Patent Application
20220090118
The present disclosure relates to conferring pathogen resistance in Cannabis plants. More particularly, the current invention pertains to producing fungal resistant Cannabis plants by controlling genes conferring susceptibility to such pathogens.
Cannabis is one of the oldest domesticated plants with evidence of being used by a vast array of ancient cultures. It is thought to have originated from central Asia from which it was spread by humans to China, Europe, the Middle East and the Americas. Thus, Cannabis has been bred by many different cultures for various uses such as food, fiber and medicine since the dawn of agricultural societies. In the last few decades, Cannabis breeding has stopped as it became illegal and non-economic to do so. With the recent legislation converting Cannabis back to legality, there is a growing need for the implementation of new and advanced breeding techniques in future Cannabis breeding programs. This will allow speeding up the long process of classical breeding and accelerate reaching new and genetically improved Cannabis varieties for fiber, food and medicine products. Developing and implementing molecular biology tools to support the breeders, will allow creating new fungal resistant traits and tracking the movement of such desired traits across breeders germplasm.
Currently, breeding of Cannabis plants is mostly done by small Cannabis growers. There is very limited if any molecular tools supporting or leading the breeding process. Traditional Cannabis breeding is done by mixing breeding material with hope to find the desired traits and phenotypes by random crosses. These methods have allowed the construction of the leading Cannabis varieties on the market today. As the cultivation of Cannabis intensifies in protected structures such as greenhouses and closed growth chambers, such an environment encourages the prevalence of certain diseases, with the lead cause being fungi.
Powdery mildew is a fungal disease that affects a wide range of plants. Powdery mildew diseases are caused by many different species of fungi in the order Erysiphales, with Podosphaera xanthii being the most commonly reported cause. Powdery mildew is one of the easier plant diseases to identify, as its symptoms are quite distinctive. Infected plants display white powdery spots on the leaves and stems. The lower leaves are the most affected, but the mildew can appear on any above-ground part of the plant. As the disease progresses, the spots get larger and denser as large numbers of asexual spores are formed, and the mildew may spread up and reduce the length of the plant.
Powdery mildew grows well in environments with high humidity and moderate temperatures. Greenhouses provide an ideal moist, temperate environment for the spread of the disease. This causes harm to agricultural and horticultural practices where powdery mildew may thrive in a greenhouse setting. In an agricultural or horticultural setting, the pathogen can be controlled using chemical methods, bio organic methods, and genetic resistance. It is important to be aware of powdery mildew and its management as the resulting disease can significantly reduce important crop yields.
MLO proteins function as negative regulators of plant defense to powdery mildew disease. Loss-of-function mlo alleles in barley, Arabidopsis and tomato have been reported to lead to broad-spectrum and durable resistance to the fungal pathogen causing powdery mildew.
U.S. Pat. Nos. 6,211,433 and 6,576,814 describe modulating the expression of Mlo genes in Maize by producing transgenic plants comprising mutation-induced recessive alleles of maize Mlo. However, such methods require genetically modifying the plant genome, particularly transforming plants with external foreign genes that enhance disease resistance.
US2018208939 discloses the generation of mutant wheat lines with mutations inactivating MLO alleles which confer heritable resistance to powdery mildew fungus.
Cannabis cultivation has always suffered from fungal diseases due to high humidity growing conditions in growth rooms or greenhouses.
In view of the above there is a heightened immediate need for the development of Cannabis plants that carry genetic resistance to fungal diseases, thereby reducing or eliminating the need for fungicide use in the cultivation of Cannabis. In addition, there is a need for non-GMO, advanced breeding programs of Cannabis for food, medicine and fiber (Hemp) production.
It is one object of the present invention to disclose a modified Cannabis plant exhibiting enhanced resistance to powdery mildew (PM), wherein said plant comprises a targeted genome modification conferring reduced expression of at least one Cannabis MLO (CsMLO) allele as compared to a Cannabis plant lacking said targeted genome modification.
It is a further object of the present invention to disclose the modified Cannabis plant as defined above, wherein said targeted genome modification is in a CsMLO allele having a wild type genomic nucleotide sequence selected from the group consisting of CsMLO1 having a sequence as set forth in SEQ ID NO:1 or a functional variant thereof, CsMLO2 having a sequence as set forth in SEQ ID NO:4 or a functional variant thereof and CsMLO3 having a sequence as set forth in SEQ ID NO:7 or a functional variant thereof.
It is a further object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said functional variant has at least 80% sequence identity to the corresponding CsMLO nucleotide sequence.
It is a further object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant has decreased expression levels of at least one Mlo protein, relative to a Cannabis plant lacking said at least one genome modification.
It is a further object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said genomic modification is introduced using mutagenesis, small interfering RNA (siRNA), microRNA (miRNA), artificial miRNA (amiRNA), DNA introgression, endonucleases or any combination thereof.
It is a further object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said genetic modification is introduced using targeted genome modification, preferably said genetic modification is introduced using an endonuclease.
It is a further object of the present invention to disclose the modified 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) gene (CRISPR/Cas), 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 modified Cannabis plant as defined in any of the above wherein said Cas gene is selected from the group consisting of Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, 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 and any combination thereof.
It is a further object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant comprises a recombinant DNA construct, said recombinant DNA construct comprising a promoter operably linked to a nucleotide sequence encoding a plant optimized Cas9 endonuclease, wherein said plant optimized Cas9 endonuclease is capable of binding to and creating a double strand break in a genomic target sequence of said plant genome.
It is a further object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said DNA construct further comprises sgRNA targeted to at least one CsMLO allele selected from the group consisting of CsMLO1, CsMLO2 and CsMLO3.
It is a further object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said sgRNA is targeted to mutate CsMLO1 gene, said sgRNA nucleotide sequence is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50.
It is a further object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant comprises at least one mutated CsMLO1 allele comprising a nucleotide sequence selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO:875, a nucleotide sequence as set forth in SEQ ID NO:877, a nucleotide sequence as set forth in SEQ ID NO:880, a homologue having at least 80% sequence identity to the nucleotide sequence of said at least one mutated CsMLO1 allele and a combination thereof.
It is a further object of the present invention to disclose the modified 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 modified Cannabis plant as defined in any of the above, wherein said genome modification is an insertion, deletion, indel or substitution.
It is a further object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutated CsMLO1 allele comprises a deletion having a nucleotide sequence as set forth in SEQ ID NO.:876, SEQ ID NO.:879 or SEQ ID NO.:881.
It is a further object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutated allele confers an enhanced resistance to powdery mildew as compared to a Cannabis plant comprising a wild type CsMLO1 allele sequence.
It is a further object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said wild type CsMLO1 allele comprises a nucleic acid sequence as set forth in at least one of SEQ ID NO:873, SEQ ID NO:876, SEQ ID NO:879 or SEQ ID NO:881.
It is a further object of the present invention to disclose the modified Cannabis plant as defined in any of the above wherein said genome modification is an induced mutation in the coding region of said allele, a mutation in the regulatory region of said allele, a mutation in a gene downstream in the MLO pathogen response pathway and/or an epigenetic factor.
It is a further object of the present invention to disclose the modified 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 modified Cannabis plant as defined in any of the above wherein said targeted genome modification is generated in planta via introduction of a construct comprising (a) Cas DNA and sgRNA sequence selected from the group consisting of SEQ ID NO:10-SEQ ID NO:870 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and sgRNA sequence selected from the group consisting of SEQ ID NO:10-870 and any combination thereof.
It is a further object of the present invention to disclose the modified Cannabis plant as defined in any of the above wherein said targeted genome modification in said CsMLO1 is generated in planta via introduction of a construct comprising (a) Cas DNA and sgRNA sequence selected from the group consisting of SEQ ID NO:10-SEQ ID NO:286 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and sgRNA sequence selected from the group consisting of SEQ ID NO:10-286 and any combination thereof.
It is a further object of the present invention to disclose the modified Cannabis plant as defined in any of the above wherein said targeted genome modification in said CsMLO2 is generated in planta via introduction of a construct comprising (a) Cas DNA and sgRNA sequence selected from the group consisting of SEQ ID NO:287-SEQ ID NO:625 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and sgRNA sequence selected from the group consisting of SEQ ID NO:287-625 and any combination thereof.
It is a further object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said targeted genome modification in said CsMLO3 is generated in planta via introduction of a construct comprising (a) Cas DNA and sgRNA sequence selected from the group consisting of SEQ ID NO:626-SEQ ID NO:870 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:626-870 and any combination thereof.
It is a further object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said sgRNA sequence comprises a 3′ Protospacer Adjacent Motif (PAM) selected from the group consisting of NGG (SpCas), NNNNGATT (NmeCas9), NNAGAAW (StCas9), NAAAAC (TdCas9) and NNGRRT (SaCas9).
It is a further object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said construct is introduced into the plant cells via Agrobacterium infiltration, virus based plasmids for delivery and/or expression 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 modified Cannabis plant as defined in any of the above, wherein said PM is selected from the group consisting of Golovinomyces cichoracearum, Golovinomyces ambrosiae and a mixture thereof.
It is a further object of the present invention to disclose the modified 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 the modified Cannabis plant as defined in any of the above, wherein said Cannabis plant does not comprise a transgene.
It is a further object of the present invention to disclose a modified Cannabis plant, progeny plant, plant part or plant cell as defined in any of the above.
It is a further object of the present invention to disclose a plant part, plant cell or plant seed of a modified 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 modified Cannabis plant as defined in any of the above.
It is a further object of the present invention to disclose the modified 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.
It is a further object of the present invention to disclose a method for producing a modified Cannabis plant with increased resistance to powdery mildew (PM) comprising introducing using targeted genome modification, at least one genomic modification conferring reduced expression of at least one Cannabis MLO (CsMLO) allele as compared to a Cannabis plant lacking 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 method comprises steps of introducing a targeted genome modification to at least one CsMLO allele having a wild type genomic nucleotide sequence selected from the group consisting of CsMLO1 comprising a sequence as set forth in SEQ ID NO:1 or a functional variant thereof, CsMLO2 comprising a sequence as set forth in SEQ ID NO:4 or a functional variant thereof and CsMLO3 comprising a sequence as set forth in SEQ ID NO:7 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 80% sequence identity to the said CsMLO nucleotide sequence.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said method comprises steps of introducing a loss of function mutation into at least one of CsMLO1, CsMLO2 and CsMLO2 nucleic acid sequence.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said method comprises steps of introducing a deletion mutation into the first exon of CsMLO1 genomic sequence to produce a mutated CsMLO1 allele comprising a nucleotide sequence selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO:875, a nucleotide sequence as set forth in SEQ ID NO:877, a nucleotide sequence as set forth in SEQ ID NO:880, a homologue having at least 80% sequence identity to the nucleotide sequence of said at least one mutated CsMLO1 allele and a combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said modified plant has decreased levels of at least one Mlo protein as compared to wild type Cannabis plant.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said modified plant has decreased levels of at least one Mlo protein as compared to a Cannabis plant comprising a wild type CsMLO1 allele sequence comprising a nucleic acid sequence as set forth in at least one of SEQ ID NO:873, SEQ ID NO:876, SEQ ID NO:879 or SEQ ID NO:881.
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 introduced using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) gene (CRISPR/Cas), 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 Cas gene is selected from the group consisting of Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, 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 and any combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, comprising steps of introducing an expression vector comprising a promoter operably linked to a nucleotide sequence encoding a plant optimized Cas9 endonuclease and sgRNA targeted to at least one CsMLO allele selected from the group consisting of CsMLO1, CsMLO2 and CsMLO3.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said sgRNA nucleotide sequence targeting CsMLO1 is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50.
It is a further object of the present invention to disclose the method as defined in any of the above, comprising steps of introducing and co-expressing in a Cannabis plant Cas9 and sgRNA targeted to at least one of CsMLO1, CsMLO2 and CsMLO3 genes and screening for induced targeted mutations in at least one of CsMLO1, CsMLO2 and CsMLO3 genes.
It is a further object of the present invention to disclose the method as defined in any of the above, comprising steps of screening for induced targeted mutations in at least one of CsMLO1, CsMLO2 and CsMLO3 genes comprising obtaining a nucleic acid sample from a transformed plant and carrying out nucleic acid amplification and optionally restriction enzyme digestion to detect a mutation in at least one of CsMLO1, CsMLO2 and CsMLO3.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said nucleic acid amplification for screening induced targeted mutations in CsMLO1 genomic sequence uses primers having nucleic acid sequence as set forth in SEQ ID NO: 871 and SEQ ID NO: 872.
It is a further object of the present invention to disclose the method as defined in any of the above, further comprising steps of assessing PCR fragments or amplicons amplified from the transformed plants using a gel electrophoresis based assay.
It is a further object of the present invention to disclose the method as defined in any of the above, further comprising steps of confirming the presence of a mutation by sequencing the at least one of CsMLO1, CsMLO2 and CsMLO3 nucleic acid fragment or amlicon.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said mutation is in the coding region of said allele, a mutation in the regulatory region of said allele, a mutation in a gene downstream in the MLO pathogen response pathway or an epigenetic factor.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said mutation is selected from the group consisting of a silencing mutation, a knockdown mutation, a knockout mutation, a loss of function mutation 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 mutation is an insertion, deletion, indel or substitution mutation.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said mutation is a deletion in the first exon of CsMLO1, said deletion comprises nucleic acid sequence selected from the group consisting of SEQ ID NO.:876, SEQ ID NO.:879 or SEQ ID NO.:881.
It is a further object of the present invention to disclose the method as defined in any of the above, further comprising steps of selecting a plant resistant to powdery mildew from transformed plants comprising mutated at least one of CsMLO1, CsMLO2 and CsMLO3 nucleic acid fragment.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said selected plant is characterized by enhanced resistance to powdery mildew as compared to a Cannabis plant comprising a CsMLO1 nucleic acid comprising a nucleic acid sequence as set forth in SEQ ID NO:873.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said genetic modification in said CsMLO1 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:10-SEQ ID NO:286 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:10-286 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 genetic modification in said CsMLO2 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:287-SEQ ID NO:625 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:287-625 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 genetic modification in said CsMLO3 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:626-SEQ ID NO:870 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:626-870 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 nucleotide sequence comprises a 3′ Protospacer Adjacent Motif (PAM), said PAM is selected from the group consisting of: NGG (SpCas9), NNNNGATT (NmeCas9), NNAGAAW (StCas9), NAAAAC (TdCas9) and NNGRRT (SaCas9).
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said construct is introduced into the plant cells using Agrobacterium infiltration, virus based plasmids for delivery of the genome editing molecules by 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 method as defined in any of the above, further comprising steps of regenerating a plant carrying said genomic modification.
It is a further object of the present invention to disclose the method as defined in any of the above, further comprising steps of screening said regenerated plants for a plant resistant to powdery mildew.
It is a further object of the present invention to disclose a method for conferring resistance to powdery mildew to a Cannabis plant comprising producing a plant as defined in any of the above.
It is a further object of the present invention to disclose a plant, plant part, plant cell, tissue culture or a seed obtained or obtainable 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 PM is selected from the group consisting of Golovinomyces cichoracearum, Golovinomyces ambrosiae and a mixture 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 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 method for producing a modified Cannabis plant with increased resistance to powdery mildew compared to a Cannabis wild type plant using targeted genome modification comprising introducing at least one genetic modification conferring reduced expression of at least one Cannabis MLO (CsMLO) allele, said method comprises steps of: (a) identifying at least one Cannabis MLO (CsMLO) orthologous allele; (b) sequencing genomic DNA of said at least one identified CsMLO; (c) synthetizing at least one guide RNA (gRNA) comprising a nucleotide sequence complementary to said at least one identified CsMLO; (d) transforming Cannabis plant cells with a construct comprising (a) Cas nucleotide sequence and said gRNA, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and said gRNA; (e) screening the genome of said transformed plant cells for induced targeted mutations in at least one of said CsMLO alleles 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 CsMLO allele; (f) confirming the presence of said genetic mutation in the genome of said plant cells by sequencing said at least one CsMLO allele; (g) regenerating plants carrying said genetic modification; and (h) screening said regenerated plants for a plant resistant to powdery mildew.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said method comprises steps of introducing a targeted genome modification to at least one CsMLO allele having a wild type genomic nucleotide sequence selected from the group consisting of CsMLO1 comprising a sequence as set forth in SEQ ID NO:1 or a functional variant thereof, CsMLO2 comprising a sequence as set forth in SEQ ID NO:4 or a functional variant thereof and CsMLO3 comprising a sequence as set forth in SEQ ID NO:7 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 80% sequence identity to the said CsMLO nucleotide sequence.
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 levels of at least one Mlo protein.
It is a further object of the present invention to disclose the method as defined in any of the above, further comprising steps of introducing into said plant sgRNA targeted to mutate CsMLO1 gene, said sgRNA nucleotide sequence is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said nucleic acid amplification for screening induced targeted mutations in CsMLO1 genomic sequence uses primers having nucleic acid sequence as set forth in SEQ ID NO: 871 and SEQ ID NO: 872.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said plant comprises at least one mutated CsMLO1 allele comprising a nucleotide sequence selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO:875, a nucleotide sequence as set forth in SEQ ID NO:877, a nucleotide sequence as set forth in SEQ ID NO:880, a homologue having at least 80% sequence identity to the nucleotide sequence of said at least one mutated CsMLO1 allele and a combination thereof.
It is a further object of the present invention to disclose the method 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 method as defined in any of the above, wherein said mutated CsMLO1 allele comprises a deletion having a nucleotide sequence as set forth in SEQ ID NO.:876, SEQ ID NO.:879 or SEQ ID NO.:881.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said mutated allele confers an enhanced resistance to powdery mildew as compared to a Cannabis plant comprising a wild type CsMLO1 allele sequence.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said wild type CsMLO1 allele comprises a nucleic acid sequence as set forth in at least one of SEQ ID NO:873, SEQ ID NO:876, SEQ ID NO:879 or SEQ ID NO:881.
It is a further object of the present invention to disclose a method of determining the presence of a mutant CsMLO1 nucleic acid in a Cannabis plant comprising assaying said Cannabis plant with primers having nucleic acid sequence as set forth in SEQ ID NO: 871 and SEQ ID NO: 872.
It is a further object of the present invention to disclose a method for determining the presence or absence of a mutant CsMLO1 nucleic acid or polypeptide in a Cannabis plant comprising detecting the presence or absence of a deletion of a nucleotide sequence as set forth in SEQ ID NO.:876, SEQ ID NO.:879 or SEQ ID NO.:881.
It is a further object of the present invention to disclose a method for identifying a Cannabis plant with resistance to powdery mildew, said method comprises steps of: (a) screening the genome of said Cannabis plant for induced targeted mutations in at least one of CsMLO1, CsMLO2 and/or CsMLO3 alleles having a wild type genomic nucleotide sequence selected from the group consisting of CsMLO1 comprising a sequence as set forth in SEQ ID NO:1 or a functional variant thereof, CsMLO2 comprising a sequence as set forth in SEQ ID NO:4 or a functional variant thereof and CsMLO3 comprising a sequence as set forth in SEQ ID NO:7 or a functional variant thereof; (b) confirming the presence of said genetic mutation in the genome of said plant cells by sequencing said at least one CsMLO allele; (c) regenerating plants carrying said genetic modification; and (d) screening said regenerated plants for a plant resistant to powdery mildew.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said screening for the presence of mutated CsMLO1 allele is carried out using a primer pair having nucleic acid sequence as set forth in SEQ ID NO: 871 and SEQ ID NO: 872.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said method comprises steps of screening for the presence of mutated CsMLO1 allele comprising a nucleic acid sequence selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO:875, a nucleotide sequence as set forth in SEQ ID NO:877, a nucleotide sequence as set forth in SEQ ID NO:880, a homologue having at least 80% sequence identity to the nucleotide sequence of said at least one mutated CsMLO1 allele and a combination thereof.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said method comprises steps of screening said Cannabis plant for the presence of a deletion in CsMLO1 comprising a nucleotide sequence selected from the group consisting of SEQ ID NO.:876, SEQ ID NO.:879 and SEQ ID NO.:881.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein the presence of at least one nucleic acid sequence selected from the group consisting of SEQ ID NO:873, SEQ ID NO:876, SEQ ID NO:879 and SEQ ID NO:881 indicates that the Cannabis plant comprises wild type CsMLO1 nucleic acid, and the presence of at least one nucleic acid sequence selected from the group consisting of SEQ ID NO:875, SEQ ID NO:877 and SEQ ID NO:880, optionally in combination with the absence of at least one nucleic acid sequence selected from the group consisting of SEQ ID NO:876, SEQ ID NO:879 and SEQ ID NO:881 indicates that the Cannabis plant comprises a mutant CsMLO1 nucleic acid.
It is a further object of the present invention to disclose the method as defined in any of the above, wherein said Cannabis plant comprising a mutant CsMLO1 nucleic acid is characterized by enhanced resistance to powdery mildew as compared to a Cannabis plant comprising said wild type CsMLO1 nucleic acid.
It is a further object of the present invention to disclose an isolated nucleotide sequence of a primer or primer pair having at least 75% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, 2, 4, 5, 7, 8 and SEQ ID NO:10-873, 875, 876, 877, 879, 880 and 881.
It is a further object of the present invention to disclose an isolated amino acid sequence having at least 75% sequence similarity 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:874, SEQ ID NO:878 and SEQ ID NO:882.
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:871 and SEQ ID NO:872 as a primer or primer pair for identifying or screening for a Cannabis plant comprising within its genome mutant CsMLO1 nucleic acid and/or polypeptide.
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:871 and SEQ ID NO:872 as a primer or primer pair for identifying or screening for a Cannabis plant resistance to powdery mildew.
It is a further object of the present invention to disclose use of a nucleotide sequence as set forth in SEQ ID NO:873, SEQ ID NO:875, SEQ ID NO:876, SEQ ID NO:877, SEQ ID NO:879, SEQ ID NO:880 and SEQ ID NO:881 for identifying and/or screening for a Cannabis plant with comprising within its genome mutant CsMLO1 nucleic acid and/or polypeptide, wherein, the presence of at least one nucleic acid sequence selected from the group consisting of SEQ ID NO:873, SEQ ID NO:876, SEQ ID NO:879 and SEQ ID NO:881 indicates that the Cannabis plant comprises wild type CsMLO1 nucleic acid, and the presence of at least one nucleic acid sequence selected from the group consisting of SEQ ID NO:875, SEQ ID NO:877 and SEQ ID NO:880, optionally in combination with the absence of at least one nucleic acid sequence selected from the group consisting of SEQ ID NO:876, SEQ ID NO:879 and SEQ ID NO:881 indicates that the Cannabis plant comprises a mutant CsMLO1 nucleic acid.
It is a further object of the present invention to disclose the use as defined in any of the above, wherein said Cannabis plant comprising a mutant CsMLO1 nucleic acid is characterized by enhanced resistance to powdery mildew as compared to a Cannabis plant comprising said wild type CsMLO1 nucleic acid.
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:10-870 and any combination thereof for targeted genome modification of at least one Cannabis MLO (CsMLO) allele.
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:10-286 and any combination thereof for targeted genome modification of Cannabis CsMLO1 allele.
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:17, SEQ ID NO:43 and SEQ ID NO:50 and any combination thereof for targeted genome modification of Cannabis CsMLO1 allele.
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:287-625 and any combination thereof for targeted genome modification of Cannabis CsMLO2 allele.
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:626-870 and any combination thereof for targeted genome modification of Cannabis CsMLO3.
It is a further object of the present invention to disclose a detection kit for determining the presence or absence of a mutant CsMLO1 nucleic acid nucleic acid or polypeptide in a Cannabis plant comprising a primer selected from SEQ ID NO:871 and SEQ ID NO:872.
It is a further object of the present invention to disclose the detection kit as defined in any of the above, wherein said kit further comprising primers or nucleic acid fragments for detection of a nucleic acid sequence selected from the group consisting of SEQ ID NO:873, SEQ ID NO:875, SEQ ID NO:876, SEQ ID NO:877, SEQ ID NO:879, SEQ ID NO:880 and SEQ ID NO:881.
It is a further object of the present invention to disclose the detection kit as defined in any of the above, wherein said kit is useful for identifying a Cannabis plant resistant to powdery mildew.
Exemplary non-limited embodiments of the disclosed subject matter will be described, with reference to the following description of the embodiments, in conjunction with the figures. The figures are generally not shown to scale and any sizes are only meant to be exemplary and not necessarily limiting. Corresponding or like elements are optionally designated by the same numerals or letters.
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 provides a modified Cannabis plant exhibiting enhanced resistance to powdery mildew (PM), wherein the plant comprises a targeted genome modification conferring reduced expression of at least one Cannabis MLO (CsMLO) allele as compared to a Cannabis plant lacking said targeted genome modification.
The present invention is aimed at showing that lack of mildew resistance loci 0 (MLO) genes in Cannabis is correlated with resistance to PM. It is herein disclosed that MLO deletions are likely to increase PM resistance in Cannabis. According to further aspects of the invention, lack of certain MLO genes is used as markers for pathogen resistance and may accelerate breeding for more resistant Cannabis lines.
According to one embodiment of the present invention, the targeted genome modification is in a CsMLO allele having a wild type genomic nucleotide sequence selected from the group consisting of CsMLO1 having a sequence as set forth in SEQ ID NO:1 or a functional variant thereof, CsMLO2 having a sequence as set forth in SEQ ID NO:4 or a functional variant thereof and CsMLO3 having a sequence as set forth in SEQ ID NO:7 or a functional variant thereof.
According to a further embodiment of the present invention, the functional variant has at least 75%, preferably 80% sequence identity to the corresponding CsMLO nucleotide sequence.
According to a further embodiment of the present invention, the modified Cannabis plant has decreased expression levels of at least one Mlo protein, relative to a Cannabis plant lacking the at least one genome modification.
According to a further embodiment of the present invention, the genome modification is introduced using mutagenesis, small interfering RNA (siRNA), microRNA (miRNA), artificial miRNA (amiRNA), DNA introgression, endonucleases or any combination thereof.
According to a further embodiment of the present invention, the genetic modification is introduced using targeted genome modification, preferably said genetic modification is introduced using an endonuclease.
According to a further embodiment of the present invention, the genome modification is introduced using guide RNA, e.g. single guide RNA (sgRNA) designed and targeted to mutate CsMLO1 gene, said sgRNA nucleotide sequence is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50.
According to a further embodiment of the present invention, the modified Cannabis plant comprises at least one mutated CsMLO1 allele comprising a nucleotide sequence selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO:875, a nucleotide sequence as set forth in SEQ ID NO:877, a nucleotide sequence as set forth in SEQ ID NO:880 or a homologue having at least 80% sequence identity to the nucleotide sequence of said at least one mutated CsMLO1 allele or a combination thereof.
According to a further embodiment of the present invention, the modified Cannabis plant comprises at least one silencing mutation, a knockdown mutation, a knockout mutation, a loss of function mutation or any combination thereof in at least one gene or allele selected from the group consisting of CsMLO1, CsMLO2 and CsMLO3.
According to a further embodiment of the present invention the mutated CsMLO1 allele comprises a deletion having a nucleotide sequence as set forth in SEQ ID NO.:876, SEQ ID NO.:879 or SEQ ID NO.:881.
According to a further embodiment of the present invention, the mutated CsMLO1 allele confers an enhanced resistance to powdery mildew as compared to a Cannabis plant comprising a wild type CsMLO1 allele sequence.
According to a further embodiment of the present invention, the wild type CsMLO1 allele comprises a nucleic acid sequence as set forth in at least one of SEQ ID NO:873, SEQ ID NO:876, SEQ ID NO:879 or SEQ ID NO:881. According to a further embodiment of the present invention the present invention provides modified Cannabis plant exhibiting enhanced resistance to powdery mildew (PM) compared to wild type Cannabis plant, wherein the modified plant comprises a genetic modification conferring reduced expression of at least one Cannabis MLO (CsMLO) allele. The present invention further provides methods for producing the aforementioned modified Cannabis plant using genome editing or modification techniques.
Powdery mildew (PM) is a major fungal disease that threatens thousands of plant species. Powdery mildew is commonly controlled by frequent applications of fungicides, having negative effects on the environment, and leading to additional costs for growers. To reduce the amount of chemicals required to control this pathogen, the development of resistant crop varieties is a priority.
It is herein acknowledged that PM pathogenesis is associated with up-regulation of specific MLO genes during early stages of infection, causing down-regulation of plant defense pathways. These up-regulated genes are responsible for PM susceptibility (S-genes) and their knock-out cause durable and broad-spectrum resistance.
As the Cannabis legal market is expanding worldwide, this agricultural crop will gradually move from indoor growing facilities to simple low cost greenhouses to enable mass production at reduced operational costs. One of the major challenges facing this transition is the lack of compatible genetics (strains) adapted for green house growth and more specifically genetic fungal resistances. Cannabis susceptibility to fungal diseases results in damages and losses to the grower and forces the widespread use of fungicides. Excessive fungicide use poses health threats to Cannabis consumers.
To date, there are no fungal disease resistant Cannabis varieties on the market. Classical breeding programs dedicated to the end of creating fungal disease resistant Cannabis varieties are virtually impossible due to limited genetic variation, legal constraints on import and export of genetic material and limited academic knowledge and gene banks involved is such projects. In addition, traditional breeding is a long process with low rates of success and certainty, as it is based on trial and error.
The solution proposed by the current invention is using genome editing such as the CRISPR/Cas system in order to create fungal disease resistant Cannabis varieties. 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. By using genome editing to generate Powdery Mildew (PM) resistant Cannabis varieties, the current disclosure will allow growers worldwide to supply a safer product to Cannabis consumers.
It is further noted that using genome editing is considered as non GMO by the Israeli regulator and in the US, the USDA has already classified a dozen of genome edited plant as non regulated and non GMO (https://www.usda.gov/media/press-releases/2018/03/28/secretary-perdue-issues-usda-statement-plant-breeding-innovation).
The Cannabis industry's value chain is based on a steady supply of high quality consistent product. Due to lack of suitable genetics adapted for intensive agriculture production, most growing methods are based on cloning as a mean of vegetative propagation in order to ensure genetic consistency of the plant material. These methods are outdated, expensive and not fit for purpose.
The lack of Cannabis strains that are disease resistant, stable and uniform, pose a threat to the ability of supplying the industry with the raw material needed to support itself.
Legal limitations and outdated breeding techniques significantly hamper the efforts of generating new and improved Cannabis varieties fit for intensive agriculture.
Cannabis legalization in certain countries has increased significantly the number of Cannabis growers and area used for growing. One possible solution is moving growing Cannabis into greenhouses (protected growing facilities) like the vegetable industry has been doing for the last few decades. Unlike the vegetable industry, Cannabis is based on vegetative propagation while vegetables are grown through seeds. In addition, Cannabis growers are using Cannabis strains that were bred for indoor cultivation and are now using those for their greenhouse operations. This situation is obviously not ideal and causes many logistic issues for the growers. For example, since Cannabis plants require short days for the induction of flowering, growers install darkening curtains in the greenhouse to control day length for the plants. This artificial darkening results in increased humidity in the greenhouse thus creating optimal conditions for fungal pathogens to spread and thrive. These conditions force growers to intensively use fungicides to control pathogen populations. With strict regulatory constraints in place across the legalized states, these conditions pose a great challenge for sustainable Cannabis production and consumer health.
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). In addition, breeding for basic agronomic traits that are completely lacking in currently available Cannabis varieties (with an emphasis on disease resistances) will significantly increase grower's productivity. This will allow growing and supplying high quality raw material for the Cannabis industry.
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.
Currently, breeding of Cannabis plants is mostly done by small Cannabis growers. There is very limited if any molecular tools supporting or leading the breeding process. Traditional Cannabis breeding is done by mixing breeding material with hope to find the desired traits and phenotypes by random crosses.
The present invention provides for the first time enhanced resistant Cannabis plants to fungal diseases. The current invention disclose the generation of non-transgenic Cannabis plant resistant to the powdery mildew fungal disease, using the genome editing technology, e.g., the CRISPR/Cas9 tool. The generated mutations can be readily introduced into elite or locally adapted Cannabis lines rapidly, with relatively minimal effort and investment.
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%.
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. reduced expression of at least one CsMLO 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.
As used herein the term “genetic modification” refers hereinafter to genetic manipulation or modulation, which is the direct manipulation of an organism's genes using biotechnology. It also refers to a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species, targeted mutagenesis and genome editing technologies to produce improved organisms. According to main embodiments of the present invention, modified Cannabis plants with increased resistance to PM are generated using genome editing mechanism. This technique enables to achieve in planta modification of specific genes that relate to and/or control the infection of powdery mildew 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:
It is noted that it is within the scope of the current invention that the term gRNA also refers to or means single guide RNA (sgRNA).
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, (2-6 nucleotides) 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
Other Cas variants and their PAM sequences (5′ to 3′) within the scope of the current invention include NmeCas9 (isolated from Neisseria meningitides) recognizing NNNNGATT, StCas9 (isolated from Streptococcus thermophiles) recognizing NNAGAAW, TdCas9 (isolated from Treponema denticola) recognizing NAAAAC and SaCas9 (isolated from Staphylococcus aureus) recognizing NNGRRT or NGRRT or NGRRN.
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” or “silence” or 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. In some embodiments of the present invention, gene silencing by targeted genome modification results in non-functional gene products, such as transcripts or proteins, for example non-functional CsMLO1 exon 1 fragments.
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).
As used herein, the term “powdery mildew” or “PM” refers hereinafter to fungi that are obligate, biotrophic parasites of the phylum Ascomycota of Kingdom Fungi. The diseases they cause are common, widespread, and easily recognizable. Infected plants display white powdery spots on the leaves and stems Infection by the fungus is favored by high humidity but not by free water. Powdery mildew fungi tend to grow superficially, or epiphytically, on plant surfaces. During the growing season, hyphae are produced preferably on both upper and lower leaf surfaces. Infections can also occur on stems, flowers, or fruit. Specialized absorption cells, termed haustoria, extend into the plant epidermal cells to obtain nutrition.
Powdery mildew fungi can reproduce both sexually and asexually. Sexual reproduction is via chasmothecia (cleistothecium), a type of ascocarp where the genetic material recombines. Within each ascocarp are several asci. Under optimal conditions, ascospores mature and are released to initiate new infections Conidia (asexual spores) are also produced on plant surfaces during the growing season. They develop either singly or in chains on specialized hyphae called conidiophores. Conidiophores arise from the epiphytic hyphae, or in the case of endophytic hyphae, the conidiophores emerge through leaf stomata. It should be noted that powdery mildew fungi must be adapted to their hosts to be able to infect them. The present invention provides for the first time Cannabis plants with enhanced resistance or tolerance to PM disease. The enhanced resistance to PM is generated by genome editing techniques targeted at silencing at least one Cannabis Mildew Locus O (MLO) gene. The modified resulted Cannabis plant exhibits enhanced resistance to PM as compared to a Cannabis plant lacking the targeted modification.
The term “MLO” or “Mlo” or “mlo” refers hereinafter to the Mildew Locus O (MLO) gene family encoding for plant-specific proteins harboring several transmembrane domains, topologically reminiscent of metazoan G-protein coupled receptors. It is within the scope of the present invention that specific homologs of the MLO family act as susceptibility genes towards PM fungi. It is emphasized that the present invention provides for the first time the identification of MLO orthologous alleles in the Cannabis plant. Three Cannabis MLO alleles or genes (i.e. MLO1, MLO2, MLO3) have been herein identified, namely CsMLO1, CsMLO2 and CsMLO3.
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 protein sequence” as used herein, for example with reference to SEQ ID NOs: 1, 2 or 3 refers to a variant gene sequence or part of the gene sequence which retains the biological function of the full non-variant allele (e.g. CsMLO allele) and hence has the activity of modulating response to PM. 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 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. In the context of the current invention, the term allele refers to the three identified Cannabis MLO genes, namely CsMLO1, CsMLO2 and CsMLO3 having the genomic nucleotide sequence as set forth in SEQ ID NOs: 1, 2 or 3, respectively.
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. In specific embodiments, the tomato plants of the present invention comprise heterozygous configuration of the genetic markers associated with the high yield characteristics.
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 each of the MLO homologs in Cannabis (nucleic acid sequences CsMLO1, CsMLO2 and CsMLO3) have been altered compared to wild type sequences using mutagenesis and/or genome editing methods as described herein. This causes inactivation of the endogenous Mlo genes and thus disables Mlo function. Such plants have an altered phenotype and show resistance or increased resistance to PM compared to wild type plants. Therefore, the resistance is conferred by the presence of at least one mutated endogenous CsMLO1, CsMLO2 and CsMLO3 genes in the Cannabis plant genome which has been specifically targeted using targeted genome modification.
According to further aspects of the present invention, the increased resistance to PM is not conferred by the presence of transgenes expressed in Cannabis.
It should be noted that nucleic acid sequences of wild type alleles are designated using capital letters namely CsMLO1, CsMLO2 and CsMLO3. Mutant mlo nucleic acid sequences use non-capitalization. Cannabis plants of the invention are modified plants compared to wild type plants which comprise and express mutant mlo alleles.
It is further within the scope of the current invention that mlo mutations that down-regulate or disrupt functional expression of the wild-type Mlo sequence may be recessive, such that they are complemented by expression of a wild-type sequence.
A mlo mutant phenotype according to the invention is characterized by the exhibition of an increased resistance against PM. In other words, a mlo mutant according to the invention confers resistance to the pathogen causing PM, which is identified as described inter alia.
It is further noted that a wild type Cannabis plant is a plant that does not have any mutant Mlo alleles.
Main aspects of the invention involve targeted mutagenesis methods, specifically genome editing, and exclude embodiments that are solely based on generating plants by traditional breeding methods. In a further embodiment of the current invention, as explained herein, the disease resistant trait is not due to the presence of a transgene.
The inventors have generated mutant Cannabis lines with mutations inactivating at least one CsMLO homoeoallele which confer heritable resistance to powdery mildew. In this way no functional CsMLO protein is made. Thus, the invention relates to these mutant Cannabis lines and related methods.
According to one embodiment, the present invention provides a modified Cannabis plant exhibiting enhanced resistance to powdery mildew (PM) compared to wild type Cannabis plant. The Cannabis plant of the present invention comprises a genetic modification conferring reduced expression of at least one Cannabis MLO (CsMLO) allele.
It is within the scope of the present invention that the CsMLO allele is selected from the group consisting of CsMLO1 having a nucleotide sequence as set forth in SEQ ID NO:1 or a fragment or a functional variant thereof, CsMLO2 having a nucleotide sequence as set forth in SEQ ID NO:4 or a fragment or a functional variant thereof and CsMLO3 having a nucleotide sequence as set forth in SEQ ID NO:7 or a fragment or a functional variant thereof.
According to a further embodiment of the present invention, the functional variant has at least 75% sequence identity to the CsMLO nucleotide sequence.
It is within the scope of the current invention that genome editing can be achieved using sequence-specific nucleases (SSNs) and results in chromosomal changes, such as nucleotide deletions, insertions or substitutions at specified genetic loci. Non limiting examples of SSNs include zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs) and, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system.
Non limiting examples Cas proteins used by the present invention include Csn1, Cpf1 Cas9, Cas12, Cas13, Cas14, CasX and any combination thereof.
According to further aspects of the invention, Cannabis plant resistant to the powdery mildew fungal pathogen using the CRISPR/Cas9 technology is generated, which is based on the Cas9 DNA nuclease guided to a specific DNA target by a single guide RNA (sgRNA).
It is herein acknowledged that wild-type alleles of MILDEW RESISTANT LOCUS 0 (Mlo), which encodes a membrane-associated protein with seven transmembrane domains, confer susceptibility to fungi causing the powdery mildew disease. Therefore, homozygous loss-of-function mutations (mlo) result in resistance to powdery mildew.
According to certain embodiments of the present invention, in planta modification of specific genes that relate to and/or control the infection of powdery mildew in the Cannabis plant is achieved for the first time by the present invention, i.e. the Cannabis MLO genes (CsMLO). More specifically, but not limited to, the use of gene editing technologies, for example the CRISPR/Cas technology (e.g. Cas9 or Cpf1), in order to generate knockout alleles of genes (i.e. MLO genes) controlling the resistance to powdery mildew (PM) is disclosed for the Cannabis plant. The above in planta modification can be based on alternative gene editing technologies such as Zinc Finger Nucleases (ZFN's), Transcription activator-like effector nucleases (TALEN's), RNA silencing (amiRNA etc.) and/or meganucleases.
The loss of function mutation may be a deletion or insertion (“indels”) with reference the wild type CsMLO allele sequence. The deletion may comprise 1-20 or more, 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, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18 or 20 or more nucleotides in one or more strand.
The plant of the invention includes plants wherein the plant is heterozygous for the each of the mutations. In a preferred embodiment however, the plant is homozygous for the mutations. Progeny that is also homozygous can be generated from these plants according to methods known in the art.
It is further within the scope that variants of a particular CsMLO 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 CsMLO nucleotide sequence of the CsMLO allele as shown in SEQ ID NO 1, 2 or 3. Sequence alignment programs to determine sequence identity are well known in the art.
Also, the various aspects of the invention encompass not only a CsMLO 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 and hence act to modulate responses to PM.
According to a further embodiment of the invention, the herein newly identified Cannabis MLO locus (CsMLO) have been targeted using the triple sgRNA strategy.
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 also possible to create a genome edited plant and use it as a rootstock. Then, the Cas protein and gRNA can be transported via the vasculature system to the top of the plant and create the genome editing event in the scion.
It is within the scope of the present invention that the usage of CRISPR/Cas system for the generation of PM resistant Cannabis plants, 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 resistance to PM in Cannabis plants is produced by generating gRNA with homology to a specific site of predetermined genes in the Cannabis genome i.e. MLO 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 MLO genes are generated thus effectively creating non-active molecules, resulting in inability of powdery mildew and similar organisms of infecting the genome edited plant.
Reference is now made to
Reference is now made to
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.
Exemplified Method for Production of Powdery Mildew Resistant Cannabis Plants by Genome Editing
Production of powdery mildew resistant Cannabis lines 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 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 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 powdery mildew resistant Cannabis plants by genome editing:
Stage 1: Identifying Cannabis sativa (C. sativa) MLO orthologues, Three MLO orthologues have herein been identified in C. sativa, namely CsMLO1, CsMLO2 and CsMLO3. These homologous genes have been sequenced and mapped. CsMLO1 has been found to be located on chromosome 5 between position 58544241 bp and position 58551241 bp and has a genomic sequence as set forth in SEQ ID NO:1. The CsMLO1 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.
CsMLO2 has been found to be located on chromosome 3 between position 92616000 bp and position 92629000 bp and has a genomic sequence as set forth in SEQ ID NO:4. The CsMLO2 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.
CsMLO3 has been found to be located on Chromosome 5 between position 23410000 bp and position 23420000 bp and has a genomic sequence as set forth in SEQ ID NO:7. The CsMLO3 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.
Stage 2: Designing and synthesizing gRNA molecules corresponding to the sequence targeted for editing, i.e. sequences of each of the genes CsMLO1, CsMLO2 and CsMLO3. 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 MLO homologues of different Cannabis strains.
Reference is now made to Tables 1, 2 and 3 presenting gRNA molecules constructed for silencing CsMLO1, CsMLO2 and CsMLO3, respectively. In Tables 1, 2 and 3 the term ‘PAM’ refers 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. The CsMLO genomic DNA sense strand is marked as “1”, and the antisense strand is marked as “−1”.
Reference is made to Table 4 summarizing sequences relating to WT CsMLO 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.
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 resistance to PM by establishing a protocol adapted for Cannabis. It is within the scope that different gRNA promoters were tested in order to maximize editing efficiency.
Identifying Powdery Mildew (PM) Pathogen Specific for Cannabis
Powdery Mildew is one of the most destructive fungal pathogens infecting Cannabis. It is an obligate biotroph that can vascularize into the plant tissue and remain invisible to a grower. Under ideal conditions, powdery mildew has a 4-7 days post inoculation (dpi) window where it remains invisible as it builds a network internally in the plant. It is herein acknowledged that the powdery mildew vascularized network in Cannabis is detectable with a PCR DNA based test prior to conidiospore generation. At later stages, powdery mildew infection and conidiospore generation results in rapid spreading of the fungus to other plants. This tends to emerge and sporulate within 2 weeks into flowering thus destroying very mature crops with severe economic consequences. DNA based tools could facilitate early detection and rapid removal of infected plant materials or screening of incoming clones.
To date, there are no fungal disease resistant Cannabis varieties on the market. Golovinomyces cichoracearum is known for causing PM on several Cucurbits and on Cannabis (Pepin et al., 2018). In order to identify the specific fungi type affecting Cannabis, a molecular analysis has been performed. Internal Transcribed Spacer (ITS) DNA of PM samples obtained from Cannabis strains growing in our greenhouse has been isolated and sequenced. The term Internal transcribed spacer (ITS) as used hereinafter refers to the spacer DNA region situated between the small-subunit ribosomal RNA (rRNA) and large-subunit rRNA genes in the chromosome or the corresponding transcribed region in the polycistronic rRNA precursor transcript. It is herein acknowledged that the internal transcribed spacer (ITS) region is considered to have the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. Thus ITS is proposed for adoption as the primary fungal barcode marker, namely as potential DNA marker or finger print for fungi (Schoch C. L. et al, PNAS, 2012 109 (16) 6241-6246). The results of the molecular analysis of PM isolated from Cannabis revealed that Golovinomyces ambrosiae or Golovinomyces cichoracearum are the cause of the disease.
A further achievement of the present invention is the establishment of an inoculation assay and index for Cannabis, or in other words establishment of bio-assay for powdery mildew inoculation in Cannabis. Such an assay establishment may include:
Production of Genome-Edited Cannabis MLO Genes
Three single guide RNAs (sgRNA) targeting the first exon (exon 1) of the CsMLO1 gene were designed and synthesized. These sgRNAs include sgRNA having nucleotide sequence as set forth in SEQ ID NO:17 (first guide), SEQ ID NO:43 (second guide) and SEQ ID NO:50 (third guide) starting at position 99, 369 and 453 of SEQ ID NO:1. The predicted Cas9 cleavage sites directed by these guide RNAs were designed to overlap with the nucleic acid recognition site of the restriction enzymes: Hinf1, BseLI and BtsI for the first, second and third gRNA, respectively (see
Two months post transformation, leaves from mature plants were sampled, and their DNA was extracted and digested with the suitable enzymes. Digested genomic DNA was used as a template for PCR using a primer pair flanking the 5′ and 3′ ends of the first exon of CsMLO1. The forward primer (fwd) (5-GAGTGGAACTAGAAGAAATGC-3) comprises a nucleotide sequence as set forth in SEQ ID NO:871, and the reverse primer (rev) (5-CCCTCCAAACACAACAGTGA-3) comprises a nucleotide sequence as set forth in SEQ ID NO:872 (see
Reference is now made to
It can be seen in
The sequencing results show that three CsMLO1 exon 1 genome edited fragments were achieved by the present invention.
Reference is now made to Table 5 summarizing sequences relating to mutated (genome edited) exon 1 fragments of CsMLO1 achieved by the current invention.
The resulted mutated CsMLO1 fragments include the following:
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL2020/050189 | 2/20/2020 | WO | 00 |
