IL295293A

IL295293A - Methods for increasing powdery mildew resistance in cannabis

Info

Publication number: IL295293A
Application number: IL295293A
Authority: IL
Inventors: SHERMAN Tal; Margalit Ido; Corem Shira
Original assignee: Betterseeds Ltd; SHERMAN Tal; Margalit Ido; Corem Shira
Priority date: 2021-08-19
Filing date: 2022-08-02
Publication date: 2023-03-01
Also published as: AU2022206708A1; CA3166209A1

Description

METHODS FOR INCREASING POWDERY MILDEW RESISTANCE IN CANNABIS FIELD OF THE INVENTION 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.

BACKGROUND OF THE INVENTION 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.

US6211433 and US6576814 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.

SUMMARY OF THE INVENTION It is therefore one object of the present invention to disclose a modified Cannabis plant exhibiting enhanced resistance to powdery mildew (PM), wherein the modified plant comprises a mutated Cannabis mlo1 (Csmlo1) allele, the mutated allele comprising a genomic modification selected from an indel of 14 bp at position corresponding to position 12 of SEQ ID NO: 882, or a fraction thereof, or a nucleic acid insertion at position corresponding to position 104-105 of SEQ ID NO: 882, or a combination thereof.

It is another object of the present invention to disclose the modified Cannabis plant as defined above, wherein the indel comprises a sequence as set forth in SEQ ID NO:883 or a fraction thereof.

It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the Csmlo1 mutant allele comprises a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:884, or a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:885, or a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:886, or a homologue having at least 80% sequence identity to the nucleic acid sequence of the mutated Csmlo1 allele, or a complementary sequence thereof, or any combination thereof.

It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the Csmlo1 mutant allele comprises a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:886, or a homologue having at least 80% sequence identity to the nucleic acid sequence of the mutated Csmlo1 allele.

It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the mutated Csmlo1 allele confers an enhanced resistance to powdery mildew as compared to a Cannabis plant comprising a wild type CsMLO1 allele having a nucleic acid sequence as set forth in SEQ ID NO:882 and/or having a nucleic acid sequence as set forth in SEQ ID NO:1 or a functional variant thereof.

It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the functional variant has at least 80% sequence identity to the corresponding CsMLO1 nucleotide sequence.

It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the mutated allele comprising a deletion of 14 bp at position 389 of SEQ ID NO: 1, or a nucleic acid insertion at position 482-483 of SEQ ID NO: 1, or a combination thereof.

It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the mutated Csmlo1 allele is generated using genome editing.

It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the plant has decreased expression levels of Mlo1 protein, relative to a Cannabis plant lacking the mutated Csmlo1 allele.

It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the mutated Csmlo1 allele is generated using mutagenesis, small interfering RNA (siRNA), microRNA (miRNA), artificial miRNA (amiRNA), DNA introgression, endonucleases or any combination thereof.

It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the endonuclease is selected from the group consisting of 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 another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the 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, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, bacteriophages Cas such as CasF (Cas-phi) and any combination thereof.

It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the plant comprises a DNA construct, the DNA construct comprising a promoter operably linked to a nucleotide sequence encoding a plant optimized Cas9 endonuclease, wherein the plant optimized Cas9 endonuclease is capable of binding to and creating a double strand break in a genomic target sequence of the plant genome.

It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the DNA construct further comprises gRNA targeted to at least one CsMLO1 allele.

It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the gRNA has a nucleic acid sequence corresponding to a sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence thereof, or any combination thereof.

It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the genome modification is a silencing mutation, a knockdown mutation, a knockout mutation, a loss of function mutation or any combination thereof.

It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the genome modification is an insertion, deletion, indel or substitution.

It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the genome modification is an induced mutation in the coding region of the allele, a mutation in the regulatory region of the allele, a mutation in a gene downstream in the MLO pathogen response pathway and/or an epigenetic factor.

It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the genome modification is generated via introduction (a) Cas DNA and gRNA sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 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:17, SEQ ID NO:and SEQ ID NO:50 and any combination thereof.

It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the gRNA sequence comprises a 3’ Protospacer Adjacent Motif (PAM) selected from the group consisting of NGG (SpCas), NNNNGATT (NmeCas9), NNAGAAW (StCas9), NAAAAC (TdCas9), NNGRRT (SaCas9) and TBN (Cas-phi).

It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the PM is selected from the group consisting of Golovinomyces cichoracearum, Golovinomyces ambrosiae and a mixture thereof.

It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the 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 another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the Cannabis plant does not comprise a transgene.

It is another 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 another 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 another 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 another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the plant genotype is obtainable by deposit under accession number with NCIMB Aberdeen AB21 9YA, Scotland, UK.

It is another object of the present invention to disclose a modified Cannabis plant exhibiting enhanced resistance to powdery mildew (PM), wherein the modified plant comprises a targeted genome modification conferring reduced expression of a Cannabis MLO1 (CsMLO1) gene as compared to a Cannabis plant lacking the targeted genome modification, the targeted genome modification generates a mutated Cannabis mlo1 (Csmlo1) allele comprising a deletion of a nucleic acid sequence as set forth in SEQ ID NO:883 or a fraction thereof as compared to the wild type CsMLO1 allele comprising a sequence as set forth in SEQ ID NO:1, or a nucleic acid insertion at position 482-483 of SEQ ID NO:1, or a combination thereof.

It is another object of the present invention to disclose a method for producing a modified Cannabis plant as defined in any of the above, the method comprises introducing using targeted genome modification, at least one genomic modification conferring reduced expression of at least one Cannabis MLO1 (CsMLO1) allele as compared to a Cannabis plant lacking the targeted genome modification, the genomic modification generates a mutated Cannabis mlo1 (Csmlo1) allele comprising an indel of 14 bp at a position corresponding to position 12 of SEQ ID NO: 882 or a fraction thereof, or a nucleic acid insertion at position corresponding to position 104-105 of SEQ ID NO: 882, or a combination thereof.

It is another object of the present invention to disclose the method as defined above, comprises steps of introducing a loss of function mutation into the CsMLO1 allele using targeted genome modification.

It is another object of the present invention to disclose the method as defined in any of the above, wherein the indel comprises a sequence as set forth in SEQ ID NO:883 or a fraction thereof.

It is another object of the present invention to disclose the method as defined in any of the above, wherein the Csmlo1 mutant allele comprises a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:884, or a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:885, or a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:886, or a homologue having at least 80% sequence identity to the nucleic acid sequence of the mutated Csmlo1 allele, or a complementary sequence thereof, or any combination thereof.

It is another object of the present invention to disclose the method as defined in any of the above, wherein the Csmlo1 mutant allele comprises a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:886, or a homologue having at least 80% sequence identity to the nucleic acid sequence of the mutated Csmlo1 allele.

It is another object of the present invention to disclose the method as defined in any of the above, wherein the mutated Csmlo1 allele confers an enhanced resistance to powdery mildew as compared to a Cannabis plant comprising a wild type CsMLO1 allele having a nucleic acid sequence as set forth in SEQ ID NO:882 and/or having a nucleic acid sequence as set forth in SEQ ID NO:1 or a functional variant thereof.

It is another object of the present invention to disclose the method as defined in any of the above, wherein the functional variant has at least 80% sequence identity to the corresponding CsMLOnucleotide sequence.

It is another object of the present invention to disclose the method as defined in any of the above, wherein the mutated allele comprising a deletion of 14 bp at position 389 of SEQ ID NO: 1, or a nucleic acid insertion at position 482-483 of SEQ ID NO: 1, or a combination thereof.

It is another object of the present invention to disclose the method as defined in any of the above, wherein the modified plant has decreased levels of at least one Mlo protein as compared to wild type Cannabis plant.

It is another object of the present invention to disclose the method as defined in any of the above, wherein the 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 another object of the present invention to disclose the method as defined in any of the above, wherein the 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, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, bacteriophages Cas such as CasF (Cas-phi) and any combination thereof.

It is another 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 gRNA targeted to at least one CsMLO1 allele.

It is another object of the present invention to disclose the method as defined in any of the above, wherein the gRNA nucleotide sequence targeting the CsMLO1 allele is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence thereof.

It is another 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 gRNA targeted to CsMLO1 gene and screening for induced targeted mutations conferring reduced expression of the CsMLO1 gene.

It is another object of the present invention to disclose the method as defined in any of the above, wherein the genomic modification is in the coding region of the allele, a mutation in the regulatory region of the allele, a mutation in a gene downstream in the MLO pathogen response pathway or an epigenetic factor.

It is another object of the present invention to disclose the method as defined in any of the above, wherein the genomic modification 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 another object of the present invention to disclose the method as defined in any of the above, wherein the genomic modification is an insertion, deletion, indel or substitution mutation.

It is another 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 plants comprising mutated Csmlo1 allele.

It is another object of the present invention to disclose the method as defined in any of the above, wherein the 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:882.

It is another object of the present invention to disclose the method as defined in any of the above, wherein the genetic modification in the 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:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence thereof, 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:17, SEQ ID NO:43 and SEQ ID NO:or a complementary sequence thereof, and any combination thereof.

It is another object of the present invention to disclose the method as defined in any of the above, wherein the gRNA nucleotide sequence comprises a 3’ Protospacer Adjacent Motif (PAM) selected from the group consisting of NGG (SpCas), NNNNGATT (NmeCas9), NNAGAAW (StCas9), NAAAAC (TdCas9), NNGRRT (SaCas9) and TBN (Cas-phi).

It is another object of the present invention to disclose the method as defined in any of the above, further comprising steps of regenerating a plant carrying the genomic modification.

It is another object of the present invention to disclose the method as defined in any of the above, further comprising steps of screening the regenerated plants for a plant resistant to powdery mildew.

It is another object of the present invention to disclose the method as defined a method for conferring powdery mildew resistance to a Cannabis plant comprising producing a plant according to the method as defined in any of the above.

It is another 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 another object of the present invention to disclose the method as defined in any of the above, wherein the powdery mildew is selected from the group of species consisting of Golovinomyces cichoracearum, Golovinomyces ambrosiae and a mixture thereof.

It is another object of the present invention to disclose the method as defined in any of the above, wherein the 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 another object of the present invention to disclose a method of determining the presence of a mutant Csmlo1 allele in a Cannabis plant comprising assaying the Cannabis plant for at least one of the presence of an indel comprising a nucleic acid sequence as set forth in SEQ ID NO:883, an insertion at position 104-105 of SEQ ID NO: 882, a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:884, a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:885, a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:886, or a homologue having at least 80% sequence identity to the nucleic acid sequence of the mutated Csmlo1 allele, a complementary sequence thereof, or any combination thereof.

It is another object of the present invention to disclose a method for identifying a Cannabis plant with resistance to powdery mildew, the method comprises steps of: (a) screening the genome of the Cannabis plant for a mutated Csmlo1 allele, the mutated allele comprises a genomic modification selected from an indel of 14 bp at a position corresponding to position 12 of SEQ ID NO: 882 or a fraction thereof, or a nucleic acid insertion at position corresponding to position 104-105 of SEQ ID NO: 882, or a combination thereof; (b) optionally, regenerating plants carrying the genetic modification; and (c) optionally, screening the regenerated plants for a plant resistant to powdery mildew.

It is another object of the present invention to disclose the method as defined in any of the above, wherein the genomic modification is a loss of function mutation.

It is another object of the present invention to disclose the method as defined in any of the above, wherein the indel comprises a sequence as set forth in SEQ ID NO:883 or a fraction and/or a complementary sequence thereof.

It is another object of the present invention to disclose the method as defined in any of the above, wherein the method comprises steps of screening the Cannabis plant for the presence of a deletion in CsMLO1 gene comprising a nucleic acid sequence as set forth in SEQ ID NO:1, the deletion comprising a nucleotide sequence as set forth in SEQ ID NO:883.

It is another object of the present invention to disclose the method as defined in any of the above, wherein the modified Cannabis plant comprising a mutant Csmlo1 nucleic acid conferring enhanced resistance to powdery mildew as compared to a Cannabis plant comprising a wild type CsMLO1 nucleic acid.

It is another object of the present invention to disclose a method for down regulation of Cannabis MLO1 (CsMLO1) gene, which comprises utilizing the nucleotide sequence as set forth in at least one of SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence thereof, and a combination thereof, for introducing a loss of function mutation into the CsMLO1 gene using targeted genome modification.

It is another object of the present invention to disclose an isolated amino acid sequence having at least 80% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:882-886, SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence or any combination thereof.

It is another object of the present invention to disclose use of a nucleotide sequence as set forth in SEQ ID NO: 883-886, SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 for generating, identifying and/or screening for a Cannabis plant comprising within its genome mutant Csmlo allele conferring resistance to PM.

It is another object of the present invention to disclose the use as defined in any of the above, wherein the presence of at least one nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:883, SEQ ID NO:882 indicates that the Cannabis plant comprises a wild type CsMLO1 allele, and the presence of at least one nucleic acid sequence selected from the group consisting of SEQ ID NO:884, SEQ ID NO:885 and SEQ ID NO:886 indicates that the Cannabis plant comprises a mutant Csmol1 allele.

It is another 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 or a complementary sequence or any combination thereof for targeted genome modification of Cannabis MLO1 (CsMLO1) gene.

It is another object of the present invention to disclose a detection kit for determining the presence or absence of a mutant Csmlo1 allele in a Cannabis plant, comprising a nucleic acid fragment comprising a sequence selected from SEQ ID NO:882-886, SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence or any combination thereof.

It is another object of the present invention to disclose the detection kit as defined above, wherein the kit is useful for identifying a Cannabis plant with enhanced resistance to powdery mildew.

BRIEF DESCRIPTION OF THE FIGURES 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.

Figs 1A-Cis presenting a photographic illustration of an infected Cannabis plant leaf exhibiting PM symptoms of white powdery spots on the leaves (Fig. 1A), an enlarged view (X4) of fungal asexual spore-carrying bodies (conidia) of Golovinomyces cichoracearum on Cannabis leaf tissue (Fig. 1B), and a microscopic imaging of Golovinomyces cichoracearum spores (Fig. 1C); Fig. 2A-B is schematically presenting WT plant cell penetrated by the fungal appressorium leading to haustorium establishment and infection by secondary hyphae (Fig. 2A), and mlo knockout plant cell into which the fungal spores are incapable of penetrating (Fig. 2B); Fig. 3 is schematically presenting CRISPR/Cas9 mode of action as depicted by Xie, Kabin, and Yinong Yang. "RNA-guided genome editing in plants using a CRISPR–Cas system." Molecular plant 6.6 (2013): 1975-1983; Fig.4 A-D is photographically presenting GUS staining after transient transformation of Cannabis axillary buds (Fig. 4A), leaves (Fig. 4B), calli (Fig. 4C), and cotyledons (Fig. 4D); Fig. 5 is presenting regenerated Cannabis tissue; Fig. 6 is photographically presenting PCR detection of Cas9 DNA in shoots of Cannabis plants transformed using biolistics; Fig. 7A-B is illustrating in vitro cleavage activity of CRISPR/Cas9; a scheme of genomic area targeted for editing is shown in Fig. 7A, and a gel showing digestion of PCR amplicon containing the gRNA sequence by RNP complex containing Cas9 and gene specific gRNA is shown in Fig. 7B; Fig. 8 is presenting a schematic illustration of a DNA plasmid containing a plant codon optimized Cas9 nuclease from Streptococcus pyogenes (pcoSpCas9) and sgRNA sequences used for transformation, as embodiments of the present invention; Fig. 9 schematically presents genomic localization of sgRNAs used for targeting CsMLO1 first exon, as embodiments of the present invention; Fig. 10 presents genomic nucleotide sequence of the first exon (exon 1) of wild type CsMLOtargeted by three gRNA sequences; Fig. 11 presents amino acid sequence of the first exon (exon 1) of wild type CsMLO1; Fig. 12 photographically presents detection of CsMLO1 PCR products showing length variation as a result of Cas9- mediated genome editing; Fig. 13 schematically presents genome edited CsMLO1 DNA fragments produced by the present invention; and Fig. 14 schematically presents nucleic acid sequence comparison of WT CsMLO1 and genome edited Csmlo1_d14i1 fragments produced by the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In the following detailed description of the preferred embodiments, reference is made to the accompanying drawings that form a part hereof, and in which are shown by way of illustration specific embodiments in which the invention may be practiced. It is understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention. The present invention may be practiced according to the claims without some or all of these specific details. For the purpose of clarity, technical material that is known in the technical fields related to the invention has not been described in detail so that the present invention is not unnecessarily obscured.

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 O (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, the present invention provides a modified Cannabis plant exhibiting enhanced resistance to powdery mildew (PM), wherein said modified plant comprises a mutated Cannabis mlo1 (Csmlo1) allele, said mutated allele comprising a genomic modification selected from an indel of 14 bp at position corresponding to position 12 of SEQ ID NO: 882, or a fraction thereof, or a nucleic acid insertion at position corresponding to position 104-105 of SEQ ID NO: 882, or a combination thereof.

According to a further embodiment of the present invention, the indel comprises a sequence as set forth in SEQ ID NO:883 or a fraction thereof.

According to a further embodiment of the present invention, the Csmlo1 mutant allele comprises a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:884, or a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:885, or a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:886, or a homologue having at least 80% sequence identity to the nucleic acid sequence of said mutated Csmlo1 allele, or a complementary sequence thereof, or any combination thereof.

According to a further embodiment of the present invention, the Csmlo1 mutant allele comprises a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:886, or a homologue having at least 80% sequence identity to the nucleic acid sequence of said mutated Csmlo1 allele.

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 having a nucleic acid sequence as set forth in SEQ ID NO:882 and/or having a nucleic acid sequence as set forth in SEQ ID NO:1 or a functional variant thereof.

According to a further embodiment of the present invention, the functional variant has at least 80% sequence identity to the corresponding CsMLO1 nucleotide sequence.

According to a further embodiment of the present invention, the mutated allele comprising a deletion of 14 bp at position 389 of SEQ ID NO: 1, or a nucleic acid insertion at position 482-4of SEQ ID NO: 1, or a combination thereof.

According to a further embodiment of the present invention, the mutated Csmlo1 allele is generated using genome editing.

It is further within the scope of the present invention to provide, a modified Cannabis plant exhibiting enhanced resistance to powdery mildew (PM), wherein said modified plant comprises a targeted genome modification conferring reduced expression of a Cannabis MLO1 (CsMLO1) gene as compared to a Cannabis plant lacking said targeted genome modification, said targeted genome modification generates a mutated Cannabis mlo1 (Csmlo1) allele comprising a deletion of a nucleic acid sequence as set forth in SEQ ID NO:883 or a fraction thereof as compared to the wild type CsMLO1 allele comprising a sequence as set forth in SEQ ID NO:1, or a nucleic acid insertion at position 482-483 of SEQ ID NO:1, or a combination thereof.

According to a further aspect of the present invention, a method for producing a modified Cannabis plant as defined in any of the above is provided. The method comprises introducing using targeted genome modification, at least one genomic modification conferring reduced expression of at least one Cannabis MLO1 (CsMLO1) allele as compared to a Cannabis plant lacking said targeted genome modification, said genomic modification generates a mutated Cannabis mlo1 (Csmlo1) allele comprising an indel of 14 bp at a position corresponding to position 12 of SEQ ID NO: 8or a fraction thereof, or a nucleic acid insertion at position corresponding to position 104-105 of SEQ ID NO: 882, or a combination thereof.

According to further aspects of the present invention, a method of determining the presence of a mutant Csmlo1 allele in a Cannabis plant is provided. The method comprising assaying the Cannabis plant for at least one of the presence of an indel comprising a nucleic acid sequence as set forth in SEQ ID NO:883, an insertion at position 104-105 of SEQ ID NO: 882, a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:884, a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:885, a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:886, or a homologue having at least 80% sequence identity to the nucleic acid sequence of said mutated Csmlo1 allele, a complementary sequence thereof, or any combination thereof.

It is further within the scope to provide 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 a mutated Csmlo1 allele, said mutated allele comprises a genomic modification selected from an indel of 14 bp at a position corresponding to position 12 of SEQ ID NO: 882 or a fraction thereof, or a nucleic acid insertion at position corresponding to position 104-105 of SEQ ID NO: 882, or a combination thereof; (b) optionally, regenerating plants carrying said genetic modification; and (c) optionally, screening said regenerated plants for a plant resistant to powdery mildew.

It is further within the scope of the present invention to provide a method for down regulation of Cannabis MLO1 (CsMLO1) gene, which comprises utilizing the nucleotide sequence as set forth in at least one of SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence thereof, and a combination thereof, for introducing a loss of function mutation into said CsMLO1 gene using targeted genome modification.

The present invention further provides an isolated amino acid sequence having at least 80% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:882-886, SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence or any combination thereof.

It is also within the scope to disclose a use of a nucleotide sequence as set forth in SEQ ID NO: 883-886, SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 for generating, identifying and/or screening for a Cannabis plant comprising within its genome mutant Csmlo allele conferring resistance to PM.

It is also within the scope to disclose a 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 or a complementary sequence or any combination thereof for targeted genome modification of Cannabis MLO1 (CsMLO1) gene.

According to further aspects, the present invention provides a detection kit for determining the presence or absence of a mutant Csmlo1 allele in a Cannabis plant, comprising a nucleic acid fragment comprising a sequence selected from SEQ ID NO:882-886, SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence or any combination thereof.

According to an 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, f uits, 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" or "genomic modification" refers hereinafter to genetic manipulation or modulation, which is the 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: Genome Editing Glossary 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 Fig. 3 schematically presenting an example of CRISPR/Casmechanism of action as depicted by Xie, Kabin, and Yinong Yang. "RNA-guided genome editing in plants using a CRISPR –Cas system." Molecular plant 6.6 (2013): 1975-1983. As shown in this figure, the Cas9 endonuclease forms a complex with a chimeric RNA (called guide RNA or gRNA), replacing the crRNA–transcrRNA heteroduplex, and the gRNA could be programmed to target specific sites. The gRNA–Cas9 should comprise at least 15-base-pairing (gRNA seed region) without mismatch between the 5'-end of engineered gRNA and targeted genomic site, and a motif called protospacer-adjacent motif or PAM that follows the base-pairing region in the complementary strand of the targeted DNA. The commonly-used Cas9 from Streptococcus pyogenes (SpCas9) recognizes the PAM sequence 5'-NGG-3' (where "N" can be any nucleotide base).

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, StCas(isolated from Streptococcus thermophiles) recognizing NNAGAAW, TdCas9 (isolated from Treponema denticola) recognizing NAAAAC, SaCas9 (isolated from Staphylococcus aureus) recognizing NNGRRT or NGRRT or NGRRN and TBN (Cas-phi).

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, 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.

The term "corresponding" or "corresponding to" or "corresponding to nucleotide sequence" or "corresponding to position" as used herein, refers in the context of the present invention to sequence homology or sequence identity. These terms relate to two or more nucleic acid or protein sequences, that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the available sequence comparison algorithms or by visual inspection. If two sequences, which are to be compared with each other, differ in length, sequence identity preferably relates to the percentage of the nucleotide residues of the shorter sequence, which are identical with the nucleotide residues of the longer sequence. As used herein, the percent of identity or homology between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of identity percent between two sequences can be accomplished using a mathematical algorithm as known in the relevant art. According to further aspects of the invention, the term "corresponding to the nucleotide sequence" or "corresponding to position", refers to variants, homologues and fragments of the indicated nucleotide sequence, which possess or perform the same biological function or correlates with the same phenotypic characteristic of the indicated nucleotide sequence.

Another indication that two nucleic acid sequences are substantially identical or that a sequence is "corresponding to the nucleotide sequence" is that the two molecules hybridize to each other under stringent conditions. High stringency conditions, such as high hybridization temperature and low salt in hybridization buffers, permits only hybridization between nucleic acid sequences that are highly similar, whereas low stringency conditions, such as lower temperature and high salt, allows hybridization when the sequences are less similar.

In other embodiments of the invention, such substantially identical sequences refer to polynucleotide or amino acid sequences that share at least about 80% similarity, preferably at least about 90% similarity, alternatively, about 95%, 96%, 97%, 98% or 99% similarity to the indicated polynucleotide or amino acid sequences.

According to other aspects of the invention, the term "corresponding" refers also to complementary sequences or base pairing such that when they are aligned antiparallel to each other, the nucleotide bases at each position in the sequences will be complementary. The degree of complementarity between two nucleic acid strands may vary.

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, CsMLOand 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: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 CasDNA 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 O (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 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 Fig. 3). The predefined gene specific gRNA’s are cloned into the same plasmid as the Cas gene and this plasmid is inserted into plant cells. Insertion of the aforementioned plasmid DNA can be done, but not limited to, using different delivery systems, biological and/or mechanical, e.g. Agrobacterium infiltration, virus based plasmids for delivery of the genome editing molecules and mechanical insertion of DNA (PEG mediated DNA transformation, biolistics, etc.).

It is further within the scope of the present invention that upon reaching the specific predetermined DNA sequence, the Cas9 nuclease cleaves both DNA strands to create double stranded breaks leaving blunt ends. This cleavage site is then repaired by the cellular non homologous end joining DNA repair mechanism resulting in insertions or deletions which eventually create a mutation at the cleavage site. For example, it is acknowledged that a deletion form of the mutation consists of at least 1 base pair deletion. As a result of this base pair deletion the gene coding sequence is disrupted and the translation of the encoded protein is compromised either by a premature stop codon or disruption of a functional or structural property of the protein. Thus DNA is cut by the Cas9 protein and re-assembled by the cell’s DNA repair mechanism.

It is further within the scope that 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 Figs 1A-C schematically present Cannabis plant infected by the fungal pathogen Golovinomyces cichoracearum, causal agent of the Powdery Mildew disease. More specifically this figure shows (A) Cannabis plant leaf exhibiting PM symptoms (B) Fungal asexual spore-carrying bodies (conidia) of Golovinomyces cichoracearum on Cannabis leaf tissue, and (C) microscopic view of Golovinomyces cichoracearum spores.

Reference is now made to Fig. 2A-B schematically presenting PM resistance suggested mode of action. This figure shows (A) a WT plant cell penetrated by the PM fungus (100). More particularly, a WT plant cell 10 is infected by PM spore 20 producing germ tubes 30 and penetrated by the PM fungal appressorium 40 which then leads to haustorium 50 establishment and infection by secondary hyphae; and (B) an mlo knockout cell 15 rendering fungal spores incapable of penetrating the plant cell (200).

It is a further aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, bacteriophages Cas such as CasF (Cas-phi) and any combination thereof.

It is a further aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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), NNGRRT (SaCas9) and TBN (Cas-phi).

It is a further aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, bacteriophages Cas such as CasF (Cas-phi) and any combination thereof.

It is a further aspect 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 aspect 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 aspect 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, CsMLOand 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 aspect 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 CsMLOgenomic sequence uses primers having nucleic acid sequence as set forth in SEQ ID NO: 871 and SEQ ID NO: 872.

It is a further aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 (SpCas), NNNNGATT (NmeCas9), NNAGAAW (StCas9), NAAAAC (TdCas9), NNGRRT (SaCas9) and TBN (Cas-phi).

It is a further aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 mutatedCsMLO1 allele and a combination thereof.

It is a further aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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:8indicates that the Cannabis plant comprises a mutant CsMLO1 nucleic acid.

It is a further aspect 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 aspect 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, 8and 881.

It is a further aspect 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:887.

It is a further aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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 aspect 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.

In order to understand the invention and to see how it may be implemented in practice, a plurality of preferred embodiments will now be described, by way of non-limiting example only, with reference to the following examples.

EXAMPLE 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: ? line stabilization by self pollination ? Generation of F6 parental lines ? Genome editing of parental lines ? Crossing edited parental lines to generate an F1 hybrid PM resistant plant Scheme 2: ? Identifying genes of interest ? Designing gRNA ? Transformation of plants with Cas9+gRNA constructs ? Screening and identifying editing events ? Genome editing of parental lines It is noted that line stabilization may be performed by the following: ? Induction of male flowering on female (XX) plants ? Self pollination 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: ? Sex markers- molecular markers are used for identification and selection of female vs male plants in the herein disclosed breeding program ? Genotyping markers- germplasm used in the current invention is genotyped using molecular markers, in order to allow a more efficient breeding process and identification of the MLO editing event.

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 between position 58544241bp and position 58551241bp 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 92616000bp and position 92629000bp and has a genomic sequence as set forth in SEQ ID NO:4. The CsMLOgene 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 23410000bp and position 23420000bp and has a genomic sequence as set forth in SEQ ID NO:7. The CsMLOgene 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".

Table 1: CsMLO1 targeted gRNA sequences Position on SEQ ID NO:1 Strand Sequence PAM SEQ ID NO 1 GTGAGTGAATGAGAGCAAGA AGG 59 -1 ATTCCGATTTCGAATTCAGA TGG 67 1 AATCCATCTGAATTCGAAAT CGG 76 1 GAATTCGAAATCGGAATGAG TGG 79 1 TTCGAAATCGGAATGAGTGG CGG 82 1 GAAATCGGAATGAGTGGCGG TGG 88 1 GGAATGAGTGGCGGTGGAGA AGG 99 1 CGGTGGAGAAGGTGAGTCCT TGG 105 -1 CCATGTGGGAGTATACTCCA AGG 116 1 CCTTGGAGTATACTCCCACA TGG 119 -1 ACGACGGCGACGATCCATGT GGG 120 -1 GACGACGGCGACGATCCATG TGG 135 -1 GACGATGACAGAGCAGACGA CGG 22 159 -1 ACGCTCCGCGGCGAGAGAAA TGG 165 1 CGTCGCCATTTCTCTCGCCG CGG 171 -1 ATAGTGGAGAAGACGCTCCG CGG 187 1 GAGCGTCTTCTCCACTATCT CGG 187 -1 TCAAAACCTGACCGAGATAG TGG 192 1 TCTTCTCCACTATCTCGGTC AGG 220 -1 AGGCCTCGTATAGAGGCTTC TGG 227 -1 TTCTGCAAGGCCTCGTATAG AGG 228 1 GAACCAGAAGCCTCTATACG AGG 240 -1 CTCCTCCTTGATCTTCTGCA AGG 246 1 CGAGGCCTTGCAGAAGATCA AGG 249 1 GGCCTTGCAGAAGATCAAGG AGG 264 1 CAAGGAGGAGTTGATGCTTT TGG 265 1 AAGGAGGAGTTGATGCTTTT GGG 292 -1 TTGTGTTCTGCGAAACAGTG AGG 332 -1 AGATTGTCGACCAAAGAAGC AGG 333 1 GTTTTGCGTACCTGCTTCTT TGG 355 -1 GATGAGGGCGCTTACAAGGG AGG 358 -1 CCTGATGAGGGCGCTTACAA GGG 359 -1 TCCTGATGAGGGCGCTTACA AGG 369 1 CCCTTGTAAGCGCCCTCATC AGG 370 -1 AATCATTAGCTTCCTGATGA GGG 371 -1 GAATCATTAGCTTCCTGATG AGG 405 -1 AGAGCCGGAGATGTGATGAG AGG 412 1 TCAACCTCTCATCACATCTC CGG 420 -1 AAGAAGGCGTCTGAAAGAGC CGG 436 -1 CAGTGGAAGTTTCTTCAAGA AGG 453 -1 GCAATAACCCAAATGAGCAG TGG 456 1 AGAAACTTCCACTGCTCATT TGG 457 1 GAAACTTCCACTGCTCATTT GGG 474 1 TTTGGGTTATTGCGCTCATA AGG 501 -1 ACAACAACAACTAAAGATAT GGG 502 -1 AACAACAACAACTAAAGATA TGG 521 1 TTAGTTGTTGTTGTTTTTTT AGG 522 1 TAGTTGTTGTTGTTTTTTTA GGG 523 1 AGTTGTTGTTGTTTTTTTAG GGG 570 1 TATAAATATACTTTCCCAAA AGG 571 1 ATAAATATACTTTCCCAAAA GGG 573 -1 TAAAGCGAATAGTCCCTTTT GGG 574 -1 TTAAAGCGAATAGTCCCTTT TGG 657 -1 ATGCTTCAACGGAATAAAAG GGG 658 -1 AATGCTTCAACGGAATAAAA GGG 659 -1 CAATGCTTCAACGGAATAAA AGG 668 -1 CAAATGGTGCAATGCTTCAA CGG 684 -1 CGAAGATAAAGATATGCAAA TGG 67 708 -1 AAGTGACATGGACAATGGCT AGG 713 -1 ACAGAAAGTGACATGGACAA TGG 720 -1 TGAGAACACAGAAAGTGACA TGG 744 1 TGTGTTCTCACTGTTGTGTT TGG 747 1 GTTCTCACTGTTGTGTTTGG AGG 755 1 TGTTGTGTTTGGAGGTGTAA AGG 779 -1 GAGAGAGAAGCATATGAATT TGG 860 1 TACACAACTAGATACGTCAA TGG 869 1 AGATACGTCAATGGAAACGT TGG 870 1 GATACGTCAATGGAAACGTT GGG 873 1 ACGTCAATGGAAACGTTGGG AGG 910 1 GAGAGTTATGACACTGAACA AGG 937 -1 TTCTATAATATTGTCAAAAG TGG 978 -1 TAAGCGATTCATATGTTAGA AGG 1017 1 TGTTCTTAAGTCTAAGAAAA AGG 1039 -1 CCTTAATGAACGCGTGTTGA TGG 1050 1 CCATCAACACGCGTTCATTA AGG 1062 1 GTTCATTAAGGACCACTTTT TGG 1063 1 TTCATTAAGGACCACTTTTT GGG 1063 -1 CTTTACCAAAACCCAAAAAG TGG 1069 1 AAGGACCACTTTTTGGGTTT TGG 1090 1 GGTAAAGACTCAGCTCTACT AGG 1094 1 AAGACTCAGCTCTACTAGGC TGG 1098 1 CTCAGCTCTACTAGGCTGGC TGG 1140 -1 ATAATCCTAATTCAGAACTT TGG 1146 1 AGTATCCAAAGTTCTGAATT AGG 1159 1 CTGAATTAGGATTATTCTTA TGG 1174 -1 ATATCAAGAGAATAAGAAAA CGG 1214 -1 AACGTTCTCAAAATAGAAAG TGG 1267 -1 CTCCAAAGGCTCAGTATCAA TGG 1276 1 CTCCATTGATACTGAGCCTT TGG 1281 -1 GTGATGGAGAAAATCTCCAA AGG 1297 -1 ATGTCCTAGTTTTCATGTGA TGG 11304 1 TTCTCCATCACATGAAAACT AGG 11327 1 ACATTTTTGTGCACATGTTA AGG 11349 1 GAGCTAGCTAACATTAACAT TGG 11356 1 CTAACATTAACATTGGAAAC AGG 11379 -1 GGGGAAAAAGAATCAAATCA TGG 11398 -1 AAAAGCATGCTGTTCACAAG GGG 11399 -1 CAAAAGCATGCTGTTCACAA GGG 11400 -1 ACAAAAGCATGCTGTTCACA AGG 11446 -1 CATGGTCGCATAATCTGATT TGG 11460 1 AATCAGATTATGCGACCATG CGG 11464 -1 CATGATGAATCCTAACCGCA TGG 11465 1 GATTATGCGACCATGCGGTT AGG 112 1476 1 CATGCGGTTAGGATTCATCA TGG 11520 1 TTACTTATAAATTACTAGAA TGG 11563 1 CTTTTTTTCTTTTCACTAAA TGG 11603 1 TTGTATTCTAGACTCACTGC AGG 11604 1 TGTATTCTAGACTCACTGCA GGG 11605 1 GTATTCTAGACTCACTGCAG GGG 11674 1 GATGATTTCAAGAAAGTTGT TGG 11675 1 ATGATTTCAAGAAAGTTGTT GGG 11681 1 TCAAGAAAGTTGTTGGGATA AGG 11691 1 TGTTGGGATAAGGTAACCCT TGG 11696 -1 GAAAATAGACTGTCAACCAA GGG 11697 -1 AGAAAATAGACTGTCAACCA AGG 11777 1 TTCTTTTAAGTCTACTGTAT CGG 11792 -1 AAAAGCTAAAAGGTCAATCT AGG 11802 -1 ACTGCAGCCAAAAAGCTAAA AGG 11806 1 AGATTGACCTTTTAGCTTTT TGG 11816 1 TTTAGCTTTTTGGCTGCAGT TGG 11825 1 TTGGCTGCAGTTGGTACCTT TGG 11826 1 TGGCTGCAGTTGGTACCTTT GGG 11830 -1 AGATGACCACAAAAACCCAA AGG 11835 1 TTGGTACCTTTGGGTTTTTG TGG 11863 1 TTCTTGTTGCTGAATGTTAA TGG 11910 -1 ATCACTTAGATCTTGAGTTA TGG 11926 1 ACTCAAGATCTAAGTGATAT TGG 11958 -1 CAAAGAACCAGACTGATTAC GGG 11959 -1 ACAAAGAACCAGACTGATTA CGG 11962 1 GCAGAATCCCGTAATCAGTC TGG 11999 1 CTTCAAGTGTGTCATCTCTT TGG 12033 -1 GCTTATTTGAAACTATAATT TGG 12101 -1 GTGGAGGCAGAGTAAGGAAT TGG 12107 -1 AGAAAAGTGGAGGCAGAGTA AGG 12117 -1 CTCCAACCATAGAAAAGTGG AGG 12120 -1 TTTCTCCAACCATAGAAAAG TGG 12122 1 ACTCTGCCTCCACTTTTCTA TGG 12126 1 TGCCTCCACTTTTCTATGGT TGG 12148 1 GAGAAAATTATACTCCAAGT TGG 12151 -1 TCCAATTCCTTACACCAACT TGG 12155 1 TTATACTCCAAGTTGGTGTA AGG 12161 1 TCCAAGTTGGTGTAAGGAAT TGG 12203 1 TCACTAGAATGCAATCAACA AGG 12204 1 CACTAGAATGCAATCAACAA GGG 12244 -1 AATTTTTTTAATCAGAATTC TGG 12293 -1 TAAAAGTAAACTAAATTTCT TGG 12347 -1 ATGTAAATTATGTTCTATAT AGG 12380 -1 GATCCAATTGAATTATCTTA AGG 157 2388 1 ACACCTTAAGATAATTCAAT TGG 12406 1 ATTGGATCTTACTCCTTGTT TGG 12408 -1 GCCTCATTGTAGTCCAAACA AGG 12418 1 TCCTTGTTTGGACTACAATG AGG 12453 -1 ATCTTGGTTTGAGTATTGAG AGG 12469 -1 ATATAAATAATGAATGATCT TGG 12497 -1 CAAAGAAGTTTAATACACAC TGG 12516 1 TATTAAACTTCTTTGTTGTA TGG 12559 1 TTTTGTCAATGTTTTGTGAT TGG 12597 1 TAATAATGTGTTATATTTGC AGG 12601 1 AATGTGTTATATTTGCAGGC TGG 12616 1 CAGGCTGGCACACATaTTTC TGG 12640 -1 CTCAATAAACTCACAAAGAA AGG 12709 1 CATGTTTCATTGTTCTTGCA TGG 12750 1 CATTTTAAGTATCATACTGA TGG 12774 1 GAAAGAGATAAAATACAGAG AGG 12775 1 AAAGAGATAAAATACAGAGA GGG 12783 1 AAAATACAGAGAGGGAGAAT CGG 12784 1 AAATACAGAGAGGGAGAATC GGG 12817 1 TTTAACACAATTTTGTAAAT AGG 12824 1 CAATTTTGTAAATAGGCAAA TGG 12837 1 AGGCAAATGGACAGCTAAGA AGG 12852 -1 TGTTCAATTAATTCTAAATT TGG 12875 1 TTAATTGAACAACATGACCT AGG 12881 -1 AAATTGCACAATATTTACCT AGG 12950 1 TAAATGTAGAGTCATGAGTC AGG 12951 1 AAATGTAGAGTCATGAGTCA GGG 12973 1 GTAGAAATTTGCACCTAGAC AGG 12975 -1 CACCTTAAAACCACCTGTCT AGG 12976 1 GAAATTTGCACCTAGACAGG TGG 12984 1 CACCTAGACAGGTGGTTTTA AGG 12987 1 CTAGACAGGTGGTTTTAAGG TGG 13011 1 ACTTCTCATCTCCAAGTCTT AGG 13011 -1 CATACATATCACCTAAGACT TGG 13046 -1 ATGTATATCACAACAGCAAA AGG 13083 -1 TTAAAAGAAAAAACAACAAG TGG 13114 1 TAAATAGCTTCTACTTGCCG TGG 13115 1 AAATAGCTTCTACTTGCCGT GGG 13120 -1 ATGCTCCAGTTTAGTGCCCA CGG 13126 1 ACTTGCCGTGGGCACTAAAC TGG 13147 1 GGAGCATGTCATTACTCAGT TGG 13184 1 GAGAAACATGTAGCAATAGA AGG 13212 -1 AACCAAAAGTGATCATCTGA TGG 23221 1 AGCCATCAGATGATCACTTT TGG 23242 -1 ATCAGGAAGAGGACAATCTG GGG 202 3243 -1 AATCAGGAAGAGGACAATCT GGG 23244 -1 GAATCAGGAAGAGGACAATC TGG 23253 -1 TGATGAAATGAATCAGGAAG AGG 23259 -1 AAAGGATGATGAAATGAATC AGG 23277 -1 TCTCAAATGAATTTTGGAAA AGG 23283 -1 ACGCAATCTCAAATGAATTT TGG 23305 1 TTGAGATTGCGTTTTTCTTC TGG 23312 1 TGCGTTTTTCTTCTGGATAT TGG 23320 1 TCTTCTGGATATTGGTAAGC TGG 23343 -1 TGGTAGAAGTAGAAGCAGAG TGG 23363 -1 AATAACAATTTGTTCTTTTT TGG 23411 1 ATCTTCTTTTCTGTGTATCT AGG 23441 1 TTCATTTAACTCCTGTATAA TGG 23441 -1 ACGAACGTGTCCCATTATAC AGG 23442 1 TCATTTAACTCCTGTATAAT GGG 23472 -1 TTTACCCAATGACAAGTCTT GGG 23473 -1 TTTTACCCAATGACAAGTCT TGG 23478 1 ATTGTCCCAAGACTTGTCAT TGG 23479 1 TTGTCCCAAGACTTGTCATT GGG 23541 -1 TAAAATAAAAGTTTCGTACT TGG 23570 -1 GAACACCCTAAAGCACAACA TGG 23575 1 TTTTTACCATGTTGTGCTTT AGG 23576 1 TTTTACCATGTTGTGCTTTA GGG 23588 1 GTGCTTTAGGGTGTTCATTC AGG 23622 -1 GTGTGACAATGGCATAGAGC GGG 23623 -1 TGTGTGACAATGGCATAGAG CGG 23633 -1 TCAACGCACCTGTGTGACAA TGG 23636 1 GCTCTATGCCATTGTCACAC AGG 23681 1 ATAATTTAATAAGTTCTAAA AGG 23689 1 ATAAGTTCTAAAAGGAAAGT AGG 23720 -1 CATTCCACAAGATTTTATTA TGG 23727 1 CTGACCATAATAAAATCTTG TGG 23743 1 CTTGTGGAATGATTTGAAGA TGG 23744 1 TTGTGGAATGATTTGAAGAT GGG 23773 1 TTACAAGAAAGCCATATTTG AGG 23773 -1 TTGCATGCGCTCCTCAAATA TGG 23789 1 TTTGAGGAGCGCATGCAAGT AGG 23802 1 TGCAAGTAGGAATTGTTAAT TGG 23803 1 GCAAGTAGGAATTGTTAATT GGG 23812 1 AATTGTTAATTGGGCTCAGA AGG 23827 1 TCAGAAGGTCAAGAAAAAGA AGG 23828 1 CAGAAGGTCAAGAAAAAGAA GGG 23849 1 GGATTTAAAGCAGCCCTCAT TGG 23851 -1 GCCAGCACCGGAACCAATGA GGG 23852 -1 AGCCAGCACCGGAACCAATG AGG 247 3855 1 AAAGCAGCCCTCATTGGTTC CGG 23861 1 GCCCTCATTGGTTCCGGTGC TGG 23863 -1 GCCTGAGCCTGAGCCAGCAC CGG 23867 1 ATTGGTTCCGGTGCTGGCTC AGG 23873 1 TCCGGTGCTGGCTCAGGCTC AGG 23879 1 GCTGGCTCAGGCTCAGGCTC AGG 23884 1 CTCAGGCTCAGGCTCAGGCT CGG 23885 1 TCAGGCTCAGGCTCAGGCTC GGG 23891 1 TCAGGCTCAGGCTCGGGATC AGG 23903 1 TCGGGATCAGGCTCTACTCC TGG 23910 -1 GTATCAGAAATTGGTTGACC AGG 23919 -1 GCAGAACCAGTATCAGAAAT TGG 23924 1 GGTCAACCAATTTCTGATAC TGG 23938 1 TGATACTGGTTCTGCATCTG TGG 23939 1 GATACTGGTTCTGCATCTGT GGG 23950 1 TGCATCTGTGGGAATTCAGC TGG 23951 1 GCATCTGTGGGAATTCAGCT GGG 23973 -1 TGCTCTGGCTTTGATGCTTT GGG 23974 -1 CTGCTCTGGCTTTGATGCTT TGG 23988 -1 TTAGAGTCATCACTCTGCTC TGG 24058 1 GAAGACATAAGTCTACCCTT AGG 24062 -1 CTAGTAGTAGTATTACCTAA GGG 24063 -1 ACTAGTAGTAGTATTACCTA AGG 24088 -1 ATCCCAGCACAGCTGGAAAG TGG 24095 -1 ATTTCTAATCCCAGCACAGC TGG 24096 1 TTGCCACTTTCCAGCTGTGC TGG 24097 1 TGCCACTTTCCAGCTGTGCT GGG 24132 1 AATTCTTCTGTCATATATTA TGG 24138 1 TCTGTCATATATTATGGCTG TGG 24141 1 GTCATATATTATGGCTGTGG TGG 24142 1 TCATATATTATGGCTGTGGT GGG 24160 -1 GTCTTGTCCATAAAAGACTT AGG 24164 1 GACTGTACCTAAGTCTTTTA TGG 24188 -1 TTATATAATATATTGATCAA AGG 24267 1 CTTCTTTCTTCTTATTATCA TGG 24280 1 ATTATCATGGTACATCCTTT TGG 24284 -1 TTCACTATTCAGTTACCAAA AGG 24312 1 AGTGAATACGTGTAGTCTCA TGG 24313 1 GTGAATACGTGTAGTCTCAT GGG 2 Table 2: CsMLO2 targeted gRNA sequences Position on SEQ ID NO:4 Strand Sequence PAM SEQ ID NO 1977 -1 GTATGAATATGAAATTAAGT TGG 22044 -1 AGAGAGAGAGAGACAGAGAG TGG 22117 -1 TTGAAATTGGGATGGAGATG TGG 22125 -1 ATTCTGTTTTGAAATTGGGA TGG 22129 -1 GTAAATTCTGTTTTGAAATT GGG 22130 -1 TGTAAATTCTGTTTTGAAAT TGG 22153 -1 GTTAGAATGAAAAGTTTGAT GGG 22154 -1 AGTTAGAATGAAAAGTTTGA TGG 22211 1 TATAATCAATTATTCCCAAG TGG 22214 -1 TAAATATAAATAGGCCACTT GGG 22215 -1 ATAAATATAAATAGGCCACT TGG 22223 -1 TAGTGATCATAAATATAAAT AGG 22278 1 AAAATTAAATTAAAAGAAGA TGG 22281 1 ATTAAATTAAAAGAAGATGG CGG 32284 1 AAATTAAAAGAAGATGGCGG TGG 32291 1 AAGAAGATGGCGGTGGCTAG CGG 32294 1 AAGATGGCGGTGGCTAGCGG AGG 32322 1 CTTTAGAACAAACACCAACA TGG 32323 1 TTTAGAACAAACACCAACAT GGG 32325 -1 ACTACGGCCACAGCCCATGT TGG 32329 1 ACAAACACCAACATGGGCTG TGG 32341 -1 TACCAAAACAAGACAAACTA CGG 32350 1 GGCCGTAGTTTGTCTTGTTT TGG 32371 -1 GATTATGTGCTCAATAATAA TGG 32393 1 GAGCACATAATCCATCTCAT TGG 32393 -1 GGTATACCTTGCCAATGAGA TGG 32398 1 CATAATCCATCTCATTGGCA AGG 32414 -1 TGAGATTAATATATATAATT GGG 32415 -1 GTGAGATTAATATATATAAT TGG 32473 1 CATTTAATTATTTAAATTAA TGG 32474 1 ATTTAATTATTTAAATTAAT GGG 317 2495 1 GGTATTTTTTTTTTTTTTAG TGG 32535 1 ACGAGCTCTTTATGAATCGT TGG 32551 1 TCGTTGGAAAAGATCAAATC AGG 32576 -1 AAAATGGGTATTCATTAATT GGG 32577 -1 AAAAATGGGTATTCATTAAT TGG 32591 -1 TTAAAAAAAAAAACAAAAAT GGG 32656 1 TTTGATAGAGCTTATGTTAT TGG 32657 1 TTGATAGAGCTTATGTTATT GGG 32658 1 TGATAGAGCTTATGTTATTG GGG 32680 1 GTTCATATCGTTGTTACTAA CGG 32683 1 CATATCGTTGTTACTAACGG TGG 32684 1 ATATCGTTGTTACTAACGGT GGG 32703 -1 GATATACAAATATTTGAGAT CGG 32726 1 ATTTGTATATCTGAGAAAAT TGG 32729 1 TGTATATCTGAGAAAATTGG AGG 32730 1 GTATATCTGAGAAAATTGGA GGG 32736 1 CTGAGAAAATTGGAGGGACA TGG 32751 -1 TCTTCTTGTTCTTTATTACA AGG 32777 1 CAAGAAGAGAAATTGAATAA AGG 32778 1 AAGAAGAGAAATTGAATAAA GGG 32779 1 AGAAGAGAAATTGAATAAAG GGG 32817 1 TCGAACATGAAAGTAACAGT CGG 32827 1 AAGTAACAGTCGGAGATTGC TGG 32839 -1 ACCGTCGCCGGACTCTAAAA AGG 32843 1 TTGCTGGCCTTTTTAGAGTC CGG 32849 1 GCCTTTTTAGAGTCCGGCGA CGG 32851 -1 GACACTAGCAGCACCGTCGC CGG 32865 1 GCGACGGTGCTGCTAGTGTC CGG 32873 -1 CGGCCGCCGCCAAAATTCGC CGG 32875 1 TGCTAGTGTCCGGCGAATTT TGG 32878 1 TAGTGTCCGGCGAATTTTGG CGG 32881 1 TGTCCGGCGAATTTTGGCGG CGG 32885 1 CGGCGAATTTTGGCGGCGGC CGG 32886 1 GGCGAATTTTGGCGGCGGCC GGG 32893 -1 TTCAGCACACTTATCAGTCC CGG 352 2908 1 GACTGATAAGTGTGCTGAAA AGG 32978 1 GTCTTTCTTATCCTTTTATT TGG 32978 -1 GACGAATATGTCCAAATAAA AGG 33000 -1 CTCCTATAATATTATATGTT TGG 33009 1 GTCCAAACATATAATATTAT AGG 33051 -1 AAATATATAAATTTAAAGGT TGG 33055 -1 AACTAAATATATAAATTTAA AGG 34125 1 AAATTATATACATATATGAA TGG 34168 1 ATATATATAATTATAATTTC AGG 34169 1 TATATATAATTATAATTTCA GGG 34187 -1 ATACCATCCGCCGAAACAAA TGG 34188 1 AGGGCAAGTTCCATTTGTTT CGG 34191 1 GCAAGTTCCATTTGTTTCGG CGG 34195 1 GTTCCATTTGTTTCGGCGGA TGG 34230 1 GCATATTTTTATCTTTGTGT TGG 34249 -1 TCATGATGCAGTAGAGAACA TGG 34272 1 CTGCATCATGACTATGTTTT TGG 34273 1 TGCATCATGACTATGTTTTT GGG 34284 1 TATGTTTTTGGGCAGACTTA AGG 34404 -1 AATTTATATATAATTATTTA GGG 34405 -1 CAATTTATATATAATTATTT AGG 34428 1 TATATAAATTGATTCCCAGA TGG 34429 1 ATATAAATTGATTCCCAGAT GGG 34431 -1 ATGCTTCCAACTTCCCATCT GGG 34432 -1 AATGCTTCCAACTTCCCATC TGG 34436 1 TTGATTCCCAGATGGGAAGT TGG 34445 1 AGATGGGAAGTTGGAAGCAT TGG 34446 1 GATGGGAAGTTGGAAGCATT GGG 34452 1 AAGTTGGAAGCATTGGGAAA AGG 34476 -1 ACCATGTGAGAATTGATATT CGG 34486 1 GCCGAATATCAATTCTCACA TGG 34548 1 CTTAATTTTAATTTTTCTAT AGG 34551 1 AATTTTAATTTTTCTATAGG TGG 34649 -1 CTATATGACATATTTGATGG TGG 34652 -1 TAACTATATGACATATTTGA TGG 387 4742 1 AATTATAAGAGCATCTTTAT TGG 34749 1 AGAGCATCTTTATTGGACAC CGG 34757 -1 TAGAAAGTGTTAAATATCAC CGG 34844 -1 TATTGGTATAATTAAGTATC AGG 34861 -1 CTTACCAATTATATTATTAT TGG 34868 1 TATACCAATAATAATATAAT TGG 34903 1 ATTTATAAGAAGTATATATA TGG 34904 1 TTTATAAGAAGTATATATAT GGG 34923 1 TGGGAGTTAGAATTAAGTAA AGG 34997 -1 CTCGCAAATCTGAATCTTTC TGG 35009 1 CAGAAAGATTCAGATTTGCG AGG 35010 1 AGAAAGATTCAGATTTGCGA GGG 35023 1 TTTGCGAGGGACACTTCTTT TGG 45045 1 GAAGAAGACATTTAAGTTTC TGG 45058 -1 CCATATTAGGAAAGGGTGTT TGG 45065 -1 TTACTATCCATATTAGGAAA GGG 45066 -1 CTTACTATCCATATTAGGAA AGG 45069 1 CCAAACACCCTTTCCTAATA TGG 45071 -1 GGGATCTTACTATCCATATT AGG 45091 -1 AAGTAAAAAGTGGGTAAAAA GGG 45092 -1 AAAGTAAAAAGTGGGTAAAA AGG 45100 -1 AATATAAAAAAGTAAAAAGT GGG 45101 -1 GAATATAAAAAAGTAAAAAG TGG 45149 -1 ATATAAGTGCATGGATATAG TGG 45158 -1 TATTAATAGATATAAGTGCA TGG 45233 -1 CATATTTATATGCATGTGAA AGG 45253 1 TGCATATAAATATGTTTGCA TGG 45269 1 TGCATGGTTTTTATACATCG TGG 47159 1 TATATATATAATATTTTTTT TGG 47213 -1 TTAATTAATAATTAAAGAGC AGG 47238 1 ATTAATTAATTATTTTTCGC AGG 47282 -1 AAAGTTAAATAATCAACTTT AGG 47302 1 GATTATTTAACTTTGAGACA TGG 47313 1 TTTGAGACATGGATTTATAA TGG 47373 1 ATTATAGCTGTAGAGATATT TGG 422 7387 -1 TAAGTATTATTAAAAATACA AGG 48017 -1 GAATGAGAATAGGAATAGAA TGG 48027 -1 ATAGGAATGGGAATGAGAAT AGG 48039 -1 ATAGGAATAGAAATAGGAAT GGG 48040 -1 TATAGGAATAGAAATAGGAA TGG 48045 -1 GAAAATATAGGAATAGAAAT AGG 48057 -1 GTTGAGAGGAATGAAAATAT AGG 48071 -1 CACAGAGGCGTTTGGTTGAG AGG 48079 -1 AATAGGCCCACAGAGGCGTT TGG 48083 1 CTCTCAACCAAACGCCTCTG TGG 48084 1 TCTCAACCAAACGCCTCTGT GGG 48086 -1 ACAAGATAATAGGCCCACAG AGG 48096 -1 TTAATACATAACAAGATAAT AGG 48150 1 ATCAATAACTAAATTAATTG AGG 48177 1 TTATAACAATTAATAATTTC AGG 48186 1 TTAATAATTTCAGGCACATT TGG 48198 -1 AAATTTTTGATGGCTTTGAG GGG 48199 -1 CAAATTTTTGATGGCTTTGA GGG 48200 -1 TCAAATTTTTGATGGCTTTG AGG 48208 -1 TTTGAAAGTCAAATTTTTGA TGG 48255 1 ATCTCTAGAAGAAGATTTCA AGG 48265 1 GAAGATTTCAAGGTCGTTGT AGG 48272 1 TCAAGGTCGTTGTAGGAATC AGG 48345 -1 ATTAAAATAAGTCATCATTT GGG 48346 -1 AATTAAAATAAGTCATCATT TGG 48399 1 TAATAATTATTATTTTGTTT TGG 48427 1 TCAATCTCAGTCCTCCTATT TGG 48427 -1 ACAGCGAAGAACCAAATAGG AGG 48430 -1 ACCACAGCGAAGAACCAAAT AGG 48440 1 TCCTATTTGGTTCTTCGCTG TGG 48465 1 TTCTTACTCTTCAATACCCA TGG 48470 -1 AATAATAAAATGCTCACCAT GGG 48471 -1 TAATAATAAAATGCTCACCA TGG 48500 -1 GGATGCATTGAAATAATTAA TGG 48521 -1 AATCTAAACTGTGATAATTA GGG 457 8522 -1 AAATCTAAACTGTGATAATT AGG 48566 -1 TTGACATATATGCACACGTT TGG 48605 1 TATATTTTTGTTTTTATTAT TGG 48618 -1 AAATGTAAACAAATTCATTA TGG 48631 1 ATAATGAATTTGTTTACATT TGG 48636 1 GAATTTGTTTACATTTGGAC AGG 48640 1 TTGTTTACATTTGGACAGGC TGG 48655 1 CAGGCTGGTATTCTTATCTT TGG 48670 -1 CTTACAATTAGAGGAATAAA AGG 48679 -1 ATATTAGTACTTACAATTAG AGG 48820 -1 GATTGTTTGAATTTTATTTT TGG 48907 -1 GTTTACAGTAAAACTTTAAA AGG 48932 -1 AATTAGCCCAATTTTTTTCA CGG 48936 1 TAAACTACCGTGAAAAAAAT TGG 48937 1 AAACTACCGTGAAAAAAATT GGG 49001 -1 CTCTTTTATTTTTTAAGAAG AGG 49053 1 TATTATAAATAAATTATGTT AGG 49065 1 ATTATGTTAGGTGATCCTAT TGG 49068 1 ATGTTAGGTGATCCTATTGG TGG 49069 1 TGTTAGGTGATCCTATTGGT GGG 49069 -1 GTAATTTCGTCCCCACCAAT AGG 49070 1 GTTAGGTGATCCTATTGGTG GGG 49101 1 ACAAGTGATTATAACAAAGA TGG 49102 1 CAAGTGATTATAACAAAGAT GGG 49103 1 AAGTGATTATAACAAAGATG GGG 49123 1 GGGCTAAGAATTCAAGAAAG AGG 49138 1 GAAAGAGGAGAAGTTGTAAA AGG 49149 1 AGTTGTAAAAGGAGTGCCTG TGG 49154 -1 TCGTCCCCAGGTTGGACCAC AGG 49159 1 GGAGTGCCTGTGGTCCAACC TGG 49160 1 GAGTGCCTGTGGTCCAACCT GGG 49161 1 AGTGCCTGTGGTCCAACCTG GGG 49162 -1 AGAAAAGGTCGTCCCCAGGT TGG 49166 -1 AACCAGAAAAGGTCGTCCCC AGG 49175 1 AACCTGGGGACGACCTTTTC TGG 492 9177 -1 GTGGGCGGTTGAACCAGAAA AGG 49192 -1 GGTAGAGAATAAGGCGTGGG CGG 49195 -1 TAAGGTAGAGAATAAGGCGT GGG 49196 -1 ATAAGGTAGAGAATAAGGCG TGG 49201 -1 AGTTAATAAGGTAGAGAATA AGG 49213 -1 GGAAGAGGACGAAGTTAATA AGG 49227 1 TATTAACTTCGTCCTCTTCC AGG 49228 -1 ATTGATTATGTACCTGGAAG AGG 59234 -1 ATTTTGATTGATTATGTACC TGG 59254 1 TAATCAATCAAAATCAGCCT TGG 59260 -1 GGTGCATTATAGAATTTCCA AGG 59281 -1 TCAATGTATTCATTTTAAGG GGG 59282 -1 ATCAATGTATTCATTTTAAG GGG 59283 -1 CATCAATGTATTCATTTTAA GGG 59284 -1 GCATCAATGTATTCATTTTA AGG 59308 -1 TTGAGTGCTAAAACAAGTAA GGG 59309 -1 TTTGAGTGCTAAAACAAGTA AGG 59350 1 TTTAGTCAAATTTTTTCTCA TGG 510632 -1 TCCATGCAAAGAACGCAAGC TGG 510642 1 TCCAGCTTGCGTTCTTTGCA TGG 510648 1 TTGCGTTCTTTGCATGGACT TGG 510649 1 TGCGTTCTTTGCATGGACTT GGG 510753 1 TTAATTTTTCAGTATGAATT TGG 510785 1 TTGCTTTCATGAACATGTTG AGG 510791 1 TCATGAACATGTTGAGGATG TGG 510809 1 TGTGGTTATCAGAATCACCA TGG 510810 1 GTGGTTATCAGAATCACCAT GGG 510811 1 TGGTTATCAGAATCACCATG GGG 510815 -1 ATATCTGTATACAGACCCCA TGG 510922 -1 AAATAAAAATTAAATATTAA TGG 510958 1 GTAAAAATTTCTAACACCGT TGG 510963 -1 CCCTGATGATCATGATCCAA CGG 510973 1 ACCGTTGGATCATGATCATC AGG 510974 1 CCGTTGGATCATGATCATCA GGG 510993 -1 TGACGTAGCTGCACAGAATC TGG 527 11016 -1 AACAAGGGCGTAGAGAGGGA GGG 511017 -1 TAACAAGGGCGTAGAGAGGG AGG 511020 -1 GTGTAACAAGGGCGTAGAGA GGG 511021 -1 TGTGTAACAAGGGCGTAGAG AGG 511031 -1 TGTAATTACTTGTGTAACAA GGG 511032 -1 GTGTAATTACTTGTGTAACA AGG 511159 -1 AGATTTTATATATTTAATTA GGG 511160 -1 TAGATTTTATATATTTAATT AGG 511524 -1 CGGACTATATTTTAATTAAA AGG 511544 -1 TAATTAAATAAAATTCTAAA CGG 511580 1 TAAAAAATATTGTCATAGTT TGG 511581 1 AAAAAATATTGTCATAGTTT GGG 511782 1 TATATATATGACACAACAGA TGG 511783 1 ATATATATGACACAACAGAT GGG 511800 -1 GTTGAATATAGTTGGTTTCA TGG 511808 -1 ACTTTGTCGTTGAATATAGT TGG 511824 1 TATATTCAACGACAAAGTAG CGG 511827 1 ATTCAACGACAAAGTAGCGG AGG 511839 -1 TGAGTGGTGCCAGTTGCGGA GGG 511840 -1 CTGAGTGGTGCCAGTTGCGG AGG 511841 1 TAGCGGAGGCCCTCCGCAAC TGG 511843 -1 GGGCTGAGTGGTGCCAGTTG CGG 511855 -1 TGATGTGCTTTCGGGCTGAG TGG 511863 -1 TTGGTGTTTGATGTGCTTTC GGG 511864 -1 TTTGGTGTTTGATGTGCTTT CGG 511881 1 GCACATCAAACACCAAAACA AGG 511882 -1 CTGACCCCGCCGCCTTGTTT TGG 511884 1 CATCAAACACCAAAACAAGG CGG 511887 1 CAAACACCAAAACAAGGCGG CGG 511888 1 AAACACCAAAACAAGGCGGC GGG 511889 1 AACACCAAAACAAGGCGGCG GGG 511910 -1 GTCGTCGGCCGGCTTGACAG CGG 511913 1 CAGTGACGCCGCTGTCAAGC CGG 511921 -1 GATGTGTGGGTGTCGTCGGC CGG 511925 -1 ATGTGATGTGTGGGTGTCGT CGG 562 11934 -1 ACCGGGGACATGTGATGTGT GGG 511935 -1 GACCGGGGACATGTGATGTG TGG 511944 1 ACCCACACATCACATGTCCC CGG 511950 -1 GTGGCGCAAGAGGTGGACCG GGG 511951 -1 AGTGGCGCAAGAGGTGGACC GGG 511952 -1 TAGTGGCGCAAGAGGTGGAC CGG 511957 -1 TGCGGTAGTGGCGCAAGAGG TGG 511960 -1 CACTGCGGTAGTGGCGCAAG AGG 511969 -1 CTGCTGCCTCACTGCGGTAG TGG 511974 1 CTTGCGCCACTACCGCAGTG AGG 511975 -1 GGCTGTCTGCTGCCTCACTG CGG 511996 -1 AGCGCCTTGGGGAGTTTTGG AGG 511999 -1 TTGAGCGCCTTGGGGAGTTT TGG 512003 1 ACAGCCTCCAAAACTCCCCA AGG 512007 -1 ATCAAAGTTTGAGCGCCTTG GGG 512008 -1 CATCAAAGTTTGAGCGCCTT GGG 512009 -1 CCATCAAAGTTTGAGCGCCT TGG 512020 1 CCAAGGCGCTCAAACTTTGA TGG 512033 1 ACTTTGATGGCGCCACTGAA CGG 512034 -1 ATCTGTCTCCCACCGTTCAG TGG 512036 1 TTGATGGCGCCACTGAACGG TGG 512037 1 TGATGGCGCCACTGAACGGT GGG 512059 -1 TGGTGGTGGTGAGATGGAGA TGG 512065 -1 CGGCCGTGGTGGTGGTGAGA TGG 512073 1 TCTCCATCTCACCACCACCA CGG 512073 -1 TCGCGAAGCGGCCGTGGTGG TGG 512076 -1 CGGTCGCGAAGCGGCCGTGG TGG 512079 -1 CCTCGGTCGCGAAGCGGCCG TGG 512085 -1 AGGAACCCTCGGTCGCGAAG CGG 512090 1 CCACGGCCGCTTCGCGACCG AGG 512091 1 CACGGCCGCTTCGCGACCGA GGG 512096 -1 ATGATGAGAGGAGGAACCCT CGG 512105 -1 ATTATTACTATGATGAGAGG AGG 512108 -1 ATTATTATTACTATGATGAG AGG 512150 1 TAAAAATCAGCAAATTGAAT TGG 597 12151 1 AAAAATCAGCAAATTGAATT GGG 512162 1 AATTGAATTGGGACAAATAA TGG 512181 1 ATGGAACAACATCATCTTCA TGG 612188 1 AACATCATCTTCATGGAGAT CGG 612204 -1 GGTTTGAGGAGGAAGCTCAT TGG 612215 -1 CTTAATGTAGTGGTTTGAGG AGG 612218 -1 TTTCTTAATGTAGTGGTTTG AGG 612225 -1 AGCTTGATTTCTTAATGTAG TGG 612267 1 TGATCAATCAGCAGCAGCAC AGG 612272 1 AATCAGCAGCAGCACAGGTG AGG 612284 -1 TTAATTTCATGGTGGGGCGG CGG 612287 -1 ATATTAATTTCATGGTGGGG CGG 612290 -1 CCAATATTAATTTCATGGTG GGG 612291 -1 TCCAATATTAATTTCATGGT GGG 612292 -1 GTCCAATATTAATTTCATGG TGG 612295 -1 TGTGTCCAATATTAATTTCA TGG 612301 1 CCCCACCATGAAATTAATAT TGG 612326 1 ACAGAGATTTCTCTTTTGAA CGG 612350 -1 CTCTCTCGTCATCAAACGCT GGG 612351 -1 TCTCTCTCGTCATCAAACGC TGG 612376 1 CGAGAGAGAATTCCGTTATT TGG 612377 -1 TTAACATTATAACCAAATAA CGG 612392 1 TATTTGGTTATAATGTTAAT CGG 612396 1 TGGTTATAATGTTAATCGGA CGG 612411 1 TCGGACGGTTCTCATTGTCT CGG 612423 -1 TCTAGCTCGTTGATCATCAG AGG 612499 -1 ATAATTAAACCGCTCATTAT TGG 612501 1 TAAGCAGCTCCAATAATGAG CGG 6 Table 3: CsMLO3 targeted gRNA sequences Position on SEQ ID NO:7 Strand Sequence PAM SEQ ID NO 777 1 TGAAACTCAAACTAAAATCA AGG 626 801 -1 TCTAACAGTTGGTATCAGAG CGG 6812 -1 ATATATAAATGTCTAACAGT TGG 6860 1 ATATGTTTAAGTATTAACTG CGG 6894 1 TATATACACTATATAACTTA AGG 6915 -1 GCTCAAGAATCAATGGCTGG AGG 6918 -1 GAAGCTCAAGAATCAATGGC TGG 6922 -1 GTTTGAAGCTCAAGAATCAA TGG 6944 -1 TTGCAGATCAAAGCTTATGT GGG 6945 -1 CTTGCAGATCAAAGCTTATG TGG 6957 1 CACATAAGCTTTGATCTGCA AGG 6958 1 ACATAAGCTTTGATCTGCAA GGG 6965 1 CTTTGATCTGCAAGGGAAAC TGG 6974 1 GCAAGGGAAACTGGTTGATG TGG 6975 1 CAAGGGAAACTGGTTGATGT GGG 6982 1 AACTGGTTGATGTGGGTAAT CGG 6983 1 ACTGGTTGATGTGGGTAATC GGG 6998 -1 TAAAGAGAGTTGAGAGAGCG AGG 61014 1 CTCTCTCAACTCTCTTTAGA TGG 61044 1 TGTTATGAACAGAATGAGTG AGG 61051 1 AACAGAATGAGTGAGGAGCT CGG 61052 1 ACAGAATGAGTGAGGAGCTC GGG 61053 1 CAGAATGAGTGAGGAGCTCG GGG 61066 -1 CACCTATAAATATAGGGTCT CGG 61072 -1 GTATCTCACCTATAAATATA GGG 61073 -1 AGTATCTCACCTATAAATAT AGG 61075 1 GACCGAGACCCTATATTTAT AGG 61096 -1 TAATGTGGCACAGATACTGA TGG 61111 -1 AAATATTCTGACAATTAATG TGG 61138 1 AATATTTTGACAATTAATTC AGG 61151 1 TTAATTCAGGAAATCAAATC AGG 61183 -1 ATTATGTAATATTCTATATA TGG 64585 1 GTTCTCACTATCAGTTATTA TGG 64595 1 TCAGTTATTATGGTTATTTA TGG 64615 1 TGGTTATTTATCTTTTTTAG TGG 64634 -1 CCTGAAGGGCTTTTTGTGTT TGG 661 4645 1 CCAAACACAAAAAGCCCTTC AGG 64648 -1 CTTCTCAAGCGCTTCCTGAA GGG 64649 -1 TCTTCTCAAGCGCTTCCTGA AGG 64670 1 GCGCTTGAGAAGATTAAATT AGG 64736 1 TTATTAGTATTTTTTTTTTT TGG 64751 1 TTTTTTGGTCTAATTTTAAT TGG 64752 1 TTTTTGGTCTAATTTTAATT GGG 64802 1 TGTTGCAGAGCTTATGCTAT TGG 64803 1 GTTGCAGAGCTTATGCTATT GGG 64842 -1 ATATGTCAGCAATGTAATCT TGG 64870 -1 CAAGTGTTTGCTGCACTTTT TGG 64882 1 CAAAAAGTGCAGCAAACACT TGG 64897 -1 TCTTCATTTTGGTATGGGCA AGG 64902 -1 TTTTCTCTTCATTTTGGTAT GGG 64903 -1 TTTTTCTCTTCATTTTGGTA TGG 64908 -1 TAGCCTTTTTCTCTTCATTT TGG 64916 1 ATACCAAAATGAAGAGAAAA AGG 64922 1 AAATGAAGAGAAAAAGGCTA AGG 64945 -1 TAATCAATTGTTTTTGATTT TGG 65012 1 TGTAATTATGTCTTAATGAT AGG 65033 1 GGACGTATACTAAAAGTGTG TGG 65078 1 AATGAGTTCTGAATTTTTGA AGG 65098 1 AGGACTTTTTGAATATTGTA TGG 65139 1 TAATATAAAATTAATATATA TGG 65181 1 TGATTTGTGTGTTTTGTGTG AGG 65187 1 GTGTGTTTTGTGTGAGGTGC AGG 65188 1 TGTGTTTTGTGTGAGGTGCA GGG 65214 1 AGTTCTTTAGTGTCTAAATA TGG 65215 1 GTTCTTTAGTGTCTAAATAT GGG 65232 -1 CAAATATGAAGATATGAAGC TGG 65249 1 TCATATCTTCATATTTGTCT TGG 65268 -1 TAGTAATGCAATATATAATA TGG 65285 1 TATATATTGCATTACTACCT TGG 65291 -1 TTTGGTTCTGCCAATAGCCA AGG 65292 1 TGCATTACTACCTTGGCTAT TGG 696 5309 -1 AACTTAAAAACTACTCACTT TGG 65361 1 CATATTCTATAAAATTAATA TGG 65401 1 TTGAATTGCAGATGAGAAAA TGG 65410 1 AGATGAGAAAATGGAAAGTT TGG 75411 1 GATGAGAAAATGGAAAGTTT GGG 75414 1 GAGAAAATGGAAAGTTTGGG AGG 75450 1 ATTGAGTACATATATAGTAA CGG 75537 1 TTGTATAATTAATTATTTTT TGG 75563 1 CACTACAACTTATCTAACTC AGG 76711 -1 ATCTTTACATTCTTACTTTT TGG 76785 1 TATATAAATATTCAATCAAA TGG 76789 1 TAAATATTCAATCAAATGGT TGG 76811 -1 CTTGTAAATCTAAATCTCTC AGG 76837 1 TTTACAAGAGACACATCATT TGG 76859 1 GAAGAAGACATTTGAACATT TGG 76873 -1 TCCAAAGTGAAATTGGTGAT TGG 76880 -1 CTTACAATCCAAAGTGAAAT TGG 76883 1 GCCAATCACCAATTTCACTT TGG 76927 -1 TTGTTTTCTTCTCTATAATA AGG 76973 1 TCAAAAGTTTTTTATTATAT AGG 77030 1 TTCTTGTTTATCAAATGATC AGG 77055 1 TGCTTTTTCAGACAATTCTT CGG 77056 1 GCTTTTTCAGACAATTCTTC GGG 77069 1 ATTCTTCGGGTCAGTCACTA AGG 77089 1 AGGTTGATTACATGACACTG AGG 77094 1 GATTACATGACACTGAGGCA TGG 77105 1 ACTGAGGCATGGATTTGTAA TGG 77126 1 GGTATGTTGCACAATGATCT TGG 77137 1 CAATGATCTTGGCCTGAAAA TGG 77138 -1 TGTAATTTGAAGCCATTTTC AGG 77203 1 AGCTATGCTTTTCCCATTTC AGG 77204 -1 GAGCCAAATGTGCCTGAAAT GGG 77205 -1 GGAGCCAAATGTGCCTGAAA TGG 77212 1 TTTCCCATTTCAGGCACATT TGG 77226 -1 TCAAATCTTGTTTCACTTTC TGG 731 7272 1 CATCAGCAAATCACTTGATC AGG 77291 1 CAGGATTTTGTAGTAATTGT TGG 77292 1 AGGATTTTGTAGTAATTGTT GGG 77323 -1 ATATTATAAGCTGATTTCAA AGG 77419 -1 CGGCAACGAACCAAATTACT GGG 77420 1 ATATATGCAGCCCAGTAATT TGG 77420 -1 ACGGCAACGAACCAAATTAC TGG 77439 -1 GTTGGACAGTAGAAACAATA CGG 77457 -1 CAATAACTTACCATATGTGT TGG 77458 1 TTTCTACTGTCCAACACATA TGG 77519 -1 CAACATTTCAGTCACTGAAA TGG 77549 1 GTTGTTCTTTTTTAATTAAC AGG 77568 1 CAGGAATATACTCTTATTTG TGG 77583 -1 CTTACAATCAAAGGTAGAAA TGG 77592 -1 TGTGTTGTACTTACAATCAA AGG 77660 -1 TTCCACACATTAGCAAATGT GGG 77661 -1 TTTCCACACATTAGCAAATG TGG 77669 1 GTCCCACATTTGCTAATGTG TGG 77699 1 TTGTGATATATAAGATGAAT AGG 77715 1 GAATAGGCTACTCCTTTTAT AGG 77716 1 AATAGGCTACTCCTTTTATA GGG 77716 -1 CCATTTGAAAACCCTATAAA AGG 77727 1 CCTTTTATAGGGTTTTCAAA TGG 77741 -1 ATTTAGGAATAAGATGAATG GGG 77742 -1 AATTTAGGAATAAGATGAAT GGG 77743 -1 GAATTTAGGAATAAGATGAA TGG 77757 -1 GACATACCATGTTAGAATTT AGG 77762 1 CTTATTCCTAAATTCTAACA TGG 77788 -1 AAAAACCCAACACTGGAAAG TGG 77793 1 TGTGTGCCACTTTCCAGTGT TGG 77794 1 GTGTGCCACTTTCCAGTGTT GGG 77795 -1 ACAGGTCAAAAACCCAACAC TGG 77813 -1 AAATTTGTAGATTTTGAAAC AGG 77849 -1 CCAAATATCGGAAAATTTGT GGG 77850 -1 GCCAAATATCGGAAAATTTG TGG 766 7860 1 CCCACAAATTTTCCGATATT TGG 77861 -1 AATCTCACAAGGCCAAATAT CGG 77872 -1 ACATTTGAAAGAATCTCACA AGG 77892 1 ATTCTTTCAAATGTCACGTT CGG 77900 1 AAATGTCACGTTCGGTCCTG TGG 77905 -1 AACGACCTTTCAGAGACCAC AGG 77911 1 TCGGTCCTGTGGTCTCTGAA AGG 77935 -1 CGTTTGGGCCTGAAAAGTGT GGG 77936 -1 ACGTTTGGGCCTGAAAAGTG TGG 77938 1 TCGTTATACCCACACTTTTC AGG 77950 -1 TTAATACACTCCTCACGTTT GGG 77951 1 ACTTTTCAGGCCCAAACGTG AGG 77951 -1 CTTAATACACTCCTCACGTT TGG 77991 1 AGTCTCACATTGCTAATGTA TGG 78020 1 ATTGTGATATATAAAATGAA TGG 78021 1 TTGTGATATATAAAATGAAT GGG 78038 -1 TAAAACTAATTGGCTGTGGG AGG 78041 -1 TCTTAAAACTAATTGGCTGT GGG 78042 -1 ATCTTAAAACTAATTGGCTG TGG 78048 -1 GGTTTTATCTTAAAACTAAT TGG 78069 -1 ATTTAGGGATAAGATGAATG GGG 78070 -1 AATTTAGGGATAAGATGAAT GGG 78071 -1 GAATTTAGGGATAAGATGAA TGG 78084 -1 ATTAAGCATGTTAGAATTTA GGG 78085 -1 GATTAAGCATGTTAGAATTT AGG 78144 1 CAAATTGCAGATAATATTAC TGG 78147 1 ATTGCAGATAATATTACTGG TGG 78148 1 TTGCAGATAATATTACTGGT GGG 78180 1 TCAAGTAATCATAACAAAGA TGG 78181 1 CAAGTAATCATAACAAAGAT GGG 78202 1 GGATTAAGCATTCAAGAGAG AGG 78210 1 CATTCAAGAGAGAGGAGATG TGG 78217 1 GAGAGAGGAGATGTGGTAAA AGG 78228 1 TGTGGTAAAAGGTGCACCAT TGG 88233 -1 TCATCTCCTGGTTGAACCAA TGG 801 8238 1 GGTGCACCATTGGTTCAACC AGG 88245 -1 AACCAGAAGAGGTCATCTCC TGG 88254 1 AACCAGGAGATGACCTCTTC TGG 88256 -1 TAGGCCGTCCGAACCAGAAG AGG 88259 1 GGAGATGACCTCTTCTGGTT CGG 88263 1 ATGACCTCTTCTGGTTCGGA CGG 88275 -1 ATGAGAAAGAGCATTAATTT AGG 88301 -1 TAAGTACCTGAAAGAGAACA AGG 88306 1 CATTCACCTTGTTCTCTTTC AGG 88413 1 AAAATGATATCTTTTCTGCT TGG 88429 1 TGCTTGGTACTAATTAATGC TGG 88487 -1 TACTGTACTCCATGCAAAAA AGG 88489 1 TTCAACTTGCCTTTTTTGCA TGG 88531 -1 TGCCTTGAAACCAAAAATCA AGG 88532 1 ATTTGACTTTCCTTGATTTT TGG 88540 1 TTCCTTGATTTTTGGTTTCA AGG 88559 1 AAGGCAATAAAATTATTACA TGG 88624 -1 TTCGTGGAAGCAAGTGTTCA AGG 88640 -1 TGATATCTTCAATTTTTTCG TGG 88669 1 TATCATCATAAGAATTTCAA TGG 88670 1 ATCATCATAAGAATTTCAAT GGG 88671 1 TCATCATAAGAATTTCAATG GGG 88805 1 TTCTCTTTTTCTTTCTTACT AGG 88819 -1 AACTGCATAGAACTTGTATG AGG 88853 -1 TGTGTGACAAGAGCATATAG AGG 88866 1 TCTATATGCTCTTGTCACAC AGG 88893 -1 GATGATAATGATGATTTAGA AGG 88954 1 ATTTGATCATATATTACAGA TGG 88955 1 TTTGATCATATATTACAGAT GGG 88956 1 TTGATCATATATTACAGATG GGG 88980 -1 ACTCTGTCATTGAAAATTAC TGG 89013 1 TAGCAACAGCATTAAAGAAC TGG 89027 -1 TGTTCTTGGTTTTGGCTGAA TGG 89035 -1 GTGTTTTTTGTTCTTGGTTT TGG 89041 -1 TCGGTTGTGTTTTTTGTTCT TGG 836 9059 1 CAAAAAACACAACCGAAATT CGG 89060 -1 GCGAGTTTGTCTCCGAATTT CGG 89082 -1 GTTGCAGGCCTACTTGAGAA TGG 89085 1 CAAACTCGCCATTCTCAAGT AGG 89097 -1 ATGCCATATGTTGGAGTTGC AGG 89105 1 AGGCCTGCAACTCCAACATA TGG 89106 -1 ACTGGAGACATGCCATATGT TGG 89124 -1 TAATTTTGCAGCAGATGAAC TGG 89156 1 TACAGAAGCACAGCAACTGA TGG 89165 1 ACAGCAACTGATGGATACTA TGG 89175 1 ATGGATACTATGGTTCTCCG AGG 89181 -1 TTTTCGACATTAGACATCCT CGG 89213 1 AACGATTACTATGAGCCTGA AGG 89214 1 ACGATTACTATGAGCCTGAA GGG 89217 -1 TTGGGAGATGGTGTCCCTTC AGG 89229 -1 GATGGTCCATTGTTGGGAGA TGG 89234 1 GGGACACCATCTCCCAACAA TGG 89235 -1 GCTGCAGATGGTCCATTGTT GGG 89236 -1 TGCTGCAGATGGTCCATTGT TGG 89247 -1 TGTATTTCACTTGCTGCAGA TGG 89284 1 GAATAACTATGAAGTTGAGA AGG 89296 1 AGTTGAGAAGGATATAAGTG AGG 89300 1 GAGAAGGATATAAGTGAGGA AGG 89311 1 AAGTGAGGAAGGACAGCCAA TGG 89316 -1 GAGCTTGGTTCCTGAACCAT TGG 89317 1 GGAAGGACAGCCAATGGTTC AGG 89331 -1 TTTTGCTGTGAGGAGGAGCT TGG 89338 -1 GACCTCATTTTGCTGTGAGG AGG 89341 -1 CTTGACCTCATTTTGCTGTG AGG 89347 1 CTCCTCCTCACAGCAAAATG AGG 89368 -1 CCTAAATGAGAAGTGAGATA AGG 89379 1 CCTTATCTCACTTCTCATTT AGG 89450 1 CTTTATTTCTTATTATCTTT TGG 89498 1 AATATGTATAAGCTTGAATT TGG 8 Reference is made to Table 4 summarizing sequences relating to WT CsMLO within the scope of the current invention.

Table 4: WT CsMLO sequence table Sequence type characterization CsMLO1 CsMLO2 CsMLO3 Genomic sequence SEQ ID NO:1 SEQ ID NO:4 SEQ ID NO:7 Coding sequence (CDS) SEQ ID NO:2 SEQ ID NO:5 SEQ ID NO:8 Amino acid sequence SEQ ID NO:3 SEQ ID NO:6 SEQ ID NO:9 gRNA sequence SEQ ID NO:10- SEQ ID NO:2(Table 1) SEQ ID NO:287- SEQ ID NO:6(Table 2) SEQ ID NO:626- SEQ ID NO:8(Table 3) 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: • DNA vectors • Ribonucleoprotein complex (RNP’s) According to further embodiments of the present invention, transformation of various Cannabis tissues was performed using Agrobacterium (Agrobacterium tumefaciens) by: ? Regeneration-based transformation ? Floral-dip transformation ? Seedling transformation 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 Fig. 4A-D photographically presenting GUS staining after transient transformation of the following Cannabis tissues (A) axillary buds (B) leaf (C) calli, and (D) cotyledons.

Fig. 4 demonstrates that various Cannabis tissues have been successfully transiently transformed using biolistics system. Transformation has been performed into calli, leaves, axillary buds and cotyledons of Cannabis.

According to further embodiments of the present invention, additional transformation tools were used in Cannabis, including, but not limited to: ? Protoplast PEG transformation ? Extend RNP use ? Directed editing screening using fluorescent tags ? Electroporation 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 Fig. 5 presenting regeneration of Cannabis tissue. In this figure, arrows indicate new meristem emergence.

Stage 5: Selection of positive transformants. Once regenerated plants appear in tissue culture, DNA is extracted from leaf sample of the transformed plant and PCR is performed using primers flanking the edited region. PCR products are then digested with enzymes recognizing the restriction site near the original gRNA sequence. If editing event occurred, the restriction site will be disrupted and the PCR product will not be cleaved. No editing event will result in a cleaved PCR product.

Reference is now made to Fig. 6 showing PCR detection of Cas9 DNA in shoots of transformed Cannabis plants. DNA extracted from shoots of plants transformed with Cas9 using biolistics. This figure shows that three weeks post transformation, Cas9 DNA was detected in shoots of transformed plants.

Screening for CRISPR/Cas9 gene editing events has been performed by at least one of the following analysis methods: ? Restriction Fragment Length Polymorphism (RFLP) ? Next Generation Sequencing (NGS) ? PCR fragment analysis ? Fluorescent-tag based screening ? High resolution melting curve analysis (HRMA) Reference is now made to Fig. 7 presenting results of in vitro analysis of CRISPR/Cas9 cleavage activity. Fig. 7A schematically shows the genomic area targeted for editing (PAM is marked in red) and amplified by the reverse and forward designed primers Fig. 7B photographically presents a gel showing successful digestion of the resulted PCR amplicon containing the gene specific gRNA sequence, by RNP complex containing Cas9. The analysis included the following steps: 1) Amplicon was isolated from two exemplified Cannabis strains by primers flanking the sequence of the gene of interest targeted by the predesigned sgRNA. 2) RNP complex was incubated with the isolated amplicon. ? 3) The reaction mix was then loaded on agarose gel to evaluate Cas9 cleavage activity at the target site.

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.

EXAMPLE 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 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 (Pépin 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: • Development of susceptibility index • Designing a protocol by testing different inoculation approaches at several plant developmental stages EXAMPLE Production of genome-edited Cannabis MLO (CsMLO) 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 Fig. 9 ). Transformation was performed using a DNA plasmid such as a plant codon optimized Streptococcus pyogenes Cas9 (pcoSpCas9) plasmid presented in Fig. 8 . The plasmid contained the plant codon optimized SpCas9 and the above mentioned at least one sgRNA.

About 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 Fig. 9 and Fig. 10 ). As shown in Fig. 10 , the aforementioned primer pair (marked with arrows) generates a 778 bp amplicon comprising the entire exon 1 of CsMLO1, having a nucleotide sequence as set forth in SEQ ID NO:873 (nucleotide positions 4-782 of SEQ ID NO:1). In Fig. 10 the three gRNA sequences used to target exon 1 of CsMLO1 genomic sequence are underlined. The translation initiation codon ATG (encoding Methionine amino acid) is marked with a square. Fig. 11 presents the amino acid sequence of CsMLO1 first exon as set forth in SEQ ID NO:874.

Reference is now made to Fig. 12 photographically presenting detection of CsMLO1 PCR products showing length variation (i.e. truncated fragments) as a result of Cas9- mediated genome editing. DNA from plants two months post transformation was used as a template for the PCR using primers having nucleic acid sequence as set forth in SEQ ID NO:871 and SEQ ID NO:872. DNA fragments shorter than the expected WT 780 bp amplicon were obtained by the PCR reaction and subcloned into a sequencing plasmid and sequenced. The sequencing results are described below.

It can be seen in Fig. 12 that WT or non-edited PCR products result in a 780 bp band, while DNA extracted from edited plants exhibit a shorter band than the expected 780 bp WT exon 1 length, i.e. samples 1 and 2 show a 450 bp fragment and samples 3 and 4 show a 350 bp fragment.

Fig. 13schematically presents sequences of WT and genome edited CsMLO1 DNA fragments obtained for the first time by the present invention. In this figure, sgRNA sequences are underlined. sgRNA having nucleotide sequence as set forth in SEQ ID NO:17 (first guide) with Hinfrestriction site appears on the left hand of exon 1, and sgRNA having nucleotide sequence as set forth in SEQ ID NO:50 (third guide) with BtsI restriction site appears on the right hand of exon fragments. PAM sequences (NGG) are in marked italics and bold and are circled. ATG codon position is marked with a square.

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.

Table 5: Sequences of mutated CsMLO1 exon 1 Sequence type Exon 1 of WT CsMLO1 65-L4 ( ? 447) fragment of CsMLO1 65-L5 (?373) fragment of CsMLO1 85-4 ( ? 456) fragment of CsMLO1 Genomic sequence (Position in SEQ ID NO:1) SEQ ID NO:8 (nucleic acid 4-782 in SEQ ID NO:1) ( Fig. 10 ) SEQ ID NO:8 (deletion of nucleic acid 109-556 in SEQ ID NO:1) ( Fig. 13 ) SEQ ID NO:8 (deletion of nucleic acid 128-501 in SEQ ID NO:1) ( Fig. 13 ) SEQ ID NO:8 (deletion of nucleic acid 96-552 in SEQ ID NO:1) ( Fig. 13 ) Deleted nucleic acid sequence SEQ ID NO:8 SEQ ID NO:8 SEQ ID NO:8 Amino acid sequence SEQ ID NO:8 ( Fig. 11 ) SEQ ID NO:8 MS SEQ ID NO:8 MSGGGEGE No amino- acid sequence is produced gRNA sequence targeted to Exon of CsMLO1 SEQ ID NO:17, SEQ ID NO:and SEQ ID NO:50 (Table 1) The resulted mutated CsMLO1 fragments include the following: (1) Fragment 1: CsMLO1 fragment marked as 65-L4 ?447 comprises a nucleotide sequence as set forth in SEQ ID NO:875 (about 330 bp). This fragment contains a deletion of 4bp (position 109-556 of SEQ ID NO:1) having a nucleotide sequence as set forth in SEQ ID NO:876. It should be noted that this fragment encodes a two amino acid peptide (SEQ ID NO:887, as shown in Table 5). The short CsMLO1 exon 1 peptide generated by the targeted genome editing is expected to result is a non-functional, silenced CsMLO1 gene or allele. (2) Fragment 2: CsMLO1 fragment marked as 65-L5 ?373 comprises a nucleotide sequence as set forth in SEQ ID NO:877 (about 405 bp). This fragment contains a deletion of 3bp (position 128-501 of SEQ ID NO:1) having a nucleotide sequence as set forth in SEQ ID NO:879. It should be noted that this fragment encodes a short peptide of eight amino acids (SEQ ID NO:878, as shown in Table 5). Such a short exon 1 fragment is expected to result in a non-functional CsMLO1 allele. (3) Fragment 3: CsMLO1 fragment marked as 85-4 ?456 comprises a nucleotide sequence as set forth in SEQ ID NO:880 (about 320 bp). This fragment contains a deletion of 456 bp (position 96-552 of SEQ ID NO:1) having a nucleotide sequence as set forth in SEQ ID NO:881. It is emphasized that fragment 3 was edited such that it lacks the ATG translation start codon, therefore no translated protein is generated. The resulted truncated CsMLOgene/protein is expected to be non-functional.

The genome-edited CsMLO1 truncated fragments of the present invention are characterized by deletion of significant parts of the first exon sequence of CsMLO1 gene. Thus these genome edited fragments produce truncated CsMLO1 proteins. The truncated proteins lack significant part of the Open Reading Frame (ORF), e.g. absent of the translation start codon or significant part of exon-protein encoding sequence, and therefore would be non-functional.

EXAMPLE

Claims

1. A modified Cannabis plant exhibiting enhanced resistance to powdery mildew (PM), wherein said modified plant comprises a mutated Cannabis mlo1 (Csmlo1) allele, said mutated allele comprising a genomic modification selected from an indel of 14 bp at position corresponding to position 12 of SEQ ID NO: 882, or a fraction thereof, or a nucleic acid insertion at position corresponding to position 104-105 of SEQ ID NO: 882, or a combination thereof.

2. The modified Cannabis plant according to claim 1, wherein said indel comprises a sequence as set forth in SEQ ID NO:883 or a fraction thereof.

3. The modified Cannabis plant according to claim 1, wherein said Csmlo1 mutant allele comprises a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:884, or a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:885, or a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:886, or a homologue having at least 80% sequence identity to the nucleic acid sequence of said mutated Csmlo1 allele, or a complementary sequence thereof, or any combination thereof.

4. The modified Cannabis plant according to claim 1, wherein said mutated Csmlo1 allele confers an enhanced resistance to powdery mildew as compared to a Cannabis plant comprising a wild type CsMLO1 allele having a nucleic acid sequence as set forth in SEQ ID NO:882 and/or having a nucleic acid sequence as set forth in SEQ ID NO:1 or a functional variant thereof.

5. The modified Cannabis plant according to claim 4, wherein said functional variant has at least 80% sequence identity to the corresponding CsMLO1 nucleotide sequence.

6. The modified Cannabis plant according to claim 1, wherein said mutated allele comprising a deletion of 14 bp at position 389 of SEQ ID NO: 1, or a nucleic acid insertion at position 482-483 of SEQ ID NO: 1, or a combination thereof.

7. The modified Cannabis plant according to claim 1 wherein said mutated Csmlo1 allele is generated using genome editing.

8. The modified Cannabis plant according to claim 1, wherein said plant has decreased expression levels of Mlo1 protein, relative to a Cannabis plant lacking said mutated Csmloallele.

9. The modified Cannabis plant according to claim 1, wherein said mutated Csmlo1 allele is generated using mutagenesis, small interfering RNA (siRNA), microRNA (miRNA), artificial miRNA (amiRNA), DNA introgression, endonucleases or any combination thereof.

10. The modified Cannabis plant according to claim 9, wherein said endonuclease is selected from the group consisting of 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.

11. The modified Cannabis plant according to claim 1, wherein said plant comprises a DNA construct, said DNA construct comprising a promoter operably linked to a nucleotide sequence encoding a plant optimized Cas9 endonuclease, wherein said plant optimized Casendonuclease is capable of binding to and creating a double strand break in a genomic target sequence of said plant genome, further wherein said DNA construct further comprises gRNA targeted to at least one CsMLO1 allele.

12. The modified Cannabis plant according to claim 11, wherein said gRNA has a nucleic acid sequence corresponding to a sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence thereof, or any combination thereof.

13. The modified Cannabis plant according to claim 1, wherein said genome modification is selected from a silencing mutation, a knockdown mutation, a knockout mutation, a loss of function mutation, an insertion, deletion, indel, substitution, 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, an epigenetic factor, or any combination thereof.

14. The modified Cannabis plant according to claim 1, wherein said genome modification is generated via introduction (a) Cas DNA and gRNA sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 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:17, SEQ ID NO:43 and SEQ ID NO:50 and any combination thereof.

15. The modified Cannabis plant according to claim 1, wherein said PM is selected from the group consisting of Golovinomyces cichoracearum, Golovinomyces ambrosiae and a mixture thereof.

16. The modified Cannabis plant according to claim 1 wherein said Cannabis plant does not comprise a transgene.

17. A progeny plant, plant part, plant cell, tissue culture of regenerable cells, protoplasts, callus or plant seed of a modified plant according to claim 1.

18. A modified Cannabis plant exhibiting enhanced resistance to powdery mildew (PM), wherein said modified plant comprises a targeted genome modification conferring reduced expression of a Cannabis MLO1 (CsMLO1) gene as compared to a Cannabis plant lacking said targeted genome modification, said targeted genome modification generates a mutated Cannabis mlo1 (Csmlo1) allele comprising a deletion of a nucleic acid sequence as set forth in SEQ ID NO:883 or a fraction thereof as compared to the wild type CsMLO1 allele comprising a sequence as set forth in SEQ ID NO:1, or a nucleic acid insertion at position 482-483 of SEQ ID NO:1, or a combination thereof.

19. The modified Cannabis plant according to claim 18, wherein said mutated allele comprising a deletion of 14 bp at position 389 of SEQ ID NO: 1, or a nucleic acid insertion at position 482-483 of SEQ ID NO: 1, or a combination thereof.

20. A method for producing a modified Cannabis plant according to any one of claims 1-and 18-19, said method comprises introducing using targeted genome modification, at least one genomic modification conferring reduced expression of at least one Cannabis MLO(CsMLO1) allele as compared to a Cannabis plant lacking said targeted genome modification, said genomic modification generates a mutated Cannabis mlo1 (Csmlo1) allele comprising an indel of 14 bp at a position corresponding to position 12 of SEQ ID NO: 882 or a fraction thereof, or a nucleic acid insertion at position corresponding to position 104-105 of SEQ ID NO: 882, or a combination thereof.

21. The method according to claim 20, comprises steps of introducing a loss of function mutation into said CsMLO1 allele using targeted genome modification.

22. The method according to claim 20, wherein said indel comprises a sequence as set forth in SEQ ID NO:883 or a fraction thereof.

23. The method according to claim 20, wherein said Csmlo1 mutant allele comprises a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:884, or a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:885, or a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:886, or a homologue having at least 80% sequence identity to the nucleic acid sequence of said mutated Csmlo1 allele, or a complementary sequence thereof, or any combination thereof.

24. The method according to claim 20, wherein said mutated Csmlo1 allele confers an enhanced resistance to powdery mildew as compared to a Cannabis plant comprising a wild type CsMLO1 allele having a nucleic acid sequence as set forth in SEQ ID NO:882 and/or having a nucleic acid sequence as set forth in SEQ ID NO:1 or a functional variant thereof, said functional variant has at least 80% sequence identity to the corresponding CsMLOnucleotide sequence.

25. The method according to claim 20, wherein said mutated allele comprising a deletion of bp at position 389 of SEQ ID NO: 1, or a nucleic acid insertion at position 482-483 of SEQ ID NO: 1, or a combination thereof.

26. The method according to claim 20, wherein said modified plant has decreased levels of at least one Mlo protein as compared to wild type Cannabis plant.

27. The method according to claim 20 comprising steps of introducing and co-expressing in a Cannabis plant Cas9 and gRNA targeted to CsMLO1 gene and screening for induced targeted mutations conferring reduced expression of said CsMLO1 gene, said gRNA nucleotide sequence targeting said CsMLO1 allele is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence thereof.

28. The method according to claim 20 wherein said genomic modification 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.

29. The method according to claim 20, wherein said genomic modification is selected from the group consisting of a silencing mutation, a knockdown mutation, a knockout mutation, a loss of function mutation, an insertion, deletion, indel or substitution mutation and any combination thereof.

30. The method according to claim 20, further comprising steps of selecting a plant resistant to powdery mildew from plants comprising mutated Csmlo1 allele.

31. The method according to claim 30, wherein said selected plant is characterized by enhanced resistance to powdery mildew as compared to a Cannabis plant comprising a CsMLOnucleic acid comprising a nucleic acid sequence as set forth in SEQ ID NO:882.

32. The method according to claim 20, 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:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence thereof, 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:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence thereof, and any combination thereof.

33. The method according to claim 20, further comprising steps of regenerating a plant carrying said genomic modification.

34. The method according to claim 33 further comprising steps of screening said regenerated plants for a plant resistant to powdery mildew.

35. A method for conferring powdery mildew resistance to a Cannabis plant comprising producing a plant according to the method of claim 20.

36. A plant, plant part, plant cell, tissue culture or a seed obtained or obtainable by the method of claim 20.

37. The method according to claim 20 wherein said powdery mildew is selected from the group of species consisting of Golovinomyces cichoracearum, Golovinomyces ambrosiae and a mixture thereof.

38. A method of determining the presence of a mutant Csmlo1 allele in a Cannabis plant comprising assaying said Cannabis plant for at least one of the presence of an indel comprising a nucleic acid sequence as set forth in SEQ ID NO:883, an insertion at position 104-105 of SEQ ID NO: 882, a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:884, a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:885, a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:886, or a homologue having at least 80% sequence identity to the nucleic acid sequence of said mutated Csmlo1 allele, a complementary sequence thereof, or any combination thereof.

39. 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 a mutated Csmlo1 allele, said mutated allele comprises a genomic modification selected from an indel of 14 bp at a position corresponding to position 12 of SEQ ID NO: 882 or a fraction thereof, or a nucleic acid insertion at position corresponding to position 104-105 of SEQ ID NO: 882, or a combination thereof; b. optionally, regenerating plants carrying said genetic modification; and c. optionally, screening said regenerated plants for a plant resistant to powdery mildew.

40. The method according to claim 39, wherein said genomic modification is a loss of function mutation.

41. The method according to claim 39, wherein said indel comprises a sequence as set forth in SEQ ID NO:883 or a fraction and/or a complementary sequence thereof.

42. The method according to claim 39, wherein said Csmlo1 mutant allele comprises a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:884, or a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:885, or a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID NO:886, or a homologue having at least 80% sequence identity to the nucleic acid sequence of said mutated Csmlo1 allele, or a complementary sequence thereof, or any combination thereof.

43. The method according to claim 39, wherein said method comprises steps of screening said Cannabis plant for the presence of a deletion in CsMLO1 gene comprising a nucleic acid sequence as set forth in SEQ ID NO:1, said deletion comprising a nucleotide sequence as set forth in SEQ ID NO:883.

44. The method according to claim 39, wherein said modified Cannabis plant comprising a mutant Csmlo1 nucleic acid conferring enhanced resistance to powdery mildew as compared to a Cannabis plant comprising a wild type CsMLO1 nucleic acid.

45. The method according to any one of claims 38 and 39, comprising steps of down regulation of Cannabis MLO1 (CsMLO1) gene, which comprises utilizing the nucleotide sequence as set forth in at least one of SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence thereof, and a combination thereof, for introducing a loss of function mutation into said CsMLO1 gene using targeted genome modification.

46. An isolated amino acid sequence having at least 80% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:882-886, SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence or any combination thereof.

47. Use of a nucleotide sequence as set forth in SEQ ID NO: 883-886, SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 for generating, identifying and/or screening for a Cannabis plant comprising within its genome mutant Csmlo allele conferring resistance to PM.

48. The use according to claim 47, wherein the presence of at least one nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:883, SEQ ID NO:8indicates that the Cannabis plant comprises a wild type CsMLO1 allele, and the presence of at least one nucleic acid sequence selected from the group consisting of SEQ ID NO:884, SEQ ID NO:885 and SEQ ID NO:886 indicates that the Cannabis plant comprises a mutant Csmol1 allele.

49. Use of a nucleotide sequence as set forth in at least one of SEQ ID NO:17, SEQ ID NO:and SEQ ID NO:50 or a complementary sequence or any combination thereof for targeted genome modification of Cannabis MLO1 (CsMLO1) gene.

50. A detection kit for determining the presence or absence of a mutant Csmlo1 allele in a Cannabis plant, comprising a nucleic acid fragment comprising a sequence selected from SEQ ID NO:882-886, SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence or any combination thereof, and/or said kit is useful for identifying a Cannabis plant with enhanced resistance to powdery mildew.