl, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl, Cs
• a construct comprising (a) Cas DNA and gRNA sequence selected from the group consisting of SEQ ID NO:22-SEQ ID NO:916 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:22- 916 and any combination thereof.
• a construct comprising (a) Cas DNA and gRNA sequence selected from the group consisting of SEQ ID NO:22-SEQ ID NO: 126 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 22- 126 and any combination thereof.
• a construct comprising (a) Cas DNA and gRNA sequence selected from the group consisting of SEQ ID NO: 127-SEQ ID NO:211 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 127-211 and any combination thereof.
• a construct comprising (a) Cas DNA and gRNA sequence selected from the group consisting of SEQ ID NO:212-SEQ ID NO:283 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:212-283 and any combination thereof.
• a construct comprising (a) Cas DNA and gRNA sequence selected from the group consisting of SEQ ID NO:284-SEQ ID NO:516 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 284-516 and any combination thereof.
• a construct comprising (a) Cas DNA and gRNA sequence selected from the group consisting of SEQ ID NO:517-SEQ ID NO:745 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:517-745 and any combination thereof.
• a construct comprising (a) Cas DNA and gRNA sequence selected from the group consisting of SEQ ID NO:746-SEQ ID NO:828 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 746-828 and any combination thereof.
• a construct comprising (a) Cas DNA and gRNA sequence selected from the group consisting of SEQ ID NO:829-SEQ ID NO:916 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 829-916 and any combination thereof.
• said gRNA sequence comprises a 3’ NGG Protospacer Adjacent Motif (PAM).
• Agrobacterium infiltration 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
• said Cannabis plant is selected from the group of species that includes, but is not limited to, Cannabis sativa (C. sativa), C. indica, C. ruderalis and any hybrid or cultivated variety of the genus Cannabis.
• SP Cannabis SELF PRUNING
• SP5G Cannabis SELF PRUNING 5G
• SP Cannabis SELF PRUNING
• SP5G Cannabis SELF PRUNING 5G
• SELF PRUNING (SP) Cannabis gene is selected from the group consisting of CsSP- 1 having a genomic nucleotide sequence as set forth in SEQ ID NO: l or a functional variant thereof, CsSP-2 having a genomic nucleotide sequence as set forth in SEQ ID NO:4 or a functional variant thereof, CsSP-3 having a genomic nucleotide sequence as set forth in SEQ ID NO:7 or a functional variant thereof and any combination thereof.
• SELF PRUNING 5G (SP5G) Cannabis gene is selected from the group consisting of CsSP5G-l having a genomic nucleotide sequence as set forth in SEQ ID NO: 10 or a functional variant thereof, CsSP5G-2 having a genomic nucleotide sequence as set forth in SEQ ID NO: 13 or a functional variant thereof, CsSP5G-3 having a genomic nucleotide sequence as set forth in SEQ ID NO: 16 or a functional variant thereof, CsSP5G-4 having a genomic nucleotide sequence as set forth in SEQ ID NO: 19 or a functional variant thereof and any combination thereof. It is another object of the present invention to disclose the method as defined in any of the above, wherein said functional variant has at least 75% sequence identity to said CsSP or said CsSP5G nucleotide sequence.
• siRNA small interfering RNA
• miRNA microRNA
• amiRNA artificial miRNA
• CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
• Cas CRISPR/Cas
• TALEN Transcription activator like effector nuclease
• ZFN Zinc Finger Nuclease
• meganuclease meganuclease
• Cas gene is selected from the group consisting of Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, CastlOd, Casl2, Casl3, Cas 14, CasX, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csnl, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl, Csbl,
• a construct comprising (a) Cas DNA and gRNA sequence selected from the group consisting of SEQ ID NO:22-SEQ ID NO:916 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:22-916 and any combination thereof.
• a construct comprising (a) Cas DNA and gRNA sequence selected from the group consisting of SEQ ID NO:22-SEQ ID NO: 126 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:22- 126 and any combination thereof.
• a construct comprising (a) Cas DNA and gRNA sequence selected from the group consisting of SEQ ID NO: 127-SEQ ID NO:211 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 127-211 and any combination thereof.
• a construct comprising (a) Cas DNA and gRNA sequence selected from the group consisting of SEQ ID NO:212-SEQ ID NO:283 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:212-283 and any combination thereof.
• a construct comprising (a) Cas DNA and gRNA sequence selected from the group consisting of SEQ ID NO:284-SEQ ID NO:516 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 284-516 and any combination thereof.
• a construct comprising (a) Cas DNA and gRNA sequence selected from the group consisting of SEQ ID NO:517-SEQ ID NO:745 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:517-745 and any combination thereof.
• a construct comprising (a) Cas DNA and gRNA sequence selected from the group consisting of SEQ ID NO:746-SEQ ID NO:828 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 746-828 and any combination thereof.
• a construct comprising (a) Cas DNA and gRNA sequence selected from the group consisting of SEQ ID NO:829-SEQ ID NO:916 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 829-916 and any combination thereof.
• said gRNA sequence comprises a 3’ NGG Protospacer Adjacent Motif (PAM).
• Agrobacterium infiltration virus based plasmids for delivery of the genome editing molecules or mechanical insertion
• PEG polyethylene glycol
• Cannabis plant is selected from the group of species that includes, but is not limited to, Cannabis sativa ( C . sativa ), C. indica, C. ruderalis and any hybrid or cultivated variety of the genus Cannabis.
• Fig. 1 is schematically presenting CRISPR/Cas9 mode 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;
• Fig. 2A-C is presenting Cannabis SP-1 gene (CsSP-1) (A) nucleotide genomic sequence as set forth in SEQ ID NO: 1, (B) nucleotide coding sequence (CDS) set forth in SEQ ID NO:2, and (C) amino acid sequence as set forth in SEQ ID NO:3;
• Fig. 3A-C is presenting Cannabis SP-2 gene (CsSP-2) (A) nucleotide genomic sequence as set forth in SEQ ID NO:4, (B) nucleotide coding sequence (CDS) set forth in SEQ ID NO:5, and (C) amino acid sequence as set forth in SEQ ID NO:6;
• Fig. 4A-C is presenting Cannabis SP-3 gene (CsSP-3) (A) nucleotide genomic sequence as set forth in SEQ ID NO:7, (B) nucleotide coding sequence (CDS) set forth in SEQ ID NO: 8, and (C) amino acid sequence as set forth in SEQ ID NO:9;
• Fig. 5A-C is presenting Cannabis SP5G-1 gene (CsSP5G-l) (A) nucleotide genomic sequence as set forth in SEQ ID NO: 10, (B) nucleotide coding sequence (CDS) set forth in SEQ ID NO: 11, and (C) amino acid sequence as set forth in SEQ ID NO: 12;
• Fig. 6A-C is presenting Cannabis SP5G-2 gene (CsSP5G-2) (A) nucleotide genomic sequence as set forth in SEQ ID NO: 13, (B) nucleotide coding sequence (CDS) set forth in SEQ ID NO: 14, and (C) amino acid sequence as set forth in SEQ ID NO: 15;
• Fig. 7A-C is presenting Cannabis SP5G-3 gene (CsSP5G-3) (A) nucleotide genomic sequence as set forth in SEQ ID NO: 16, (B) nucleotide coding sequence (CDS) set forth in SEQ ID NO: 17, and (C) amino acid sequence as set forth in SEQ ID NO: 18;
• Fig. 8A-C is presenting Cannabis SP5G-4 gene (CsSP5G-3) (A) nucleotide genomic sequence as set forth in SEQ ID NO: 19, (B) nucleotide coding sequence (CDS) set forth in SEQ ID NO:20, and (C) amino acid sequence as set forth in SEQ ID NO:21 ;
• Figs 9A-D is photographically presenting staining of Cannabis plants after transient GUS transformation of (A) axillary buds (B) leaf (C) calli, and (D) cotyledons;
• Figs 10A-C is presenting regenerated transformed Cannabis tissue
• Fig. 11 is photographically presenting PCR detection of Cas9 DNA in shoots of Cannabis plants transformed using biolistics.
• Figs 12A-B is illustrating in vitro cleavage activity of CRISPR/Cas9; (A) a scheme of genomic area targeted for editing, and (B) a gel showing digestion of PCR amplicon containing RNP complex of Cas9 and gene specific gRNA.
• the present invention provides a modified Cannabis plant exhibiting at least one improved domestication trait compared with wild type Cannabis, wherein said modified plant comprises at least one mutated Cannabis SELF PRUNING (SP) (CsSP) gene and/or at least one mutated Cannabis SELF PRUNING 5G (SP5G) (CsSP5G) gene.
• the present invention further provides methods for producing the aforementioned modified Cannabis plant using genome editing or other genome modification techniques.
• Cannabis legal market As the Cannabis legal market is expanding worldwide, this agricultural crop will gradually move from indoor growing facilities to simple low cost greenhouses to enable mass production at reduced operational costs.
• One of the major challenges facing this transition is the lack of compatible genetics (strains) adapted for green house growth.
• strains strains
• Classical breeding programs dedicated to the end are virtually impossible due to limited genetic variation, legal constraints on import and export of genetic material and limited academic knowledge and gene banks involved is such projects.
• traditional breeding is a long process with low rates of success and certainty, as it is based on trial and error.
• the solution proposed by the current invention is using genome editing such as the CRISPR/Cas system in order to create cultivated Cannabis plants with improved yield and more specifically with determinate growth habit and with significantly reduced requirement for short days period needed for induction of flowering. 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 varieties faster and at a lower cost.
• Cannabis growers are using Cannabis strains that were bred for indoor cultivation and are now using those for their greenhouse operations. This situation is obviously not ideal and causes many logistic issues for the growers. For example, since Cannabis plants require short days for the induction of flowering, growers install darkening curtains in the greenhouse to control day length for the plants. This artificial darkening results in increased humidity in the greenhouse thus creating optimal conditions for fungal pathogens to spread and thrive. These conditions force growers to intensively use fungicides to control pathogen populations. With strict regulatory constraints in place across the legalized states, these conditions pose a great challenge for sustainable Cannabis production and consumer health.
• Cannabis growers are currently using vegetative propagation (cloning or tissue culture).
• vegetative propagation cloning or tissue culture
• FI hybrid seeds are used to maintain FI hybrid seeds. These hybrids are generated by crossing homozygous parental lines.
• the next step for the Cannabis industry is the adoption and use of hybrid seeds for propagation, which is common practice in the conventional seed industry (from field crops to vegetables).
• breeding for basic agronomic traits that are completely lacking in currently available Cannabis varieties will significantly increase grower’s productivity. This will allow growing and supplying high quality raw material for the Cannabis industry.
• Cannabis plants are mostly done by small Cannabis growers. There is very limited if any molecular tools supporting or leading the breeding process. Traditional Cannabis breeding is done by mixing breeding material with hope to find the desired traits and phenotypes by random crosses.
• the present invention provides for the first time Cannabis plants with improved domestication traits such as plant architecture and day length sensitivity.
• the current invention discloses the generation of non-transgenic Cannabis plants with improved yield traits, using the genome editing technology, e.g., the CRISPR/Cas9 highly precise tool.
• the generated mutations can be introduced into elite or locally adapted Cannabis lines rapidly, with relatively minimal effort and investment.
• Genome editing is an efficient and useful tool for increasing crop productivity, and there is particular interest in advancing manipulation of domestication genes in Cannabis 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 these deficiencies, with the aims of increasing quality and yield, improve adaptation and expand geographical ranges of cultivation.
• CRISPR clustered regularly interspaced short palindromic repeats
• Cas9 CRISPR-associated protein-9 nuclease
• 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 surprisingly provides transformed and regenerated Cannabis plants with modified desirable domestication genes.
• Precise editing of SELFPRUNING (SP) and SELF-PRUNING 5G (SP5G) in wild Cannabis species should serve as a first step towards generating commercially cultivable lines, without causing an associated linkage drag on other useful traits. Genome engineering could thus be applied for de novo domestication of wild species to create climate-smart crops.
• gRNAs guide RNAs
• similar denotes a correspondence or resemblance range of about ⁇ 20%, particularly ⁇ 15%, more particularly about ⁇ 10% and even more particularly about ⁇ 5%.
• 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 it refers to plants of the same Cannabis 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, plant protoplasts, plant cell tissue culture from which tomato 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, roots, root tips and the like.
• 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 a form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.
• 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 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, 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 a root, stem, leaf, flower, flower bud, 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, whole plants, plant organs, plant seeds, 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 non limiting 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 CsSP gene or at least one CsSP5G gene.
• Cannabisbis refers hereinafter to a genus of flowering plants in the family Cannabaceae.
• Cannabis is an annual, dioecious, flowering herb that includes, but is not limited to three different species, Cannabis sativa, Cannabis indica and Cannabis ruderalis.
• the term also refers to hemp.
• Cannabis plants produce a group of chemicals called cannabinoids. Cannabinoids, terpenoids, and other compounds are secreted by glandular trichomes that occur most abundantly on the floral calyxes and bracts of female Cannabis plants.
• '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 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 of, for example, tomato and thus conditions the 'determinate' (sp/sp) and 'indeterminate' (SP) growth habits of the plant. SP is a developmental regulator which is homologous to CENTRORADIALIS (CEN) from Antirrhinum and TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT) from Arabidopsis.
• CEN CENTRORADIALIS
• FLOWER 1 FLOWERING LOCUS T
• the present invention discloses that SP is a member of a gene family in Cannabis composed of at least three genes.
• the newly revealed Cannabis SP genes comprise CsSP-1, CsSP-2 and CsSP-3, encoded by genomic sequence as set forth in SEQ. ID. NO: 1, 4 and 7, coding sequence as set forth in SEQ. ID. NO:2, 5 and 8, and amino acid sequence as set forth in SEQ. ID. NOG, 6 and 9, respectively.
• genome editing- targeted mutation in at least one of the aforementioned CsSP genes which reduces the functional expression of the gene, affect the plant sympodial growth habit which plays a key role in determining plant architecture.
• 'SELF-PRUNING 5G' or 'SP5G' in the context of the present invention refers to a gene encoding florigen paralog and flowering repressor responsible for loss of day-length-sensitive flowering. It is further acknowledged that SP5G expression is induced to high levels during long days in wild species. It is within the scope of the current invention that CRISPR/Cas9-engineered mutations in SP5G cause rapid flowering and enhance the compact determinate growth habit of Cannabis plants, resulting in a quick burst of flower production that turns to an early yield. The findings of the current invention suggest that variation in SP and/or SP5G facilitate the production of cultivated Cannabis strains with improved demonstration traits. The inventors of the present invention use gene editing techniques to rapidly improve yield traits in crop such as Cannabis.
• 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 Cannabis plants with improved domestication traits are generated using genome editing mechanism. This technique enables to achieve in planta modification of specific genes that relate to and/or control the flowering time and plant architecture in the Cannabis plant.
• genome editing or “genome/genetic modification” or “genome engineering” 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 to site specific locations.
• 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 end-joining (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 (TAFEN), and the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system.
• ZFNs zinc finger nucleases
• TAFEN transcription activator-like effector-based nucleases
• CRISPR/Cas9 clustered regularly interspaced short palindromic repeats
• Csn I a CRISPR-associated protein
• HDR Homology-Directed Repair Effector Nuclease
• HNH an endonuclease domain named ZFN ⁇ Zinc-Finger Nuclease
• the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are used for the first time for generating genome modification in targeted genes in the Cannabis plant. 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 select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material. These repeats were initially discovered in the 1980s in E. coli.
• 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.
• 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.
• PAM protospacer-associated motif
• 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.
• sgRNA single guide RNA
• Cas9 nuclease variants include wild-type Cas9, Cas9D10A and nuclease-deficient Cas9 (dCas9).
• Fig. 1 schematically presenting 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.
• the Cas9 endonuclease forms a complex with a chimeric RNA (called guide RNA or gRNA), replacing the crRNA-transcrRNA 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
• PAM protospacer adjacent motif
• 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.
• 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.
• 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 or at the level of protein.
• gene knockdown also refers to a loss of function mutation and /or gene knockout mutation in which an organism's genes 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.
• miRNAs refers hereinafter to small non-coding RNAs that have been found in most of the eukaryotic organisms. They are involved in the regulation of gene expression at the post-transcriptional level in a sequence specific manner. MiRNAs are produced from their precursors by Dicer-dependent small RNA biogenesis pathway. MiRNAs are candidates for studying gene function using different RNA-based gene silencing techniques. For example, artificial miRNAs (amiRNAs) targeting one or several genes of interest is a potential tool in functional genomics.
• miRNAs amiRNAs
• 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. It also used to describe conditions present in a non-laboratory environment (e.g. in vivo).
• 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 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', and 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 accelerates the termination of sympodial units but does not change the sympodial habit. The result is a progressive reduction in the number of vegetative nodes between inflorescences in a pattern that depends on light intensity and genetic background.
• earsliness refers hereinafter to early flowering and/or rapid transition from the vegetative to reproductive stages, or reduced‘time to initiation of flowering’ and more generally to earlier completion of the life-cycle.
• reduced flowering time' refers to time to production of first inflorescence. Such a trait can be evaluated or measured, for example, with reference to the number of leaves produced prior to appearance of the first inflorescence.
• 'harvest index' can be herein defined as the total yield per plant weight.
• the term 'day length' or 'day length sensitivity' as used in the context of the present invention generally refers to photoperiodism, which is the physiological reaction of organisms to the length of day or night. Photoperiodism can also be defined as the developmental responses of plants to the relative lengths of light and dark periods. Plants are classified under three groups according to the photoperiods: short-day plants, long-day plants, and day-neutral plants. Photoperiodism affects flowering by inducing the shoot to produce floral buds instead of leaves and lateral buds. It is within the scope of the present invention that Cannabis is included within the short-day facultative plants. The Cannabis plants of the present invention are genetically modified so as to exhibit loss of day-length sensitivity, which is highly desirable agronomical trait enabling enhanced yield of the cultivated crop.
• 'determinate' or 'determinate growth' 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.
• '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.
• 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, for example with reference to SEQ ID NOs: 1, 4 or 7 refers to a variant of a sequence or part of a sequence which retains the biological function of the full non-variant allele (e.g. CsSP or CsSP5G allele) and hence has the activity of SP or SP5G 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.
• plant or “cultivar” 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 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 single nucleotide polymorphisms, insertions, inversions, translocations or deletions, or the consequence of gene regulation caused by, for example, by chemical or structural modification, transcription regulation or post-translational modification/regulation.
• An allele associated with a qualitative trait may comprise alternative or variant forms of various genetic units including those mat 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.
• 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. A wild type allele is a naturally occurring allele.
• the term allele refers to the three identified SP genes in Cannabis, namely CsSP-1, CsSP-2 and CsSP-3 having the genomic nucleotide sequence as set forth in SEQ ID NOs: 1, 4 and 7, respectively.
• the term SP5G genes in Cannabis namely CsSP5G-l, CsSP5G-2, and CsSP5G-4 having the genomic nucleotide sequence as set forth in SEQ ID NOs: 10, 13, 18 and 19, respectively.
• locus means a specific place or places or region or a site 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.
• the Cannabis plants of the present invention comprise homozygous configuration of at least one of the mutated Cssp genes (i.e. Cssp-1, Cssp-2 and Cssp-3) and/or the mutated Cssp5g genes (i.e. Cssp5g-1, Cssp5g-2, Cssp5g-3 and Cssp5g-4).
• the mutated Cssp genes i.e. Cssp-1, Cssp-2 and Cssp-3
• the mutated Cssp5g genes i.e. Cssp5g-1, Cssp5g-2, Cssp5g-3 and Cssp5g-4.
• 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.
• 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.
• 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.
• hybrid hybrid plant
• hybrid progeny used herein refers to an individual produced from genetically different parents (e.g., a genetically heterozygous or mostly heterozygous individual).
• 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.
• 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.
• 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 is providing 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.
• nucleic acid 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.
• genes are used broadly to refer to a DNA nucleic acid associated with a biological function.
• genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in combination with regulatory sequences.
• genomic DNA, cDNA or coding DNA may be used.
• the nucleic acid is cDNA or coding DNA.
• peptide refers to amino acids in a polymeric form of any length, linked together by peptide bonds.
• a "modified” or a “mutant” plant is a plant that has been altered compared to the naturally occurring wild type (WT) plant.
• WT wild type
• the endogenous nucleic acid sequences of each of the SP or SP5G homologs in Cannabis have been altered compared to wild type sequences using mutagenesis and/or genome editing methods as described herein. This causes inactivation of the endogenous SP and/or SP5G genes and thus disables SP and/or SP5G function.
• Such plants have an altered phenotype and show improved domestication traits such as determinant plant architecture, synchronous and/or early flowering and loss of day length sensitivity compared to wild type plants. Therefore, the improved domestication phenotype is conferred by the presence of at least one mutated endogenous Cssp and /or Cssp5g gene in the Cannabis plant genome which has been specifically targeted using genome editing technique.
• the at least one improved domestication trait is not conferred by the presence of transgenes expressed in Cannabis.
• nucleic acid sequences of wild type alleles are designated using capital letters namely CsSP-1, CsMSP-2 and CsSP-3; and CsSP5G-l, CsMSP5G-2, CsSP5G-3 and CsSP5G-4.
• Mutant sp and sp5g nucleic acid sequences use non-capitalization.
• Cannabis plants of the invention are modified plants compared to wild type plants which comprise and express mutant Cssp and/or Cssp5g alleles.
• sp and/or sp5g mutations that down- regulate or disrupt functional expression of the wild-type SP and/or SP5G sequence respectively may be recessive, such that they are complemented by expression of a wild-type sequence.
• a wild type Cannabis plant is a plant that does not have any mutant sp and/or sp5g alleles.
• 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.
• the improved domestication at least one trait is not due to the presence of a transgene.
• the inventors have generated mutant Cannabis lines with mutations inactivating at least one CsSP and/or CsSP5G homoeoallele which confer heritable improved domestication trait(s). In this way no functional CsSP and/or CsSP5G protein is made.
• the invention relates to these mutant Cannabis lines and related methods.
• modifying Cannabis shoot architecture by selection for mutations in florigen flowering pathway genes allowed major improvements in plant architecture and yield.
• a mutation in the antiflorigen SELFPRUNING (SP) gene (sp classic) provided compact‘determinate’ growth that translated to a burst of flowers, thereby enabling largescale field production.
• SELFPRUNING (SP) homologues and related florigen family members such as SELF-PRUNING 5G (SP5G) have been identified in both genome and transcriptome in Cannabis.
• CRISPR/Cas9 can be used to create heritable mutations in florigen pathway family members that result in desirable phenotypic effects.
• Csy4 multi-gRNA system To edit multiple domestication genes simultaneously and stack the resulting allelic variants, on option is that several gRNAs can be assembled to edit several genes into one construct, by using the Csy4 multi-gRNA system. The construct is then transformed via an appropriate vector into several wild-Cannabis accessions.
• Cannabis SP genes namely CsSP-1, CsSP-2 and CsSP-3 having genomic nucleotide sequence as set forth in SEQ. ID. NO.: l, 4 and 7 respectively, were targeted using guide RNAs as set forth in SEQ ID NO:22-126, 127-211 and 212-283, respectively.
• Several mutated alleles have been identified. Notably, the plants with mutated sp alleles were more compact than the wild type plants lacking the mutated allele.
• SP5G represses flowering predominantly in primary and canonical axillary shoots.
• SP5G was shown to control primary and canonical axillary shoot flowering time and to contribute to controlling day-length sensitivity.
• reduced SP5G activity was shown to mitigate day-length sensitivity.
• CRISPR-Cas9-induced null sp5g mutations in Cannabis eliminate day-length sensitivity and causing faster primary and axillary shoot flowering.
• the present invention discloses that the combination of both mutations sp5g and sp results in faster-flowering Cannabis plants with typical sp determinate growth. Such Cannabis plants could have agronomic value, particularly for the desirable trait of earliness of yield.
• Cs-sp5g/ sp double mutants are not simply additive but substantially more compact than mutant sp Cannabis plants owing to faster axillary shoot flowering and earlier termination of sympodial cycling. According to further aspects of the present invention, compared with wild type and/or sp Cannabis plants, Cs-sp5g/ sp double mutant plants provide a more rapid flowering burst, and reach final harvest sooner.
• the harvest index (defined as the total yield per plant weight) of the Cs-sp5g/ sp double mutant plants is higher than that for wild type and/or sp mutant Cannabis plants.
• large-scale Cannabis production based on sp determinate growth may be achieved only in the absence of day-length sensitivity, i.e. by loss of function mutation in at least one of Cssp5g-1, Cssp5g-2, Cssp5g-3 or Cssp5g-4 as set forth in SEQ ID NO.: 10, 13 16 and 19, respectively.
• targeting SP5G homologs and/or other diurnally regulated CENTRORADIALIS/TERMINAL FLOWER 1 /SELF-PRUNING (CETS) genes may allow immediate customization of day-length sensitivity in Cannabis elite germplasm to expand the geographical range of cultivation, and could serve as a first step toward engineering the domestication of wild Cannabis species with agricultural potential.
• the loss of function mutation may be a deletion or insertion ("indels") with reference the wild type CsSP and/or CsSP5G allele sequence.
• the deletion 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 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 the each of the mutations. In a preferred embodiment however, the plant is homozygous for the mutations. Progeny that is also homozygous can be generated from these plants according to methods known in the art.
• variants of a particular CsSP and/or CsSP5G nucleotide or amino acid sequence 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 CsSP and/or CsSP5G nucleotide sequence of the CsSP and/or CsSP5G allele as shown in SEQ ID NO 1, 4 or 7; and/or SEQ ID NO 10, 13, 18 or 19, respectively. Sequence alignment programs to determine sequence identity are well known in the art.
• fragments are intended 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 domestication trait.
• the herein newly identified Cannabis SP and/or SP5G locus have been targeted using the double sgRNA strategy.
• 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
• the Cas protein and gRNA can be transported via the vasculature system to the top of the plant and create the genome editing event in the scion .
• CRISPR/Cas system for the generation of Cannabis plants with at least one improved domestication trait, 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 (for example see Fig. 1).
• 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 domestication traits in Cannabis plants is herein produced by generating gRNA with homology to a specific site of predetermined genes in the Cannabis genome i.e. SP and/or SP5G genes, sub cloning this gRNA into a plasmid containing the Cas9 gene, and insertion of the plasmid into the Cannabis plant cells.
• site specific mutations in the SP and/or SP5G genes are generated thus effectively creating non-active molecules, resulting in loss of day length sensitivity, reduced flowering time and determinant growth habit of the genome edited plant.
• Production of Cannabis lines with mutated sp and/or sp5g gene may be achieved by at least one of the following breeding/cultivation schemes:
• line stabilization may be performed by the following:
• line stabilization requires about 6 self crossing (6 generations) and done through a single seed descent (SSD) approach.
• FI hybrid seed production Novel hybrids are produced by crosses between different Cannabis strains.
• shortening line stabilization is performed by Doubled Haploids (DH). More specifically, the CRISPR-Cas9 system is transformed into microspores to achieve DH homozygous parental lines.
• a doubled haploid (DH) is a genotype formed when haploid cells undergo chromosome doubling. Artificial production of doubled haploids is important in plant breeding. It is herein acknowledged that conventional inbreeding procedures take about six generations to achieve approximately complete homozygosity, whereas doubled haploidy achieves it in one generation.
• Sex markers- molecular markers are used for identification and selection of female vs male plants in the herein disclosed breeding program • Genotyping markers- germplasm used in the current invention is genotyped using molecular markers, in order to allow a more efficient breeding process and identification of the SP and/or SP5G editing event.
• Stage 1 Identifying Cannabis sativa (C. sativa), C. indica and C. ruderalis SP and SP5G orthologues.
• CsSP-1 has been mapped to CM011605.1:71328589-71330978 and has a genomic sequence as set forth in SEQ ID NO: l (see Fig. 2A).
• the CsSP-1 gene has a coding sequence as set forth in SEQ ID NO:2 (see Fig. 2B) and it encodes an amino acid sequence as set forth in SEQ ID NOG (see Fig. 2C).
• CsSP-2 has been mapped to CM011605.1 :25325478-25326672 and has a genomic sequence as set forth in SEQ ID NO:4 (see Fig. 3A).
• the CsSP-2 gene has a coding sequence as set forth in SEQ ID NOG (see Fig. 3B) and it encodes an amino acid sequence as set forth in SEQ ID NOG (see Fig. 3C).
• CsSP-3 has been mapped to CM011608.1 :9602945-9603900 and has a genomic sequence as set forth in SEQ ID NOG (see Fig. 4A).
• the CsSP-3 gene has a coding sequence as set forth in SEQ ID NOG (see Fig. 4B) and it encodes an amino acid sequence as set forth in SEQ ID NO:9 (see Fig. 4C).
• CsSP5G-l has been mapped to CMOl 1610.1 :5735300-5738406 and has a genomic sequence as set forth in SEQ ID NO: 10 (see Fig. 5A).
• the CsSP-1 gene has a coding sequence as set forth in SEQ ID NO: 11 (see Fig. 5B) and it encodes an amino acid sequence as set forth in SEQ ID NO: 12 (see Fig. 5C).
• CsSP5G-2 has been mapped to CMOl 1610.1:6032638-6035504 and has a genomic sequence as set forth in SEQ ID NO: 13 (see Fig. 6A).
• the CsSP5G-2 gene has a coding sequence as set forth in SEQ ID NO: 14 (see Fig. 6B) and it encodes an amino acid sequence as set forth in SEQ ID NO: 15 (see Fig. 6C).
• CsSP5G-3 has been mapped to CMOl 1607.1:79899046-79900718 and has a genomic sequence as set forth in SEQ ID NO: 16 (see Fig. 7A).
• the CsSP5G-3 gene has a coding sequence as set forth in SEQ ID NO: 17 (see Fig. 7B) and it encodes an amino acid sequence as set forth in SEQ ID NO: 18 (see Fig. 7C).
• CsSP5G-4 has been mapped to CMOl 1614.1:9255475-9256908 and has a genomic sequence as set forth in SEQ ID NO: 19 (see Fig. 8A).
• the CsSP5G-4 gene has a coding sequence as set forth in SEQ ID NO:20 (see Fig. 8B) and it encodes an amino acid sequence as set forth in SEQ ID NO:21 (see Fig. 8C).
• Stage 2 Designing and synthesizing gRNA molecules corresponding to the sequence targeted for editing, i.e. sequences of each of the genes CsSP-1, CsSP-2 and CsSP-3; and CsSP5G-l, CsSP5G- 2, CsSP5G-3 and CsSP5G-4.
• the editing event is preferably targeted to a unique restriction site sequence to allow easier screening for plants carrying an editing event within their genome.
• the nucleotide sequence of the gRNAs should be completely compatible with the genomic sequence of the target gene. Therefore, for example, suitable gRNA molecules should be constructed for different SP and/or SP5G homologues of different Cannabis strains.
• Tables 1-3 presenting gRNA molecules targeted for silencing CsSP-1, CsSP-2 and CsSP-3, respectively; and Tables 4-7 presenting gRNA molecules targeted for silencing CsSP5G-l, CsSP5G-2, CsSP5G-3 and CsSP5G-4 respectively.
• the term 'PAM' refers hereinafter to Protospacer Adjacent Motif, which is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system.
• Table 1 gRNA sequences targeted for CsSP-1
• gRNA molecules have been cloned into suitable vectors and their sequence has been verified.
• different Cas9 versions have been analyzed for optimal compatibility between the Cas9 protein activity and the gRNA molecule in the Cannabis plant.
• the efficiency of the designed gRNA molecules have been validated by transiently transforming Cannabis tissue culture.
• a plasmid carrying a gRNA sequence together with the Cas9 gene has been transformed into Cannabis protoplasts.
• the protoplast cells have been grown for a short period of time and then were analyzed for existence of genome editing events.
• the positive constructs have been subjected to the herein established stable transformation protocol into Cannabis plant tissue for producing genome edited Cannabis plants in SP and/or SP5G genes.
• Stage 3 Transforming Cannabis plants using Agrobacterium or biolistics (gene gun) methods.
• Agrobacterium and bioloistics a DNA plasmid carrying (Cas9 + gene specific gRNA) can be used.
• a vector containing a selection marker, Cas9 gene and relevant gene specific gRNA’s is constructed.
• Ribonucleoprotein (RNP) complexes carrying (Cas9 protein + gene specific gRNA) are used. RNP complexes are created by mixing the Cas9 protein with relevant gene specific gRNA’s.
• transformation of various Cannabis tissues was performed using particle bombardment of:
• RNP Ribonucleoprotein complex
• transformation of various Cannabis tissues was performed using Agrobacterium (.
• Transformation efficiency by A. tumefaciens has been compared to the bombardment method by transient GUS transformation experiment. After transformation, GUS staining of the transformants has been performed.
• FIG. 9 photographically presenting GUS staining after transient transformation of the following Cannabis tissues (A) axillary buds (B) leaf (C) calli, and (D) cotyledons.
• Fig. 9 demonstrates that various Cannabis tissues have been successfully transiently transformed using biolistics system. Transformation has been performed into calli, leaves, axillary buds and cotyledons of Cannabis.
• additional transformation tools were used in Cannabis, including, but not limited to:
• Stage 4 Regeneration in tissue-culture. When transforming DNA constructs into the plant, antibiotics is used for selection of positive transformed plants. An improved regeneration protocol was herein established for the Cannabis plant.
• Fig. 10A-C presenting regeneration of Cannabis tissue.
• arrows indicate new meristem emergence.
• Stage 5 Selection of positive transformants. Once regenerated plants appear in tissue culture, DNA is extracted from leaf sample of the transformed plant and PCR is performed using primers flanking the edited region. PCR products are then digested with enzymes recognizing the restriction site near the original gRNA sequence. If editing event occurred, the restriction site will be disrupted and the PCR product will not be cleaved. No editing event will result in a cleaved PCR product.
• FIG. 11 showing PCR detection of Cas9 DNA in shoots of transformed Cannabis plants. DNA extracted from shoots of plants transformed with Cas9 using biolistics. This figure shows that three weeks post transformation, Cas9 DNA was detected in shoots of transformed plants.
• FIG. 12 presenting results of in vitro analysis of CRISPR/Cas9 cleavage activity.
• Fig. 12A schematically shows the genomic area targeted for editing (PAM is marked in red) and amplified by the reverse and forward designed primers
• Fig. 12B photographically presents a gel showing successful digestion of the resulted PCR amplicon containing the gene specific gRNA sequence, by RNP complex containing Cas9. The analysis included the following steps:
• Amplicon was isolated from two exemplified Cannabis strains by primers flanking the sequence of the gene of interest targeted by the predesigned sgRNA.
• Stage 6 Selection of transformed Cannabis plants presenting sp and sp5g related phenotypes as described above. It is within the scope that different gRNA promoters were tested in order to maximize editing efficiency.
• RNA-guided genome editing in plants using a CRISPR-Cas system Molecular plant, 2013 6 (6), 1975-1983.

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• 2020
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