Patent Application
20220002742
The Sequence Listing submitted in text format (.txt) filed on Jun. 30, 2021, named “SequenceListing.txt”, created on Jun. 25, 2021 (329 KB), is incorporated herein by reference.
The present invention relates generally to Cannabis plants with altered expression of cannabinoids and/or altered expression of cannabinoid synthesizing enzymes. More specifically, the present disclosure relates to methods for controlling genes associated with cannabinoids synthesis in Cannabis plants.
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.
According to some publications, the Cannabis plant chemical profile is composed of at least 483 known chemical compounds, which include cannabinoids, terpenoids, flavonoids, nitrogenous compounds, amino acids, proteins, glycoproteins, enzymes, sugars and related compounds, hydrocarbons, alcohols, aldehydes, ketones, acids, fatty acids, esters, lactones, steroids, terpenes, non-cannabinoid phenols, vitamins, and pigments.
Cannabinoids are of particular interest for research and commercialization. There are at least 113 different cannabinoids isolated from Cannabis, exhibiting varied effects. The classical cannabinoids are concentrated in a viscous resin produced in structures known as glandular trichomes. The most notable cannabinoid is the phytocannabinoid delta 9 tetrahydrocannabinol (THC), the primary psychoactive compound in Cannabis. Cannabidiol (CBD) is another major constituent of the plant.
Other cannabinoids of interest include, Cannabigerol (CBG), Cannabigerolic Acid (CBGA), Cannabinol (CBN), Cannabichromene (CBC), Tetrahydrocannabivarin (THCV), Cannabigerovarin (CBGV), Cannabigerovarinic Acid (CBGVA), tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), cannabichromene (CBC), cannabicyclol (CBL), cannabivarin (CBV), cannabidivarin (CBDV), cannabichromevarin (CBCV), cannabigerol monomethyl ether (CBGM), cannabielsoin (CBE) and cannabicitran (CBT).
Cannabis plants can exhibit wide variation in the quantity and type of cannabinoids they produce. The mixture of cannabinoids produced by a plant is known as the plant's cannabinoid profile. Selective breeding has been used to control the genetics of plants and modify the cannabinoid profile. For example, strains that are used as fiber (commonly called hemp) are usually bred such that they are low in psychoactive chemicals like THC. Strains used in medicine are often bred for high CBD content, and strains used for recreational purposes are usually bred for high THC content or for a specific chemical balance.
Quantitative analysis of a plant's cannabinoid profile is often determined by analytical methods such as gas chromatography (GC), gas chromatography combined with mass spectrometry (GC/MS) and liquid chromatography (LC) techniques.
A variety of growing and cultivating techniques have been developed for increasing the production of secondary compounds within plants of genus Cannabis. These techniques include outdoor cultivation, indoor cultivation, hydroponics, fertilization, atmospheric manipulation, cloning, crossbreeding etc. 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.
As the cultivation of Cannabis intensifies, breeding and farming techniques fail to provide the level of control of cannabinoid production and yield needed. Cannabinoid research is still new and having plants producing modified levels of certain cannabinoids would be advantageous for research and medical purposes. Furthermore, separating and isolating specific molecules of the plant out of hundreds could be challenging and time consuming.
In view of the above there is an unmet and long felt need for non-GMO, advanced breeding of Cannabis plants producing particular amounts of predetermined cannabinoids. In particular, there is a need for Cannabis plants selectively producing predetermined ratios and/or concentrations of cannabinoids for medical use.
It is one object of the present invention to disclose a Cannabis plant with reduced delta-9-tetrahydrocannabinol (THC) content, or reduced cannabidiol (CBD) content, or reduced THC and CBD content, wherein said plant comprises at least one targeted genome modification effective in decreasing expression of a at least one Cannabis gene encoding a cannabinoid biosynthesis enzyme selected from the group consisting of Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS), Cannabis cannabidiolic acid synthase (CsCBDAS), Cannabis aromatic prenyltransferase (CsPT), Cannabis olivetol synthase (CsOLS), Cannabis acyl-activating enzyme 1 (CsAAE1) and any combination thereof.
It is a further object of the present invention to disclose the Cannabis plant as defined above, wherein said plant comprises reduced THC content, or reduced CBD content, or reduced THC and CBD content relative to a Cannabis plant of a similar genotype or genetic background that does not comprise said targeted genome modification.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said targeted genome modification is located in the cannabinoid biosynthesis enzyme gene locus.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said targeted genome modification interrupts or interferes with or down regulate or silence transcription and/or translation of said Cannabis gene encoding said cannabinoid biosynthesis enzyme.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said plant comprises an endonuclease enzyme targeting a nucleic acid sequence coding for said at least one cannabinoid biosynthesis enzyme.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said plant does not comprise within its genome exogenous genetic material and said plant is a non-naturally occurring Cannabis plant or cell thereof.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein the nucleotide sequence encoding said at least one cannabinoid biosynthesis enzyme is selected from the group consisting of: CsTHCAS having a genomic nucleotide sequence as set forth in SEQ ID NO:1 or a functional variant thereof, CsCBDAS having a genomic nucleotide sequence as set forth in SEQ ID NO:4 or a functional variant thereof, CsPT having a genomic nucleotide sequence as set forth in SEQ ID NO:7 or a functional variant thereof, CsOLS having a genomic nucleotide sequence as set forth in SEQ ID NO:10 or a functional variant thereof and CsAAE1 having a genomic nucleotide sequence as set forth in SEQ ID NO:13 or a functional variant thereof.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said functional variant has at least 75% sequence identity to the nucleotide sequence of said cannabinoid biosynthesis enzyme or a codon degenerate nucleotide sequence thereof.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said plant has decreased expression levels of at least one of CsTHCAS protein having an amino acid sequence with at least 75% identity to the amino acid sequence as set forth in SEQ ID NO:3 or a conservatively substituted amino acid sequence thereof, CsCBDAS protein having an amino acid sequence with at least 75% identity to the amino acid sequence as set forth in SEQ ID NO:6 or a conservatively substituted amino acid sequence thereof, CsPT protein having an amino acid sequence with at least 75% identity to the amino acid sequence as set forth in SEQ ID NO:9 or a conservatively substituted amino acid sequence thereof, CsOLS protein having an amino acid sequence with at least 75% identity to the amino acid sequence as set forth in SEQ ID NO:12 or a conservatively substituted amino acid sequence thereof, and CsAAE1 protein having an amino acid sequence with at least 75% identity to the amino acid sequence as set forth in SEQ ID NO:15 or a conservatively substituted amino acid sequence thereof.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said Cannabis plant has modulated expression of one or more of the cannabinoids, optionally cannabigerolic acid, cannabigerol, DELTA.9-tetrahydrocannabinolic acid, cannabidiolic acid, cannabichromenic acid, DELTA.9-tetrahydrocannabinol, cannabidiol, cannabichromene, THC, D9-THC, D8-THC, THCA, THCV, D8-THCV, D9-THCV, THCVA, CBD, CBDA, CBDV, CBDVA, CBC, CBCA, CBCV, CBCVA, CBG, CBGA, CBGV, CBGVA, CBN, CBNA, CBNV, CBNVA, CBND, CBNDA, CBNDV, CBNDVA, CBE, CBEA, CBEV, CBEVA, CBL, CBLA, CBLV, or CBLVA and cannabidiol.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said Cannabis plant has reduced expression of one or more of the cannabinoids, optionally cannabigerolic acid, cannabigerol, DELTA.9-tetrahydrocannabinolic acid, cannabidiolic acid, cannabichromenic acid, DELTA.9-tetrahydrocannabinol, cannabidiol, cannabichromene, THC, D9-THC, D8-THC, THCA, THCV, D8-THCV, D9-THCV, THCVA, CBD, CBDA, CBDV, CBDVA, CBC, CBCA, CBCV, CBCVA, CBG, CBGA, CBGV, CBGVA, CBN, CBNA, CBNV, CBNVA, CBND, CBNDA, CBNDV, CBNDVA, CBE, CBEA, CBEV, CBEVA, CBL, CBLA, CBLV, or CBLVA and cannabidiol, as compared to a Cannabis plant of a similar genotype or genetic background that does not comprise said targeted genome modification.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said targeted genome modification is introduced using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes (CRISPR/Cas) system, Transcription activator-like effector nuclease (TALEN), Zinc Finger Nuclease (ZFN), meganuclease or any combination thereof.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said CRISPR/Cas genes or proteins are selected from the group consisting of Cas3, Cas4, Cas5, Cas5e (or CasD), Cash, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cast 0, Castl Od, Cas12, Cas13, Cas14, CasX, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn1, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb 1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said targeted genome modification is introduced via (i) at least one RNA-guided (gRNA) endonuclease or nucleic acid encoding at least one RNA-guided endonuclease, and (ii) at least one guide RNA (gRNA) or DNA encoding at least one guide RNA (gRNA), further wherein each of said at least one gRNA directs said endonuclease to a targeted site located in the genomic sequence of said at least one Cannabis gene encoding a cannabinoid biosynthesis enzyme.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein the gRNA nucleotide sequence targeted to CsTHCAS genomic sequence is as set forth in nucleotide sequence selected from the group consisting of SEQ. ID. NO.: 16-167 and any combination thereof.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein the gRNA nucleotide sequence targeted to CsCBDAS genomic sequence is as set forth in nucleotide sequence selected from the group consisting of SEQ. ID. NO.: 168-304, SEQ. ID. NO.: 824-825, and any combination thereof.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein the gRNA nucleotide sequence targeted to CsPT genomic sequence is as set forth in nucleotide sequence selected from the group consisting of SEQ. ID. NO.: 305-458 and any combination thereof.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein the gRNA nucleotide sequence targeted to CsOLS genomic sequence is as set forth in nucleotide sequence selected from the group consisting of SEQ. ID. NO.: 459-509 and any combination thereof.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein the gRNA nucleotide sequence targeted to CsAAE1 genomic sequence is as set forth in nucleotide sequence selected from the group consisting of SEQ. ID. NO.: 510-823, SEQ. ID. NO.: 826, and any combination thereof.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said plant is mutated in a gene selected from the group consisting of CsTHCAS, CsSP, CsCBDAS, CsPT, CsOLS, CsAAE1 and any combination thereof.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said mutated CsTHCAS, CsSP, CsCBDAS, CsPT, CsOLS and/or CsAAE1 gene is a CRISPR/Cas9-induced heritable mutated allele.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said genome modification is a missense mutation, nonsense mutation, insertion, deletion, indel, substitution or duplication.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein the insertion or the deletion produces a gene comprising a frameshift.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said plant is homozygous for said at least one mutated gene.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said mutation is in the coding region of said gene, a mutation in the regulatory region of said gene, a mutation in a gene downstream of said gene in the cannabinoid biosynthesis pathway or an epigenetic factor.
It is a further object of the present invention to disclose the Cannabis 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 or any combination thereof.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said genome modification is generated in planta.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above wherein said genome modification is generated in planta via introduction of a construct comprising (a) Cas DNA and gRNA sequence selected from the group consisting of SEQ ID NO:16-SEQ ID NO:826 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:16-826 and any combination thereof.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above wherein said gRNA sequence comprises a 3′ NGG Protospacer Adjacent Motif (PAM).
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above wherein said gRNA is introduced into the plant cells via 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.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above wherein 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.
It is a further object of the present invention to disclose a Cannabis plant, plant part or plant cell as defined in any of the above wherein said plant does not comprise a transgene.
It is a further object of the present invention to disclose a plant part, plant cell or plant seed of a plant as defined in any of the above.
It is a further object of the present invention to disclose a tissue culture of regenerable cells, protoplasts or callus obtained from the Cannabis plant as defined in any of the above.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above wherein said plant genotype is obtainable by deposit under accession number with NCIMB Aberdeen AB21 9YA, Scotland, UK, or wherein said plant genotype is obtainable by deposit under accession number with ATCC, LGC Standards, Queens Road Teddington Middlesex TW11 OLY UK.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said Cannabis plant comprises a DNA encoding an antisense RNA or a siRNA effective to suppress expression of CsTHCAS, CsSP, CsCBDAS, CsPT, CsOLS, CsAAE1 and any combination thereof, the DNA operably linked to a heterologous promoter, wherein the Cannabis plant comprises reduced THC content, reduced CBD content, or decreased THC and CBD content relative to a Cannabis plant of a similar genotype that does not comprise the DNA.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said Cannabis plant has a THC content of up to 30% by weight, particularly between about %0.1 to about 30% by weight, more particularly between about %0.3 to about 30%, even more particularly between about %0.3 to about 10% by weight.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said Cannabis plant has a CBD content of up to 30% by weight, particularly between about %0.1 to about 30% by weight, more particularly between about %0.3 to about 30%, even more particularly between about %0.3 to about 10% by weight.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said plant has a THC and/or CBD content of not more than about 0.5% by weight.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said plant is THC free.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said plant has at least one targeted genome modification in at least one Cannabis gene encoding cannabinoid precursor synthesis enzyme selected from the group consisting of CsAAE, CsPT and CsOLS.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said plant exhibits reduced expression of THC, CBD or both relative to a Cannabis plant of a similar genotype or genetic background lacking said targeted genome modification.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said plant has a THC and/or CBD content of up to 30% by weight, particularly between about %0.1 to about 30% by weight, more particularly between about %0.3 to about 30%, even more particularly between about %0.3 to about 10% by weight.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said plant has a THC and/or CBD content of not more than about 0.5% by weight.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said plant has at least one targeted genome modification in at least one Cannabis gene encoding cannabinoid synthesis enzyme selected from the group consisting of CsTHCAS and CsCBDAS.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said plant has at least one targeted genome modification in CsTHCAS and said plant exhibits reduced expression of THCA or THC, and elevated expression of CBD or CBDA relative to a Cannabis plant of a similar genotype or genetic background lacking said targeted genome modification.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said plant has a THC and/or THCA content of up to 30% by weight, particularly between about %0.1 to about 30% by weight, more particularly between about %0.3 to about 30%, even more particularly between about %0.3 to about 10% by weight.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said plant has a THC and/or THCA content of not more than about 0.5% by weight.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said plant has at least one targeted genome modification in CsCBDAS and said plant exhibits reduced expression of CBDA or CBD, and elevated expression of THC or THCA relative to a Cannabis plant of a similar genotype or genetic background lacking said targeted genome modification.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said plant has a THC and/or CBD content of up to 30% by weight, particularly between about %0.1 to about 30% by weight, more particularly between about %0.3 to about 30%, even more particularly between about %0.3 to about 10% by weight.
It is a further object of the present invention to disclose the Cannabis plant as defined in any of the above, wherein said plant has a CBD and/or CBDA content of not more than about 0.5% by weight.
It is a further object of the present invention to disclose a Cannabis plant derived product from the plant as defined in any of the above.
It is a further object of the present invention to disclose the Cannabis plant derived product as defined above, comprising a combined cannabidiolic acid and cannabidiol concentration of about 0.3% to about 30% by weight.
It is a further object of the present invention to disclose the Cannabis plant derived product as defined in any of the above, comprising a combined delta-9-tetrahydrocannabinol and tetrahydrocannabinolic acid concentration of between about 0.3% to about 30% by weight.
It is a further object of the present invention to disclose the Cannabis plant derived product as defined in any of the above, comprising Cannabis oil, Cannabis tincture, dried Cannabis flowers, and/or dried Cannabis leaves.
It is a further object of the present invention to disclose the Cannabis plant derived product as defined in any of the above, for medical use.
It is a further object of the present invention to disclose the Cannabis plant derived product as defined in any of the above, formulated for inhalation, oral consumption, sublingual consumption, or topical consumption.
It is a further object of the present invention to disclose a medical composition derived from the plant as defined in any of the above.
It is a further object of the present invention to disclose a method for producing a Cannabis plant with reduced delta-9-tetrahydrocannabinol (THC) content, or reduced cannabidiol (CBD) content, or reduced THC and CBD content, wherein said method comprises steps of introducing into said Cannabis plant genome or a cell thereof at least one targeted genome modification effective in decreasing expression of a at least one Cannabis gene encoding a cannabinoid biosynthesis enzyme selected from the group consisting of tetrahydrocannabinolic acid synthase (THCAS), cannabidiolic acid synthase (CBDAS), aromatic prenyltransferase (PT), olivetol synthase (OLS), acyl-activating enzyme 1 (AAE1) and any combination thereof.
It is a further object of the present invention to disclose the method as defined above, further comprising steps of: (a) constructing an endonuclease enzyme targeting a nucleic acid sequence coding for a cannabinoid biosynthesis enzyme selected from the group consisting of CsTHCAS, CsCBDAS, CsPT, CsOLS, CsAAE1 and any combination thereof; (b) introducing the endonuclease enzyme into the genome of the Cannabis plant of; and (c) decreasing expression of the at least one cannabinoid biosynthesis enzyme within the genome.
It is a further object of the present invention to disclose the method as defined in any of the above, further comprising steps of: (a) introducing into the Cannabis plant or a cell thereof (i) at least one RNA-guided endonuclease or nucleic acid encoding at least one RNA-guided endonuclease, and (ii) at least one guide RNA (gRNA) or DNA encoding at least one gRNA; (b) assaying the Cannabis plant or a cell thereof for an endonuclease-mediated modification in the DNA of said at least one Cannabis cannabinoid biosynthesis enzyme gene locus; and (c) identifying the Cannabis plant, or a cell thereof, or a progeny cell thereof as comprising a modification in said at least one gene locus.
It is a further object of the present invention to disclose the method as defined in any of the above, further comprising steps of culturing the Cannabis plant or cell thereof such that each guide RNA directs an RNA-guided endonuclease to a targeted site in the chromosomal sequence of said at least one Cannabis cannabinoid biosynthesis enzyme, enabling the RNA-guided endonuclease introduce a double-stranded break in the targeted site, and repair of the double-stranded break by a DNA repair process such that the chromosomal sequence is modified, wherein the targeted site is located in the gene locus of the at least one Cannabis cannabinoid biosynthesis enzyme and the chromosomal modification interrupts or interferes with transcription and/or translation of said at least one gene encoding Cannabis cannabinoid biosynthesis enzyme.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein the endonuclease enzyme is a CRISPR/Cas9 system.
It is a further object of the present invention to disclose the method as defined in any of the above, comprising steps of interfering with expression of a cannabinoid biosynthesis enzyme selected from the group consisting of CsTHCAS, CsCBDAS, CsPT, CsOLS, CsAAE1 and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said plant comprises reduced THC content, or reduced CBD content, or reduced THC and CBD content relative to a Cannabis plant of a similar genotype or genetic background that does not comprise said targeted genome modification.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said targeted genome modification is located in the cannabinoid biosynthesis enzyme gene locus.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said targeted genome modification interrupts or interferes with or down regulate or silence transcription and/or translation of said Cannabis gene encoding said cannabinoid biosynthesis enzyme.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said plant comprises an endonuclease enzyme targeting a nucleic acid sequence coding for said at least one cannabinoid biosynthesis enzyme.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said plant does not comprise within its genome exogenous genetic material and said plant is a non-naturally occurring Cannabis plant or cell thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein the nucleotide sequence encoding said at least one cannabinoid biosynthesis enzyme is selected from the group consisting of: CsTHCAS having a genomic nucleotide sequence as set forth in SEQ ID NO:1 or a functional variant thereof, CsCBDAS having a genomic nucleotide sequence as set forth in SEQ ID NO:4 or a functional variant thereof, CsPT having a genomic nucleotide sequence as set forth in SEQ ID NO:7 or a functional variant thereof, CsOLS having a genomic nucleotide sequence as set forth in SEQ ID NO:10 or a functional variant thereof and CsAAE1 having a genomic nucleotide sequence as set forth in SEQ ID NO:13 or a functional variant thereof.
It is a further 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 the nucleotide sequence of said cannabinoid biosynthesis enzyme or a codon degenerate nucleotide sequence thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said plant has decreased expression levels of at least one of CsTHCAS protein having an amino acid sequence with at least 75% identity to the amino acid sequence as set forth in SEQ ID NO:3 or a conservatively substituted amino acid sequence thereof, CsCBDAS protein having an amino acid sequence with at least 75% identity to the amino acid sequence as set forth in SEQ ID NO:6 or a conservatively substituted amino acid sequence thereof, CsPT protein having an amino acid sequence with at least 75% identity to the amino acid sequence as set forth in SEQ ID NO:9 or a conservatively substituted amino acid sequence thereof, CsOLS protein having an amino acid sequence with at least 75% identity to the amino acid sequence as set forth in SEQ ID NO:12 or a conservatively substituted amino acid sequence thereof, and CsAAE1 protein having an amino acid sequence with at least 75% identity to the amino acid sequence as set forth in SEQ ID NO:15 or a conservatively substituted amino acid sequence thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said Cannabis plant has modulated expression of one or more of the cannabinoids, optionally cannabigerolic acid, cannabigerol, DELTA. 9-tetrahydrocannabinolic acid, cannabidiolic acid, cannabichromenic acid, DELTA. 9-tetrahydrocannabinol, cannabidiol, cannabichromene, THC, D9-THC, D8-THC, THCA, THCV, D8-THCV, D9-THCV, THCVA, CBD, CBDA, CBDV, CBDVA, CBC, CBCA, CBCV, CBCVA, CBG, CBGA, CBGV, CBGVA, CBN, CBNA, CBNV, CBNVA, CBND, CBNDA, CBNDV, CBNDVA, CBE, CBEA, CBEV, CBEVA, CBL, CBLA, CBLV, or CBLVA and cannabidiol.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said Cannabis plant has reduced expression of one or more of the cannabinoids, optionally cannabigerolic acid, cannabigerol, DELTA. 9-tetrahydrocannabinolic acid, cannabidiolic acid, cannabichromenic acid, DELTA. 9-tetrahydrocannabinol, cannabidiol, cannabichromene, THC, D9-THC, D8-THC, THCA, THCV, D8-THCV, D9-THCV, THCVA, CBD, CBDA, CBDV, CBDVA, CBC, CBCA, CBCV, CBCVA, CBG, CBGA, CBGV, CBGVA, CBN, CBNA, CBNV, CBNVA, CBND, CBNDA, CBNDV, CBNDVA, CBE, CBEA, CBEV, CBEVA, CBL, CBLA, CBLV, or CBLVA and cannabidiol, as compared to a Cannabis plant of a similar genotype or genetic background that does not comprise said targeted genome modification.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said targeted genome modification is introduced using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes (CRISPR/Cas) system, Transcription activator-like effector nuclease (TALEN), Zinc Finger Nuclease (ZFN), meganuclease or any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said CRISPR/Cas genes or proteins are selected from the group consisting of Cas3, Cas4, Cas5, Cas5e (or CasD), Cash, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cast10d, Cas12, Cas13, Cas14, CasX, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn1, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said targeted genome modification is introduced via (i) at least one RNA-guided (gRNA) endonuclease or nucleic acid encoding at least one RNA-guided endonuclease, and (ii) at least one guide RNA (gRNA) or DNA encoding at least one guide RNA (gRNA), further wherein each of said at least one gRNA directs said endonuclease to a targeted site located in the genomic sequence of said at least one Cannabis gene encoding a cannabinoid biosynthesis enzyme.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein (a) the gRNA nucleotide sequence targeted to CsTHCAS genomic sequence is as set forth in nucleotide sequence selected from the group consisting of SEQ. ID. NO.: 16-167 and any combination thereof; (b) the gRNA nucleotide sequence targeted to CsCBDAS genomic sequence is as set forth in nucleotide sequence selected from the group consisting of SEQ. ID. NO.: 168-304, SEQ. ID. NO.: 824-825, and any combination thereof; (c) the gRNA nucleotide sequence targeted to CsPT genomic sequence is as set forth in nucleotide sequence selected from the group consisting of SEQ. ID. NO.: 305-458 and any combination thereof; (d) the gRNA nucleotide sequence targeted to CsOLS genomic sequence is as set forth in nucleotide sequence selected from the group consisting of SEQ. ID. NO.: 459-509 and any combination thereof; and (e) the gRNA nucleotide sequence targeted to CsAAE1 genomic sequence is as set forth in nucleotide sequence selected from the group consisting of SEQ. ID. NO.: 510-823, SEQ. ID. NO.: 826, and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said plant is mutated in a gene selected from the group consisting of CsTHCAS, CsSP, CsCBDAS, CsPT, CsOLS, CsAAE1 and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said at least one genome modification is generated in planta.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said genome modification is generated in planta via introduction of a construct comprising (a) Cas DNA and gRNA sequence selected from the group consisting of SEQ ID NO:16-SEQ ID NO:826 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:16-826 and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said gRNA is introduced into the plant cells via 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.
It is a further object of the present invention to disclose a plant part, plant cell or plant seed produced by the method as defined in any of the above.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said Cannabis plant comprises a DNA encoding an antisense RNA or a siRNA effective to suppress expression of CsTHCAS, CsSP, CsCBDAS, CsPT, CsOLS, CsAAE1 and any combination thereof, the DNA operably linked to a heterologous promoter, wherein the Cannabis plant comprises reduced THC content, reduced CBD content, or decreased THC and CBD content relative to a Cannabis plant of a similar genotype that does not comprise the DNA.
It is a further object of the present invention to disclose a method for producing a medical Cannabis composition, the method comprising: (a) obtaining the Cannabis plant of claim 1; and (b) formulating a medical Cannabis composition from said plant.
It is a further object of the present invention to disclose a method for manipulating a content of one or more cannabinoids in a Cannabis plant, the method comprising down-regulating activity of at least one Cannabis gene encoding a cannabinoid biosynthesis enzyme selected from the group consisting of tetrahydrocannabinolic acid synthase (THCAS), cannabidiolic acid synthase (CBDAS), aromatic prenyltransferase (PT), olivetol synthase (OLS), acyl-activating enzyme 1 (AAE1) and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, comprises steps of: (a) identifying at least one Cannabis gene locus encoding a cannabinoid biosynthesis enzyme selected from the group consisting of tetrahydrocannabinolic acid synthase (THCAS), cannabidiolic acid synthase (CBDAS), aromatic prenyltransferase (PT), olivetol synthase (OLS), acyl-activating enzyme 1 (AAE1) and any combination thereof; (b) identifying at least one endonuclease recognition sequence in or proximal to the at least one cannabinoid biosynthesis enzyme gene locus; (c) providing at least one guide RNA (gRNA) comprising a nucleotide sequence at least partially complementary to said at least one identified gene locus; (d) introducing into Cannabis plant cells a construct comprising (i) an endonuclease nucleotide sequence operably linked to said gRNA, or (ii) a ribonucleoprotein (RNP) complex comprising an endonuclease protein and said gRNA; (e) assaying the Cannabis plant or a cell thereof for an endonuclease-mediated modification in the DNA of the at least one cannabinoid biosynthesis enzyme gene locus; and (f) identifying the Cannabis plant, a cell thereof, or a progeny cell thereof as comprising a modification in the at least one cannabinoid biosynthesis enzyme gene locus.
It is a further object of the present invention to disclose the method as defined in any of the above, further comprises steps of (a) screening the genome of the transformed Cannabis plant cells for induced targeted mutations in at least one of said cannabinoid biosynthesis enzyme gene locus comprising obtaining a nucleic acid sample from said transformed plant and carrying out nucleic acid amplification and optionally restriction enzyme digestion to detect a mutation in said at least one of said cannabinoid biosynthesis enzyme gene locus; (b) confirming the presence of said genetic mutation in the genome of said plant cells by sequencing said at least one cannabinoid biosynthesis enzyme gene locus; (c) regenerating plants carrying said genetic modification; and (d) screening said regenerated plants for a plant with modified cannabinoid content.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein the endonuclease is expressed transiently or stably in the Cannabis plant.
It is a further object of the present invention to disclose an isolated nucleotide sequence having at least 75% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, and SEQ ID NO:13.
It is a further object of the present invention to disclose an isolated nucleotide sequence having at least 75% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11 and SEQ ID NO:14.
It is a further object of the present invention to disclose an isolated amino acid sequence having at least 75% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12 and SEQ ID NO:15.
It is a further object of the present invention to disclose an isolated nucleotide sequence having at least 75% sequence identity to a nucleotide sequence as set forth in SEQ ID NO:16-826.
It is a further object of the present invention to disclose a vector, construct or expression system or cassette comprising the nucleic acid sequence as defined in any one of claims 90-93.
It is a further object of the present invention to disclose use of a nucleotide sequence as set forth in at least one of SEQ ID NO:16-826 and any combination thereof for down regulation of at least one Cannabis cannabinoid biosynthesis enzyme gene selected from the group consisting of Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS), Cannabis cannabidiolic acid synthase (CsCBDAS), Cannabis aromatic prenyltransferase (CsPT), Cannabis olivetol synthase (CsOLS), Cannabis acyl-activating enzyme 1 (CsAAE1) and any combination thereof.
It is a further object of the present invention to disclose use of a nucleotide sequence as set forth in at least one of SEQ ID NO:16-167 and any combination thereof for targeted genome modification of Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS) gene.
It is a further object of the present invention to disclose use of a nucleotide sequence as set forth in at least one of SEQ ID NO:168-304, SEQ ID NO:824-825, and any combination thereof for targeted genome modification of Cannabis cannabidiolic acid synthase (CsCBDAS).
It is a further object of the present invention to disclose use of a nucleotide sequence as set forth in at least one of SEQ ID NO:305-458 and any combination thereof for targeted genome modification of Cannabis aromatic prenyltransferase (CsPT).
It is a further object of the present invention to disclose use of a nucleotide sequence as set forth in at least one of SEQ ID NO:459-509 and any combination thereof for targeted genome modification of Cannabis olivetol synthase (CsOLS).
It is a further object of the present invention to disclose use of a nucleotide sequence as set forth in at least one of SEQ ID NO:510-823, SEQ ID NO:826, and any combination thereof for targeted genome modification of Cannabis acyl-activating enzyme 1 (CsAAE1).
Non-limiting examples of embodiments of the disclosure are described below with reference to figures attached hereto that are listed following this paragraph. Identical features that appear in more than one figure are generally labeled with a same label in all the figures in which they appear. A label labeling an icon representing a given feature of an embodiment of the disclosure in a figure may be used to reference the same given feature in other embodiments. Dimensions of features shown in the figures are chosen for convenience and clarity of presentation and are not necessarily shown to scale.
The invention, features and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanied drawings. Embodiments of the invention are illustrated by way of example and not limitation in the figures of the accompanying drawings, in which like reference numerals indicate corresponding, analogous or similar elements, and in which:
It will be appreciated that, for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn accurately or to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity, or several physical components may be included in one functional block or element. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.
In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components, modules, units and/or circuits have not been described in detail so as not to obscure the invention. Some features or elements described with respect to one embodiment may be combined with features or elements described with respect to other embodiments. For the sake of clarity, discussion of same or similar features or elements may not be repeated.
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.
The present invention discloses manipulation of the biosynthesis pathways of a Cannabis plant of genus Cannabis. Accordingly, Cannabis plants of the present invention having a modified therapeutic component(s) profile may be useful in the production of medical Cannabis and/or may also be useful in the production of specific components or therapeutic formulations derived therefrom.
According to other main aspects of the present invention, THC free (e,g, hemp) plants for seeds, fiber and/or medical use are produced.
According to a main embodiment, the present invention provides a Cannabis plant with reduced delta-9-tetrahydrocannabinol (THC) content, or reduced cannabidiol (CBD) content, or reduced THC and CBD content, wherein said plant comprises at least one targeted genome modification effective in decreasing expression of a at least one Cannabis gene encoding a cannabinoid biosynthesis enzyme selected from the group consisting of Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS), Cannabis cannabidiolic acid synthase (CsCBDAS), Cannabis aromatic prenyltransferase (CsPT), Cannabis olivetol synthase (CsOLS), Cannabis acyl-activating enzyme 1 (CsAAE1) and any combination thereof.
According to one embodiment of the present invention, genes encoding cannabinoid precursor synthesis enzymes, namely AAE, PT and OLS are down regulated using targeted genome modification (e.g. gene editing techniques as inter alia presented). These enzymes are responsible for the production of a main cannabinoid precursor cannabigerolic acid (CBGA).
The final steps catalysing the synthesis of major active cannabinoids, namely, cannabichromenic acid (CBCA), cannabidiolic acid (CBDA) and Δ9-tetrahydrocannabinolic acid (THCA), are performed by oxidocyclases, namely, CBCA synthase (CBCAS), CBDA synthase (CBDAS) and THCA synthase (THCAS).
According to a further embodiment of the present invention, cannabinoid biosynthesis enzymes namely cannabidiolic acid (CBDA) synthase (CBDAS) and/or 49-tetrahydrocannabinolic acid (THCA) synthase (THCAS) are down regulated using targeted genome modification (e.g. gene editing techniques as inter alia presented). As a result, the production of the major cannabinoids CBDA (converted into CBD by decarboxylation) and/or THCA (converted into THC by decarboxylation) is significantly reduced and/or totally abolished.
Thus, according to one embodiment, targeted genome modification of one or more of the herein identified Cannabis genes encoding cannabinoid precursor synthesis enzymes, namely, CsAAE, CsPT and CsOLS, negatively affects the production of the cannabinoid precursor CBGA. As a result Cannabis plants with reduced content, or free of, THCA (and/or THC) and reduced content or free of CBDA (and/or CBD) are provided by the present invention.
According to a further embodiment of the present invention, targeted genome modification of the herein identified Cannabis gene encoding cannabinoid synthesis enzyme CsTHCAS results in reduced or no production of THCA and thus Cannabis plants with reduced content, or free of, THCA and/or THC are provided by the present invention.
According to a further embodiment of the present invention, targeted genome modification of the herein identified Cannabis gene encoding cannabinoid synthesis enzyme CsCBDAS results in reduced or no production of CBDA and thus Cannabis plants with reduced content, or free of, CBDA and/or CBD are provided by the present invention.
Breeding Cannabis plants is currently 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 in random crosses. These methods have allowed the construction of the leading Cannabis varieties on the market today. During the last few decades, most of the breeding was focused on the psychoactive phytochemicals of the Cannabis plant. These phytochemicals, known as cannabinoids, are the compounds responsible for the medical attributes of the Cannabis plant.
As the medical Cannabis pharmaceutical industry is focusing on developing new cannabinoid based drugs, and these are mostly extracted from the Cannabis plant, there is a growing need for Cannabis plants bred for producing high levels of specific cannabinoids. In addition, there is a need for advanced breeding programs for food and fiber (Hemp) as well.
The present invention is aimed at enhancing cannabinoid breeding capabilities by using advanced molecular genome editing technologies in order to maximize the plants' phyto-chemical molecules production potential.
According to a further aspect of the present invention, a method or a tool is provided that enables the regulation in planta or the production of specific cannabinoid molecules.
It is further within the scope of the present invention to provide means and methods for in planta modification of specific genes that relate to and/or control the cannabinoid biosynthesis pathways (as indicated in
According to some embodiments of the present invention, the above in planta modification can be based on alternative gene silencing technologies such as Zinc Finger Nucleases (ZFN's), Transcription activator-like effector nucleases (TALEN's), RNA silencing, amiRNA or any other gene silencing technique known in the art.
According to some other embodiments of the present invention, DNA introduction into the plant cells can be done by Agrobacterium infiltration, viral based plasmids for virus induced gene silencing (VIGS) and by mechanical insertion of DNA (PEG, gene gun etc).
According to further aspects of the present invention, it is possible to directly insert the Cas9 protein together with a gRNA in order to bypass the need for in vivo transcription and translation of the Cas9+gRNA plasmid in planta in order to achieve the same desired outcome.
It is a core aspect of the present invention that the above CRISPR/Cas system allows the modification of specific DNA sequences. This is achieved by combining the Cas nuclease (Cas9, Cpf1 or the like) with a guide RNA molecule (gRNA). The gRNA is designed such that it should be complementary to a specific DNA sequence targeted for editing in the plant genome and which guides the Cas nuclease to a specific nucleotide sequence (see
Without wishing to be bound by theory, according to further specific aspects of the present invention, upon reaching the specific 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 creates a mutation around the cleavage site. The 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.
It is further within the scope that by introducing a gRNA with homology to a specific site of a gene described in
It is herein acknowledged that as the pharma industry is interested in extracting the cannabinoids from the Cannabis plant, individual Cannabis plants or strains or varieties containing modulated levels of such cannabinoids can be developed, tailored to the specific needs of the pharma industry thereby increasing the cost effectiveness and attractiveness of this crop.
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 modulated levels or ratios of cannabinoids. More specifically alternation of specific cannabinoids, i.e. THC and CBD is achieved by using genome editing techniques to reduce the expression of enzymes in the cannabinoid biosynthesis pathway.
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.
In order to generate a reproducible product, Cannabis growers are currently using vegetative propagation (cloning or tissue culture). However, in conventional agricultural, genetic stability of field crops and vegetables is maintained by using F1 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). This will allow growing and supplying high quality and reproducible raw material for the pharmaceutical industry.
The current invention discloses the generation of non GMO Cannabis plants with manipulated and controlled cannabinoid content, 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 traits, and there is particular interest in advancing manipulation of genes controlling cannabinoids biosynthesis in Cannabis species, to produce strains which are adapted to specific therapeutic or regulatory needs.
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.
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 cannabinoids content.
To that end, guide RNAs (gRNAs) were designed for each of the target genes herein identified in Cannabis to induce mutations in at least one cannabinoid biosynthesis enzyme including Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS), Cannabis cannabidiolic acid synthase (CsCBDAS), Cannabis aromatic prenyltransferase (CsPT), Cannabis olivetol synthase (CsOLS), Cannabis acyl-activating enzyme 1 (CsAAE1) and any combination thereof.
The present invention shows that Cannabis plants which contain genome editing events with at least one of the CsAAE1, CsOLS, CsPT genes or any combination thereof, express not more than 0.5% THC (or THCA) and CBD (or THCA) by weight. In specific embodiments, such plants express less than 0.5%, preferably less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1% or 0% THC and CBD by weight (e.g. by dry weight).
It is further within the scope that Cannabis plants which contain genome editing events with at least one of the CsAAE1, CsOLS, CsPT genes or any combination thereof, express not more than 0.5% THC (or THCA) and/or CBD (or THCA) by weight. In specific embodiments, such plants express less than 0.5%, preferably less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1% or 0% THC and/or CBD by weight (e.g. by dry weight).
The present invention further shows that Cannabis plants containing genome editing events within the CsTHCAS gene express higher levels of CBD (or CBDA) compared to non-edited plants. In a further embodiment, the CsTHCAS edited plants contain very low levels of THCA (or THC), preferably not more than 0.5% by weight.
The present invention further shows that plants containing genome editing events within the CsCBDAS gene express higher levels of THC (or THCA) as compared to non-edited plants. In a further embodiment, the CsCBDAS edited plants contain very low levels of CBDA (or CBD), preferably not more than 0.5% by weight.
It is a further aspect of the present invention to provide the Cannabis plant as defined in any of the above, wherein said plant has a THC and/or CBD content of up to 30% by weight, particularly between about %0.1 to about 30% by weight, more particularly between about %0.3 to about 30%, even more particularly between about %0.3 to about 10% by weight.
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 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.
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 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.
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 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, 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.
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, 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.
As used herein, the term “progeny” or “progenies” refers in a non limiting 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 CsSP gene or at least one CsSP5G gene.
The term “Cannabis” 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.
The term “nonpsychoactive” refers hereinafter to products or compositions or elements or components of Cannabis not significantly affecting the mind or mental processes.
The term “cannabinoid” refers hereinafter to a class of diverse chemical compounds that act on cannabinoid receptors on cells that repress neurotransmitter release in the brain. These receptor proteins include the endocannabinoids (produced naturally in the body by humans and animals), the phytocannabinoids (found in Cannabis and some other plants), and synthetic cannabinoids.
The main cannabinoids are concentrated in a viscous resin produced in structures known as glandular trichomes. Up until now, at least 113 different cannabinoids have been isolated from the Cannabis plant. The main classes of cannabinoids from Cannabis are THC (tetrahydrocannabinol), THCA (tetrahydrocannabinolic acid), CBD (cannabidiol), CBDA (cannabidiolic acid), CBN (cannabinol), CBG (cannabigerol), CBC (cannabichromene), CBL (cannabicyclol), CBV (cannabivarin), THCV (tetrahydrocannabivarin), CBDV (cannabidivarin), CBCV (cannabichromevarin), CBGV (cannabigerovarin), CBGM (cannabigerol monomethyl ether), CBE (cannabielsoin), CBT (cannabicitran) and any combination thereof.
The best studied cannabinoids include tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN).
Reference is now made to Tetrahydrocannabinol (THC), the primary psychoactive component of the Cannabis plant. Delta-9-tetrahydrocannabinol (Δ9-THC, THC) and delta-8-tetrahydrocannabinol (Δ8-THC), through intracellular CB1 activation, induce anandamide and 2-arachidonoylglycerol synthesis produced naturally in the body and brain. These cannabinoids produce the effects associated with Cannabis by binding to the CB1 cannabinoid receptors in the brain.
Tetrahydrocannabinolic acid (THCA, 2-COOH-THC; conjugate base tetrahydrocannabinolate) is a precursor of tetrahydrocannabinol (THC), the active component of cannabis. THCA is found in variable quantities in fresh, undried cannabis, but is progressively decarboxylated to THC with drying, and especially under intense heating such as when cannabis is smoked or cooked into cannabis edibles. In the context of the present invention, the term THC also refers to THCA and vice versa.
Reference is now made to Cannabidiol (CBD) which is considered as non-psychotropic. Cannabidiol has little affinity for CB1 and CB2 receptors but acts as an indirect antagonist of cannabinoid agonists. It is further acknowledged herein that it is an antagonist at the putative cannabinoid receptor, GPR55, a GPCR expressed in the caudate nucleus and putamen. Cannabidiol has also been shown to act as a 5-HT1A receptor agonist. Cannabis produces CBD-carboxylic acid through the same metabolic pathway as THC, until the next to last step, where CBDA synthase performs catalysis instead of THCA synthase. CBDA is converted into CBD by decarboxylation. In the context of the present invention, the term CBD also refers to CBDA and vice versa.
CBD shares a precursor with THC and is the main cannabinoid in CBD-dominant Cannabis strains.
In the context of the current invention, enzymes within the biosynthetic pathway of THC and CBD, especially AAE, OLS, PT, CBDAS and THCAS (depicted in
Reference is now made to
Cannabigerolic acid (CBGA), the precursor to all natural cannabinoids, is cyclized into tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) by THCA and CBDA synthase (THCAS and CBDAS in
A more specific and detailed description of the biosynthetic pathway of cannabinoids in the Cannabis plant follows below:
The isoprenoid and prenyl precursors for cannabigerolic acid (CBGA), are provided by the hexanoate and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, respectively. Geranyl diphosphate (GPP), is a key intermediate metabolite and building block for both cannabinoid and terpenoid biosynthesis. The seven-step mevalonate (MVA) pathway converts pyruvate and glyceraldehyde-3-phosphate (G-3-P) into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Key catalytic enzymes controlling flux through this pathway include the first two steps, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 1-deoxy-D-xylulose 5-phosphate reductase (DXR). In the six-step MEP pathway, three units of acetyl coenzyme A (CoA) are converted to IPP, which is isomerized with DMAPP by IPP isomerase. The enzyme catalysing the synthesis of MEV, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), is considered to control flux through this pathway. The number of consecutive condensations of the five-carbon monomer isopentenyl diphosphate (IPP) to its isomer, dimethylallyl diphosphate (DMAPP) is indicated by 1x, 2x, 3x. Longer-chain isoprenoids, GPP, farnesyl diphosphate (FPP) and geranyl geranyl diphosphate (GGPP), are the products of IPP and DMAPP condensation catalysed by GPP synthase, FPP synthase and GGPP synthase, respectively. GPP, FPP and geranyl-geranyl diphosphate (GGPP) are the precursors for mono-, sequi-, and di-terpines, respectively. The final steps catalysing the synthesis of major active cannabinoids, cannabichromenic acid (CBCA), cannabidiolic acid (CBDA) and Δ9-tetrahydrocannabinolic acid (THCA), are oxidocyclases, CBCA synthase (CBCAS), CBDA synthase (CBDAS) and THCA synthase (THCAS).
In the current invention, AAE, refers to acyl-activating enzyme; CBD: cannabidiol; CYP76F39, α/β-santalene monooxygenase; GPP synthase small subunit; OLS, olivetol synthase; P450: haemoprotein cytochrome P450; PT, prenyltransferase; STS, santalene synthase; TS, gamma-terpinene synthase; and TXS, taxadiene synthase.
As used herein the term “genetic modification” or “genome 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 altered cannabinoid content traits are generated using genome editing mechanism. This technique enables to achieve in planta modification of specific genes that control the biosynthesis of main cannabinoids, namely, THC and/or CBD in the Cannabis plant.
The term “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.
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 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 (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system.
Reference is now made to exemplary genome editing terms used by the current disclosure:
Cas=CRISPR-associated genes
Cas9, Csn1=a CRISPR-associated protein containing two nuclcease domains, that is programmed by small RNAs to cleave DNA
crRNA=CRISPR RNA
dCAS9=nuclease-deficient Cas9
DSB=Double-Stranded Break
gRNA=guide RNA
HDR=Homology-Directed Repair
HNH=an endonuclease domain named for characteristic histidine and asparagine residues
Indel=insertion and/or deletion
NHEJ=Non-Homologous End Joining
PAM=Protospacer-Adjacent Motif
RuvC=an endonuclease domain named for an E. coli protein involved to DNA repair
sgRNA=single guide RNA
tracrRNA, trRNA=trans-activating crRNA
TALEN=Transcription-Activator Like Effector Nuclease
ZFN=Zinc-Finger Nuclease
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 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. 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 Csn1) 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
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 “protospacer 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 Cas9 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. 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. It is within the scope of the present invention that the term 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.
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 synonyms of the term included within the scope of the present invention is null mutation.
The term “microRNAs” or “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.
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. It also used to describe conditions present in a non-laboratory environment (e.g. in vivo).
The term “genotype” or “genetic background” refers hereinafter to the genetic constitution of a cell or organism. An individual's genotype includes the specific alleles, for one or more genetic marker loci, present in the individual's haplotype. As is known in the art, a genotype can relate to a single locus or to multiple loci, whether the loci are related or unrelated and/or are linked or unlinked. In some embodiments, an individual's genotype relates to one or more genes that are related in that the one or more of the genes are involved in the expression of a phenotype of interest. Thus, in some embodiments a genotype comprises a summary of one or more alleles present within an individual at one or more genetic loci. In some embodiments, a genotype is expressed in terms of a haplotype. It further refers to any inbreeding group, including taxonomic subgroups such as subspecies, taxonomically subordinate to species and superordinate to a race or subrace and marked by a pre-determined profile of latent factors of hereditary traits.
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, 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 and hence has the activity of the 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.
The term “variety” 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.
The term “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. 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. A wild type allele is a naturally occurring allele.
As used herein, the term “locus” (loci plural) 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.
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.
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 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 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 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.
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, 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” plant is a plant that has been altered compared to the naturally occurring wild type (WT) plant. Specifically, the endogenous nucleic acid sequences of one or more of the Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS), Cannabis cannabidiolic acid synthase (CsCBDAS), Cannabis aromatic prenyltransferase (CsPT), Cannabis olivetol synthase (CsOLS), Cannabis acyl-activating enzyme 1 (CsAAE1), and any combination thereof, 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 at least one of these endogenous genes and thus disables their function in production of THC and/or CBD, depending on the gene or combination of genes down regulated.
Such plants have an altered cannabinoid profile which may be suitable for treatment of different medical conditions or diseases. Therefore, the cannabinoid profile is affected by the presence of at least one mutated endogenous cannabinoid biosynthesis enzyme gene in the Cannabis plant genome which has been specifically targeted using genome editing technique.
As used herein, the term “cannabinoid biosynthesis enzyme” refers to a protein acting as a catalyst for producing one or more cannabinoids in a plant of genus Cannabis.
Examples of cannabinoid biosynthesis enzymes within the context of this disclosure include, but are not limited to: tetrahydrocannabinolic acid synthase (THCAS), cannabidiolic acid synthase (CBDAS), aromatic prenyltransferase (PT), olivetol synthase (OLS), acyl-activating enzyme 1 (AAE1), polyketide synthase (PKS), olivetolic acid cyclase (OAC), tetraketide synthase (TKS), type III PKS, chalcone synthase (CHS), prenyltransferase, CBCA synthase, GPP synthase, FPP synthase, Limonene synthase, aromatic prenyltransferase, and geranylphosphate: olivetolate geranyltrasferase.
Disclosed herein, is a method of controlling cannabinoid synthesis in a plant of genus Cannabis. In some embodiments, the method comprising: Manipulating expression of a gene coding for a cannabinoid biosynthesis enzyme selected from the group consisting of Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS), Cannabis cannabidiolic acid synthase (CsCBDAS), Cannabis aromatic prenyltransferase (CsPT), Cannabis olivetol synthase (CsOLS), Cannabis acyl-activating enzyme 1 (CsAAE1) and any combination thereof.
As used herein, the term “controlling” refers to directing, governing, steering, and/or manipulating, specifically reducing, decreasing or down regulating or silencing the amount of a cannabinoid or cannabinoids produced in a plant of genus Cannabis. In one embodiment, controlling comprises modifying a plant of genus Cannabis to produce an unnaturally occurring concentration of a first cannabinoid. In one embodiment, controlling comprises modifying a plant of genus Cannabis to produce an unnaturally occurring ratio of a first cannabinoid. In one embodiment, controlling comprises modifying a plant of genus Cannabis to produce an unnaturally occurring concentration of a second cannabinoid. In one embodiment, controlling comprises modifying a plant of genus Cannabis to produce an unnaturally occurring ratio of a second cannabinoid.
As used herein, the term “expression of a gene” refers to a plant's ability to utilize information from genetic material for producing functional gene products. Within the context of this disclosure, expression is meant to encompass the plant's ability to produce proteins, such as enzymes, and various other molecules from the plant's genetic material. In one embodiment, the plant expresses mutated or modified cannabinoid biosynthesis enzymes for cannabinoid biosynthesis. In one embodiment it refers to transcription (RNA) or translation (protein) levels of gene expression.
As used herein, the term “manipulating expression of a gene” refers to intentionally changing the genome of a plant of genus Cannabis to control the expression of certain features.
In one embodiment, the plant's genome is manipulated to express less CBDA synthase.
In one embodiment, the plant's genome is manipulated to express less THCA synthase.
In one embodiment, the plant's genome is manipulated to express less aromatic prenyltransferase (PT).
In one embodiment, the plant's genome is manipulated to express less olivetol synthase (OLS).
In one embodiment, the plant's genome is manipulated to express less acyl-activating enzyme 1 (AAE1).
According to a further embodiment, the plant's genome is manipulated to express less of any combination of the above mentioned cannabinoid biosynthesis enzymes.
As used herein, the term “coding” refers to storing genetic information and accessing the genetic information for producing functional gene products.
According to further aspects of the present invention, the altered THC and/or CBD content trait is not conferred by the presence of transgenes expressed in Cannabis.
Cannabis plants of the invention are modified plants compared to wild type plants which comprise and express at least one mutant Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS), Cannabis cannabidiolic acid synthase (CsCBDAS), Cannabis aromatic prenyltransferase (CsPT), Cannabis olivetol synthase (CsOLS), Cannabis acyl-activating enzyme 1 (CsAAE1) and any combination thereof 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.
Described are polynucleotides as well as methods for modifying metabolite biosynthesis pathways in Cannabis plants and/or Cannabis plant cells, Cannabis plants and/or plant cells exhibiting modified metabolite biosynthesis pathways. In particular, described are methods for modifying production of THC and/or CBD in Cannabis plants by modulating the expression and/or activity of at least one of Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS), Cannabis cannabidiolic acid synthase (CsCBDAS), Cannabis aromatic prenyltransferase (CsPT), Cannabis olivetol synthase (CsOLS), Cannabis acyl-activating enzyme 1 (CsAAE1) and any combination thereof and Cannabis plants having modified expression and/or activity of at least one of these genes/proteins.
Accordingly, in certain embodiments, the present invention provides methods of downregulating production of THC and/or CBD. In particular embodiments, there is provided methods of downregulating expression and/or activity THCA synthase and/or CBDA synthase.
Also provided are plants and/or plant cells having modified production of THC and/or CBD. In certain embodiments, there are provided Cannabis plants and/or cells having down-regulated expression of and/or activity of at least one of Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS), Cannabis cannabidiolic acid synthase (CsCBDAS), Cannabis aromatic prenyltransferase (CsPT), Cannabis olivetol synthase (CsOLS), Cannabis acyl-activating enzyme 1 (CsAAE1) and any combination thereof.
Down regulation of key steps in metabolic pathway re-directs intermediates and energy to alternative metabolic pathways and results in increased production and accumulation or reduced production and elimination of other end products. THC and other Cannabis metabolites share a biosynthetic pathway; that cannabigerolic acid is a precursor of THC, CBD and Cannabichromene. In particular, THCA synthase catalyzes the production of delta-9-tetrahydrocannabinolic acid from cannabigerolic acid; delta-9-tetrahydrocannabinolic undergoes thermal conversion to form THC. CBDA synthase catalyzes the production of cannabidiolic acid from cannabigerolic acid; cannabidiolic acid undergoes thermal conversion to CBD. CBCA synthase catalyzes the production of cannabichromenic acid from cannabigerolic acid; cannabichromenic acid undergoes thermal conversion to cannabichromene.
A reduction in the production of THC, CBD, or Cannabichromene will enhance production of the remaining metabolites in this shared pathway. For example, production of CBD and/or Cannabichromene is enhanced by inhibiting production of THC. THC production may be inhibited by inhibiting expression and/or activity of tetrahydrocannabinolic acid (THCA) synthase enzyme.
Described are certain embodiments of enhancing production of one or more secondary metabolites by downregulation of the production of one or more metabolites having a shared biosynthetic pathway. Certain embodiments provide methods of enhancing production of one or more secondary metabolites that share steps and intermediates in the THC and/or CBD biosynthetic pathway by downregulation of THC and/or CBD production. In specific embodiments, there are provided methods of enhancing production of CBD and/or Cannabichromene by inhibiting production of THC. In other specific embodiments, there are provided methods of enhancing production of THC by inhibiting production of CBD.
In other specific embodiments, both the production of CBD and THC is inhibited by targeting at least one of the herein identified genes CsAAE1 (SEQ ID NO:13), or CsOLS (SEQ ID NO:10), or CsPT (SEQ ID NO:7) or any combination thereof.
In other specific embodiments, the production of CBD is inhibited (and THC is induced or not affected) by targeting the herein identified gene CsCBDAS (SEQ ID NO:4).
In other specific embodiments, the production of THC is inhibited (and CBD is induced or not affected) by targeting the herein identified gene CsTHCAS (SEQ ID NO:1).
Certain embodiments provide methods of enhancing production of one or more secondary metabolites which share steps and intermediates in the THC biosynthetic pathway by downregulation of expression and/or activity of CsTHCA synthase (SEQ ID NO:1).
In specific embodiments, there are provided methods of enhancing production of CBD and/or Cannabichromene by downregulation of expression and/or activity of THCA synthase.
Also provided are plants and plant cells having modified production of one or more metabolites having a shared biosynthetic pathway. In certain embodiments, there are provided Cannabis plants and cells enhanced production of one or more secondary metabolites and downregulation of one or more other metabolites having a shared biosynthetic pathway. In certain embodiments, there are provided Cannabis plants and cells having enhanced production of one or more secondary metabolites and downregulation of one or more other metabolites in the THC and or CBD biosynthetic pathway. In certain embodiments, there are provided Cannabis plants and cells having enhanced production of one or more secondary metabolites in the THC biosynthetic pathway and downregulated THC production. In specific embodiments, there are provided Cannabis plants and cells having enhanced production of CBD and/or Cannabichromene and downregulated THC production.
In specific embodiments, there are provided Cannabis plants and/or cells having enhanced production of CBD and/or Cannabichromene and downregulated expression and/or activity of THCA synthase.
The loss of function mutation may be a deletion or insertion (“indels”) with reference the wild type 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 other 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.
It is further within the scope that variants of a particular nucleotide or amino acid 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 nucleotide sequence of the Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS), Cannabis cannabidiolic acid synthase (CsCBDAS), Cannabis aromatic prenyltransferase (CsPT), Cannabis olivetol synthase (CsOLS), Cannabis acyl-activating enzyme 1 (CsAAE1) allele as shown in SEQ ID NO 1, 4, 7, 10 or 13; and/or SEQ ID NO 2, 5, 8, 11 or 14, respectively. Sequence alignment programs to determine sequence identity are well known in the art.
Also, the various aspects of the invention encompass not only a Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS), Cannabis cannabidiolic acid synthase (CsCBDAS), Cannabis aromatic prenyltransferase (CsPT), Cannabis olivetol synthase (CsOLS), Cannabis acyl-activating enzyme 1 (CsAAE1) nucleic acid sequence or amino acid sequence, but also fragments thereof. By “fragment” is 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 cannabinoid biosynthesis enzymes.
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 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. According to one embodiment of the present invention, this is achieved by combining the Cas nuclease (e.g. Cas9, Cpf1 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
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 manipulation of cannabinoid biosynthesis enzymes in Cannabis plants is herein achieved by generating gRNA with homology to a specific site of predetermined genes in the Cannabis genome i.e. Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS), Cannabis cannabidiolic acid synthase (CsCBDAS), Cannabis aromatic prenyltransferase (CsPT), Cannabis olivetol synthase (CsOLS), Cannabis acyl-activating enzyme 1 (CsAAE1) genes, sub cloning this gRNA into a plasmid containing the Cas9 gene, and insertion of the plasmid into the Cannabis plant cells. In this way site specific mutations in the aforementioned genes are generated thus effectively creating non-active molecules, resulting in loss of function of at least one of the enzymes, reduced content of THC, CBD or both of the cannabinoids in the genome edited plant.
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.
Production of Cannabis lines with mutated Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS) or Cannabis cannabidiolic acid synthase (CsCBDAS) or Cannabis aromatic prenyltransferase (CsPT) or Cannabis olivetol synthase (CsOLS) or Cannabis acyl-activating enzyme 1 (CsAAE1) gene or any combination thereof may be achieved by at least one of the following breeding/cultivation schemes:
Scheme 1:
Scheme 2:
It is noted that line stabilization may be performed by the following:
According to some embodiments of the present invention, line stabilization requires about 6 self-crossing (6 generations) and done through a single seed descent (SSD) approach.
F1 hybrid seed production: Novel hybrids are produced by crosses between different Cannabis strains.
According to a further aspect of the current invention, 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.
It is within the scope of the current invention that genetic markers specific for Cannabis are developed and provided by the current invention:
It is further within the scope of the current invention that allele and genetic variation is analysed for the Cannabis strains used.
Reference is now made to optional stages that have been used for the production of mutated Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS), Cannabis cannabidiolic acid synthase (CsCBDAS), Cannabis aromatic prenyltransferase (CsPT), Cannabis olivetol synthase (CsOLS), Cannabis acyl-activating enzyme 1 (CsAAE1) Cannabis plants by genome editing:
Stage 1: Identifying Cannabis sativa (C. sativa), C. indica and C. ruderalis tetrahydrocannabinolic acid synthase (THCAS), cannabidiolic acid synthase (CBDAS), aromatic prenyltransferase (PT), olivetol synthase (OLS), acyl-activating enzyme 1 (AAE1) orthologues/homologs.
The following homologs have herein been identified in Cannabis sativa (C. sativa), C. indica and C. ruderalis, namely Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS), Cannabis cannabidiolic acid synthase (CsCBDAS), Cannabis aromatic prenyltransferase (CsPT), Cannabis olivetol synthase (CsOLS) and Cannabis acyl-activating enzyme 1 (CsAAE1). These homologous genes have been sequenced and mapped.
CsTHCAS has been mapped to CM011610.1:22243181-22246809 and has a genomic sequence as set forth in SEQ ID NO:1. The CsTHCAS gene has a coding sequence as set forth in SEQ ID NO:2 and it encodes an amino acid sequence as set forth in SEQ ID NO:3.
CsCBDAS has been mapped to CM011610.1:21836038-21839672 and has a genomic sequence as set forth in SEQ ID NO:4. The CsCBDAS gene has a coding sequence as set forth in SEQ ID NO:5 and it encodes an amino acid sequence as set forth in SEQ ID NO:6.
CsPT has been mapped to CM011614.1:1184501-1186728 and has a genomic sequence as set forth in SEQ ID NO:7. The CsPT gene has a coding sequence as set forth in SEQ ID NO:8 and it encodes an amino acid sequence as set forth in SEQ ID NO:9.
CsOLS has been mapped to CM011613.1:2335391-2338392 and has a genomic sequence as set forth in SEQ ID NO:10. The CsOLS gene has a coding sequence as set forth in SEQ ID NO:11 and it encodes an amino acid sequence as set forth in SEQ ID NO:12.
CsAAE1 has been mapped to CM011611.1:1210973-1228229 and has a genomic sequence as set forth in SEQ ID NO:13. The CsAAElgene has a coding sequence as set forth in SEQ ID NO:14 and it encodes an amino acid sequence as set forth in SEQ ID NO:15.
Stage 2: Designing and synthesizing gRNA molecules corresponding to the sequence targeted for editing, i.e. sequences of each of the genes Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS), Cannabis cannabidiolic acid synthase (CsCBDAS), Cannabis aromatic prenyltransferase (CsPT), Cannabis olivetol synthase (CsOLS), Cannabis acyl-activating enzyme 1 (CsAAE1). It is noted that 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.
According to some aspects of the invention, 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 Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS), Cannabis cannabidiolic acid synthase (CsCBDAS), Cannabis aromatic prenyltransferase (CsPT), Cannabis olivetol synthase (CsOLS), Cannabis acyl-activating enzyme 1 (CsAAE1) homologues of different Cannabis strains.
Reference is now made to Tables 1-5 presenting gRNA molecules targeted for silencing Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS), Cannabis cannabidiolic acid synthase (CsCBDAS), Cannabis aromatic prenyltransferase (CsPT), Cannabis olivetol synthase (CsOLS), Cannabis acyl-activating enzyme 1 (CsAAE1), 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.
Reference is made to Table 6 presenting a summary of the sequences within the scope of the current invention.
The above gRNA molecules have been cloned into suitable vectors and their sequence has been verified. In addition 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 Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS), Cannabis cannabidiolic acid synthase (CsCBDAS), Cannabis aromatic prenyltransferase (CsPT), Cannabis olivetol synthase (CsOLS), Cannabis acyl-activating enzyme 1 (CsAAE1) genes.
Stage 3: Transforming Cannabis plants using Agrobacterium or biolistics (gene gun) methods. For 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. For biolistics, 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.
According to some embodiments of the present invention, transformation of various Cannabis tissues was performed using particle bombardment of:
According to further embodiments of the present invention, transformation of various Cannabis tissues was performed using Agrobacterium (Agrobacterium tumefaciens) by:
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.
Reference is now made to
According to further embodiments of the present invention, 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.
Reference is now made to
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.
Reference is now made to
Screening for CRISPR/Cas9 gene editing events has been performed by at least one of the following analysis methods:
Reference is now made to
Stage 6: Selection of transformed Cannabis plants presenting reduced expression of at least one of Cannabis tetrahydrocannabinolic acid synthase (CsTHCAS), Cannabis cannabidiolic acid synthase (CsCBDAS), Cannabis aromatic prenyltransferase (CsPT), Cannabis olivetol synthase (CsOLS), Cannabis acyl-activating enzyme 1 (CsAAE1) as described above. It is within the scope that different gRNA promoters were tested in order to maximize editing efficiency.
Various embodiments have been presented. Each of these embodiments may of course include features from other embodiments presented, and embodiments not specifically described may include various features described herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL2019/050920 | 8/15/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62719151 | Aug 2018 | US |
