CA3142241A1 - Cannabis plants with improved yield - Google Patents
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- CA3142241A1 CA3142241A1 CA3142241A CA3142241A CA3142241A1 CA 3142241 A1 CA3142241 A1 CA 3142241A1 CA 3142241 A CA3142241 A CA 3142241A CA 3142241 A CA3142241 A CA 3142241A CA 3142241 A1 CA3142241 A1 CA 3142241A1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/02—Flowers
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/28—Cannabaceae, e.g. cannabis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
Abstract
The present invention discloses a modified Cannabis plant exhibiting at least one improved domestication trait compared with wild type Cannabis, wherein the modified plant comprises at least one mutated Cannabis SELF PRUNING (SP) (CsSP) gene and/or at least one mutated Cannabis SELF PRUNING 5G (SP5G) (CsSPSG) gene. The present invention further discloses methods for production of the aforementioned modified Cannabis plant using genome modification.
Description
CANNABIS PLANTS WITH IMPROVED YIELD
FIELD OF THE INVENTION
The present disclosure relates to conferring desirable agronomic traits in Cannabis plants. More particularly, the current invention pertains to producing Cannabis plants with improved yield traits by manipulating genes controlling day-length sensitivity and plant architecture.
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 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.
One of the most important determinants of crop productivity is plant architecture. For many crops, artificial selection for modified shoot architectures provided critical steps towards improving yield, followed by innovations enabling large- scale field production. A prominent example is tomato, in which the discovery of a mutation in the antiflorigen-encoding self-pruning gene (sp), led to determinate plants that provided a burst of flowering and synchronized fruit ripening, permitting mechanical harvesting.
In addition, day-length sensitivity in crops limits their geographical range of cultivation, and thus modification of the photoperiod response was critical for their domestication.
PCT application W02017180474 discloses a tomato plant that is a sp5g sp double mutant and that flowers earlier than the corresponding sp [sibling] tomato plant, as measured with reference to the number of leaves produced prior to appearance of first inflorescence.
The publication of Soyk et al (2016), Nature Genetics, "Variation in the flowering gene SELF
PRUNING 5G promotes day-neutrality and early yield in tomato" shows that loss of day-length-sensitive flowering in tomato was driven by the florigen paralog and flowering repressor SELF-PRUNING 5G (SP5G). This publication reports that CRISPR/Cas9-engineered mutations in SP5G
cause rapid flowering and enhance the compact determinate growth habit of field tomatoes, resulting in a quick burst of flower production that translates to an early yield. However, these engineered tomato plants showed total yield reduction as compared to sp mutated tomato plants.
The publication of Li et al (2018), nature biotechnology, "Domestication of wild tomato is accelerated by genome editing" teach the assembly of a set of six gRNAs to edit four genes (S1CLV3, SlWUS, SP and SP5G) into one construct. The construct was transformed into four S.
pimpinellifolium accessions, all of which are resistant to bacterial spot disease, and two of which are salt tolerant. Small indels and large insertions have been identified in the targeted regulatory regions of 51CLV3 and SlWUS in TO and their Ti mutant plants. It was reported in this publication that although SP and SP5G are crucial for improving the harvest index, the limited allelic variation has hampered efforts to optimize this trait. It was further reported that locule number was not increased in TO and Ti plants with large insertions and inversions in the targeted 51CLV3 promoter region. One explanation for this finding is that the targeted region of the 51CLV3 promoter may not be essential for regulating 51CLV3 transcription. Alternatively, it was suggested that disruption of regions (gRNA-5) flanking the CArG element downstream of SlWUS may have decreased its transcription and counteracted the effects of mutation of 51CLV3, owing to a negative feedback loop of CLV3¨WUS in controlling stem cell proliferation.
FIELD OF THE INVENTION
The present disclosure relates to conferring desirable agronomic traits in Cannabis plants. More particularly, the current invention pertains to producing Cannabis plants with improved yield traits by manipulating genes controlling day-length sensitivity and plant architecture.
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 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.
One of the most important determinants of crop productivity is plant architecture. For many crops, artificial selection for modified shoot architectures provided critical steps towards improving yield, followed by innovations enabling large- scale field production. A prominent example is tomato, in which the discovery of a mutation in the antiflorigen-encoding self-pruning gene (sp), led to determinate plants that provided a burst of flowering and synchronized fruit ripening, permitting mechanical harvesting.
In addition, day-length sensitivity in crops limits their geographical range of cultivation, and thus modification of the photoperiod response was critical for their domestication.
PCT application W02017180474 discloses a tomato plant that is a sp5g sp double mutant and that flowers earlier than the corresponding sp [sibling] tomato plant, as measured with reference to the number of leaves produced prior to appearance of first inflorescence.
The publication of Soyk et al (2016), Nature Genetics, "Variation in the flowering gene SELF
PRUNING 5G promotes day-neutrality and early yield in tomato" shows that loss of day-length-sensitive flowering in tomato was driven by the florigen paralog and flowering repressor SELF-PRUNING 5G (SP5G). This publication reports that CRISPR/Cas9-engineered mutations in SP5G
cause rapid flowering and enhance the compact determinate growth habit of field tomatoes, resulting in a quick burst of flower production that translates to an early yield. However, these engineered tomato plants showed total yield reduction as compared to sp mutated tomato plants.
The publication of Li et al (2018), nature biotechnology, "Domestication of wild tomato is accelerated by genome editing" teach the assembly of a set of six gRNAs to edit four genes (S1CLV3, SlWUS, SP and SP5G) into one construct. The construct was transformed into four S.
pimpinellifolium accessions, all of which are resistant to bacterial spot disease, and two of which are salt tolerant. Small indels and large insertions have been identified in the targeted regulatory regions of 51CLV3 and SlWUS in TO and their Ti mutant plants. It was reported in this publication that although SP and SP5G are crucial for improving the harvest index, the limited allelic variation has hampered efforts to optimize this trait. It was further reported that locule number was not increased in TO and Ti plants with large insertions and inversions in the targeted 51CLV3 promoter region. One explanation for this finding is that the targeted region of the 51CLV3 promoter may not be essential for regulating 51CLV3 transcription. Alternatively, it was suggested that disruption of regions (gRNA-5) flanking the CArG element downstream of SlWUS may have decreased its transcription and counteracted the effects of mutation of 51CLV3, owing to a negative feedback loop of CLV3¨WUS in controlling stem cell proliferation.
2 The publication of Zsogon et al (2018), nature biotechnology, "De novo domestication of wild tomato using genome editing" discloses a devised CRISPR¨Cas9 genome engineering strategy to combine agronomically desirable traits with useful traits presented in Solanum pimpinellifolium wild lines. The four edited genes were SELF-PRUNING (SP), OVATE (0), FRUIT
WEIGHT 2.2 (FW2.2) and LYCOPENE BETA CYCLASE (CycB).
Lemmon et al (2018), Nature Plants, "Rapid improvement of domestication traits in an orphan crop by genome editing" describes the usage of CRISPR¨Cas9 to mutate orthologues of tomato domestication and improvement genes that control plant architecture, flower production and fruit size in the orphan Solanaceae crop `groundcherry' (Physalis pruinosa).
It is noted, however that Cannabis architecture or earliness traits were not targeted during domestication.
In view of the above there is still a long felt and unmet need to manipulate Cannabis plant architecture and flower production in a rapid and efficient way to improve yield and reduce production costs.
SUMMARY OF THE INVENTION
It is therefore one object of the present invention to disclose a modified Cannabis plant exhibiting at least one improved domestication trait compared with wild type Cannabis, wherein said modified plant comprises at least one mutated Cannabis SELF PRUNING (SP) (CsSP) gene and/or at least one mutated Cannabis SELF PRUNING 5G (SP5G) (CsSP5G) gene.
It is another object of the present invention to disclose the modified Cannabis plant as defined above, wherein said SELF PRUNING (SP) Cannabis gene is selected from the group consisting of CsSP-1 having a genomic nucleotide sequence as set forth in SEQ ID NO:1 or a functional variant thereof, CsSP-2 having a genomic nucleotide sequence as set forth in SEQ ID NO:4 or a functional variant thereof, CsSP-3 having a genomic nucleotide sequence as set forth in SEQ ID
NO:7 or a functional variant thereof and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said SELF PRUNING 5G (SP5G) Cannabis gene is selected from the group consisting of CsSP5G-1 having a genomic nucleotide sequence as set forth in SEQ ID
NO:10 or a functional variant thereof, CsSP5G-2 having a genomic nucleotide sequence as set
WEIGHT 2.2 (FW2.2) and LYCOPENE BETA CYCLASE (CycB).
Lemmon et al (2018), Nature Plants, "Rapid improvement of domestication traits in an orphan crop by genome editing" describes the usage of CRISPR¨Cas9 to mutate orthologues of tomato domestication and improvement genes that control plant architecture, flower production and fruit size in the orphan Solanaceae crop `groundcherry' (Physalis pruinosa).
It is noted, however that Cannabis architecture or earliness traits were not targeted during domestication.
In view of the above there is still a long felt and unmet need to manipulate Cannabis plant architecture and flower production in a rapid and efficient way to improve yield and reduce production costs.
SUMMARY OF THE INVENTION
It is therefore one object of the present invention to disclose a modified Cannabis plant exhibiting at least one improved domestication trait compared with wild type Cannabis, wherein said modified plant comprises at least one mutated Cannabis SELF PRUNING (SP) (CsSP) gene and/or at least one mutated Cannabis SELF PRUNING 5G (SP5G) (CsSP5G) gene.
It is another object of the present invention to disclose the modified Cannabis plant as defined above, wherein said SELF PRUNING (SP) Cannabis gene is selected from the group consisting of CsSP-1 having a genomic nucleotide sequence as set forth in SEQ ID NO:1 or a functional variant thereof, CsSP-2 having a genomic nucleotide sequence as set forth in SEQ ID NO:4 or a functional variant thereof, CsSP-3 having a genomic nucleotide sequence as set forth in SEQ ID
NO:7 or a functional variant thereof and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said SELF PRUNING 5G (SP5G) Cannabis gene is selected from the group consisting of CsSP5G-1 having a genomic nucleotide sequence as set forth in SEQ ID
NO:10 or a functional variant thereof, CsSP5G-2 having a genomic nucleotide sequence as set
3 forth in SEQ ID NO:13 or a functional variant thereof, CsSP5G-3 having a genomic nucleotide sequence as set forth in SEQ ID NO:16 or a functional variant thereof, CsSP5G-
4 having a genomic nucleotide sequence as set forth in SEQ ID NO:19 or a functional variant thereof and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said functional variant has at least 75% sequence identity to said CsSP
or said CsSP5G nucleotide sequence.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation is introduced 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 said mutation is introduced using targeted genome modification.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation 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 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, Castl0d, Cas12, Cas13, Cas14, CasX, CasF, CasG, CasH, Csyl, Csy2, Csy3, Cse 1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc 1, Csc2, Csa5, Csnl, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz 1, Csx15, Csfl, Csf2, Csf3, Csf4, and Cu 1966and 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 mutated CsSP or CsSP5G gene is a CRISPR/Cas9-induced heritable mutated allele.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation is a missense mutation, nonsense mutation, insertion, deletion, indel, substitution or duplication.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the insertion or the deletion produces a gene comprising a frameshift.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant is homozygous for said at list one CsSP
mutated gene.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant is homozygous for said at list one CsSP5G
mutated gene.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant is a Cssp Cssp5g double mutant.
It is another object of the present invention to disclose the modified Cannabis plant 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, 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 said mutation 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 said mutation is generated in planta.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation 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:22-SEQ ID NO:916 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:22-916 and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation in said CsSP-1 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:22-SEQ ID NO:126 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ
ID NO:22-126 and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation in said CsSP-2 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:127-SEQ ID NO:211 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ
ID NO:127-211 and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation in said CsSP-3 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:212-SEQ ID NO:283 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ
ID NO:212-283 and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation in said CsSP5G-1 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:284-SEQ ID NO:516 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ
ID NO: 284-516 and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation in said CsSP5G-2 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:517-SEQ ID NO:745 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ
ID NO:517-745 and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation in said CsSP5G-3 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:746-SEQ ID NO:828 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ
ID NO: 746-828 and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation in said CsSP5G-4 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:829-SEQ ID NO:916 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ
ID NO: 829-916 and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said gRNA sequence comprises a 3' NGG Protospacer Adjacent Motif (PAM).
It is another object of the present invention to disclose the modified 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 of the genome editing molecules or mechanical insertion such as polyethylene glycol (PEG) mediated DNA transformation, electroporation or gene gun biolistics.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant has decreased expression levels of at least one of said CsSP
genes.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the sequence of said expressed CsSP gene is selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:9 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 said plant has decreased expression levels of at least one of said CsSP5G
genes.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the sequence of said expressed CsSP5G gene is selected from the group consisting of: SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:21 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 said plant is semi-determinant.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant has determinant growth habit.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant flowers earlier than a corresponding wild type cannabis plant.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant exhibits improved earliness as compared to a corresponding wild type cannabis plant.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant exhibits suppressed sympodial shoot termination as compared to a corresponding wild type cannabis plant.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant exhibits similar sympodial shoot termination as compared to a corresponding wild type cannabis plant.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant exhibits suppressed or reduced day-length sensitivity as compared to a corresponding wild type cannabis plant.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said Cannabis plant is selected from the group of species that includes, but is not limited to, Cannabis sativa (C. sativa), C. indica, C. ruderalis and any hybrid or cultivated variety of the genus Cannabis.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said domestication trait is selected from the group consisting of reduced flowering time, earliness, synchronous flowering, reduced day-length sensitivity, determinant or semi-determinant architecture, early termination of sympodial cycling, earlier axillary shoot flowering, compact growth habit, reduced height, reduced number of sympodial units, adaptation to mechanical harvest, higher harvest index and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said Cssp Cssp5g double mutant is characterized by having a more than additive effect on a trait selected from the group consisting of compactness, earlier axillary shoot flowering, earlier termination of sympodial cycling, harvest index and any combination thereof as compared to wild type and/or sp mutant Cannabis plants.
It is another object of the present invention to disclose a modified Cannabis plant, plant part or plant cell as defined in any of the above, wherein said plant does not comprise a transgene.
It is another object of the present invention to disclose a plant part, plant cell or plant seed of a 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 said plant genotype is obtainable by deposit under accession number with NCIMB Aberdeen AB21 9YA, Scotland, UK or with ATCC.
It is another object of the present invention to disclose a method for producing a modified Cannabis plant exhibiting at least one improved domestication trait compared with wild type Cannabis, said method comprises steps of genetically modifying at least one Cannabis SELF
PRUNING (SP) (CsSP) gene and/or at least one Cannabis SELF PRUNING 5G (SP5G) (CsSP5G) gene.
It is another object of the present invention to disclose a method for producing a modified Cannabis plant exhibiting at least one improved domestication trait compared with wild type Cannabis by targeted genome modification, said method comprises steps of genetically introducing a loss of function mutation in at least one Cannabis SELF PRUNING (SP) (CsSP) gene and/or at least one Cannabis SELF PRUNING 5G (SP5G) (CsSP5G) gene.
It is another object of the present invention to disclose a method of improving at least one domestication trait compared with wild type Cannabis, comprising steps of producing a modified Cannabis plant, seed or plant part thereof, that is homozygous for at least one mutated CsSP5G
gene in an sp background and enabling growth of said Cannabis plant, seed or plant part thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said method comprises steps of: (a) identifying at least one Cannabis SP (CsSP) and/or at least one Cannabis SP5G (CsSP5G) allele; (b) synthetizing at least one guide RNA (gRNA) comprising a nucleotide sequence complementary to said at least one identified CsSP and/or CsSP5G allele; (c) transforming Cannabis plant cells with a construct comprising (a) Cas nucleotide sequence operably linked to said at least one gRNA, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and said at least one gRNA; (d) screening the genome of said transformed plant cells for induced targeted loss of function mutation in at least one of said CsSP
and/or CsSP5G allele; (e) regenerating Cannabis plants carrying said loss of function mutation in at least one of said CsSP and/or CsSP5G allele; and (f) screening said regenerated plants for a Cannabis plant with improved domestication trait.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said step of screening the genome of said transformed plant cells for induced targeted loss of function mutation further comprises steps of obtaining a nucleic acid sample of said transformed plant and performing a nucleic acid amplification and optionally restriction enzyme digestion to detect a mutation in said at least one of said CsSP and/or CsSP5G allele.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said SELF PRUNING (SP) Cannabis gene is selected from the group consisting of CsSP-1 having a genomic nucleotide sequence as set forth in SEQ ID NO:1 or a functional variant thereof, CsSP-2 having a genomic nucleotide sequence as set forth in SEQ ID
NO:4 or a functional variant thereof, CsSP-3 having a genomic nucleotide sequence as set forth in SEQ ID NO:7 or a functional variant thereof and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said SELF PRUNING 5G (SP5G) Cannabis gene is selected from the group consisting of CsSP5G-1 having a genomic nucleotide sequence as set forth in SEQ ID NO:10 or a functional variant thereof, CsSP5G-2 having a genomic nucleotide sequence as set forth in SEQ ID NO:13 or a functional variant thereof, CsSP5G-3 having a genomic nucleotide sequence as set forth in SEQ ID NO:16 or a functional variant thereof, CsSP5G-4 having a genomic nucleotide sequence as set forth in SEQ ID NO:19 or a functional variant thereof and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said functional variant has at least 75% sequence identity to said CsSP or said CsSP5G
nucleotide sequence.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation is introduced 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 method as defined in any of the above, wherein said mutation is introduced using targeted genome modification.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation 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 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, Castl0d, Cas12, Cas13, Cas14, CasX, CasF, CasG, CasH, Csyl, Csy2, Csy3, Cse 1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc 1, Csc2, Csa5, Csn 1, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Cszl, Csx15, Csfl, Csf2, Csf3, Csf4, and Cu1966and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein the mutated CsSP or CsSP5G gene is a CRISPR/Cas9- induced heritable mutated allele.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation is a missense mutation, nonsense mutation, insertion, deletion, indel, substitution or duplication.
It is another object of the present invention to disclose the method as defined in any of the above, wherein the insertion or the deletion produces a gene comprising a frameshift.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said plant is homozygous for said at list one CsSP mutated gene.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said plant is homozygous for said at list one CsSP5G mutated gene.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said plant is a Cssp Cssp5g double mutant.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation is in the coding region of said allele, a mutation in the regulatory region of said allele, or an epigenetic factor.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation is a silencing mutation, a knockdown mutation, a knockout mutation, a loss of function mutation or any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation is generated in planta.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation 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:22-SEQ
ID NO:916 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:22-916 and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation in said CsSP-1 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:22-SEQ ID NO:126 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:22-126 and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation in said CsSP-2 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:127-SEQ ID NO:211 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID
NO:127-211 and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation in said CsSP-3 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:212-SEQ ID NO:283 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID
NO:212-283 and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation in said CsSP5G-1 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:284-SEQ ID NO:516 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:
284-516 and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation in said CsSP5G-2 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:517-SEQ ID NO:745 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID
NO:517-745 and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation in said CsSP5G-3 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:746-SEQ ID NO:828 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:
746-828 and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation in said CsSP5G-4 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:829-SEQ ID NO:916 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:
829-916 and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said gRNA sequence comprises a 3' NGG Protospacer Adjacent Motif (PAM).
It is another object of the present invention to disclose the method as defined in any of the above, wherein said construct is introduced into the plant cells via Agrobacterium infiltration, virus based plasmids for delivery of the genome editing molecules or mechanical insertion such as polyethylene glycol (PEG) mediated DNA transformation, electroporation or gene gun biolistics.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said plant has decreased expression levels of at least one of said CsSP genes.
It is another object of the present invention to disclose the method as defined in any of the above, wherein the sequence of said expressed CsSP gene is selected from the group consisting of: SEQ
ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:9 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 said plant has decreased expression levels of at least one of said CsSP5G genes.
It is another object of the present invention to disclose the method as defined in any of the above, wherein the sequence of said expressed CsSP5G gene is selected from the group consisting of:
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID
NO:18, SEQ ID NO:20 and SEQ ID NO:21 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 said plant is semi-determinant.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said plant has determinant growth habit.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said plant flowers earlier than a corresponding wild type cannabis plant.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said plant exhibits improved earliness as compared to a corresponding wild type cannabis plant.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said plant exhibits suppressed sympodial shoot termination as compared to a corresponding wild type cannabis plant.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said plant exhibits similar sympodial shoot termination as compared to a corresponding wild type cannabis plant.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said plant exhibits suppressed or reduced day-length sensitivity as compared to a corresponding wild type cannabis plant.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said Cannabis plant is selected from the group of species that includes, but is not limited to, Cannabis sativa (C. sativa), C. indica, C. ruderalis and any hybrid or cultivated variety of the genus Cannabis.
It is another object of the present invention to disclose a modified Cannabis plant, plant part or plant cell produced by the method as defined in any of the above, wherein said plant does not comprise a transgene.
It is another object of the present invention to disclose a plant part, plant cell or plant seed of a plant produced by the method 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 produced 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 said plant genotype is obtainable by deposit under accession number with NCIMB
Aberdeen AB21 9YA, Scotland, UK or with ATCC.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said at least one domestication trait is selected from the group consisting of reduced flowering time, earliness, synchronous flowering, reduced day-length sensitivity, determinant or semi-determinant architecture, early termination of sympodial cycling, earlier axillary shoot flowering, compact growth habit, reduced height, reduced number of sympodial units, adaptation to mechanical harvest, higher harvest index and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said Cssp Cssp5g double mutant is characterized by having a more than additive effect on a trait selected from the group consisting of compactness, earlier axillary shoot flowering, earlier termination of sympodial cycling, harvest index and any combination thereof as compared to wild type and/or sp mutant Cannabis plants.
It is another object of the present invention to disclose an isolated nucleotide sequence having at least 75% sequence identity to a CsSP genomic nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4 and SEQ ID NO:7.
It is another object of the present invention to disclose an isolated nucleotide sequence having at least 75% sequence identity to a CsSP5G genomic nucleotide sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16 and SEQ ID NO:19.
It is another object of the present invention to disclose an isolated nucleotide sequence having at least 75% sequence identity to a CsSP nucleotide coding sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.
It is another object of the present invention to disclose an isolated nucleotide sequence having at least 75% sequence identity to a CsSP5G nucleotide coding sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17 and SEQ ID NO:20.
It is another object of the present invention to disclose an isolated amino acid sequence having at least 75% sequence similarity to a CsSP amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:9.
It is another object of the present invention to disclose an isolated amino acid sequence having at least 75% sequence similarity to a CsSP5G amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18 and SEQ ID NO:21.
It is another object of the present invention to disclose an isolated nucleotide sequence having at least 75% sequence identity to a CsSP-targeted gRNA nucleotide sequence as set forth in SEQ ID
NO:22-283.
It is another object of the present invention to disclose an isolated nucleotide sequence having at least 75% sequence identity to a CsSP5G-targeted gRNA nucleotide sequence as set forth in SEQ
ID NO:284-916.
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:22-126 and any combination thereof for targeted genome modification of Cannabis SP-1 (CsSP-1) 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:127-211 and any combination thereof for targeted genome modification of Cannabis SP-2 (CsSP-2) 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:212-283 and any combination thereof for targeted genome modification of Cannabis SP-3 (CsSP-3) 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:284-516 and any combination thereof for targeted genome modification of Cannabis SP5G-1 (CsSP5G-1) 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:517-745 and any combination thereof for targeted genome modification of Cannabis SP5G-2 (CsSP5G-2) 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:746-828 and any combination thereof for targeted genome modification of Cannabis SP5G-3 (CsSP5G-3) 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:829-916 and any combination thereof for targeted genome modification of Cannabis SP5G-4 (CsSP5G-4) allele.
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.
Fig. 1 is schematically presenting CRISPR/Cas9 mode of action as depicted by Xie, Kabin, and Yinong Yang. "RNA-guided genome editing in plants using a CRISPR¨Cas system."
Molecular plant 6.6 (2013): 1975-1983;
Fig. 2A-C is presenting Cannabis SP-1 gene (CsSP-1) (A) nucleotide genomic sequence as set forth in SEQ ID NO:1, (B) nucleotide coding sequence (CDS) set forth in SEQ ID
NO:2, and (C) amino acid sequence as set forth in SEQ ID NO:3;
Fig. 3A-C is presenting Cannabis SP-2 gene (CsSP-2) (A) nucleotide genomic sequence as set forth in SEQ ID NO:4, (B) nucleotide coding sequence (CDS) set forth in SEQ ID
NO:5, and (C) amino acid sequence as set forth in SEQ ID NO:6;
Fig. 4A-C is presenting Cannabis SP-3 gene (CsSP-3) (A) nucleotide genomic sequence as set forth in SEQ ID NO:7, (B) nucleotide coding sequence (CDS) set forth in SEQ ID
NO:8, and (C) amino acid sequence as set forth in SEQ ID NO:9;
Fig. 5A-C is presenting Cannabis SP5G-1 gene (CsSP5G-1) (A) nucleotide genomic sequence as set forth in SEQ ID NO:10, (B) nucleotide coding sequence (CDS) set forth in SEQ ID NO:11, and (C) amino acid sequence as set forth in SEQ ID NO:12;
Fig. 6A-C is presenting Cannabis SP5G-2 gene (CsSP5G-2) (A) nucleotide genomic sequence as set forth in SEQ ID NO:13, (B) nucleotide coding sequence (CDS) set forth in SEQ ID NO:14, and (C) amino acid sequence as set forth in SEQ ID NO:15;
Fig. 7A-C is presenting Cannabis SP5G-3 gene (CsSP5G-3) (A) nucleotide genomic sequence as set forth in SEQ ID NO:16, (B) nucleotide coding sequence (CDS) set forth in SEQ ID NO:17, and (C) amino acid sequence as set forth in SEQ ID NO:18;
Fig. 8A-C is presenting Cannabis SP5G-4 gene (CsSP5G-3) (A) nucleotide genomic sequence as set forth in SEQ ID NO:19, (B) nucleotide coding sequence (CDS) set forth in SEQ ID NO:20, and (C) amino acid sequence as set forth in SEQ ID NO:21;
Figs 9A-D is photographically presenting staining of Cannabis plants after transient GUS
transformation of (A) axillary buds (B) leaf (C) calli, and (D) cotyledons;
Figs 10A-C is presenting regenerated transformed Cannabis tissue;
Fig. 11 is photographically presenting PCR detection of Cas9 DNA in shoots of Cannabis plants transformed using biolistics; and Figs 12A-B is illustrating in vitro cleavage activity of CRISPR/Cas9; (A) a scheme of genomic area targeted for editing, and (B) a gel showing digestion of PCR amplicon containing RNP
complex of Cas9 and gene specific gRNA.
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 at least one improved domestication trait compared with wild type Cannabis, wherein said modified plant comprises at least one mutated Cannabis SELF PRUNING (SP) (CsSP) gene and/or at least one mutated Cannabis SELF PRUNING 5G (SP5G) (CsSP5G) gene. The present invention further provides methods for producing the aforementioned modified Cannabis plant using genome editing or other genome modification techniques.
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.
To date, there are no Cannabis varieties with improved domestication traits on the market.
Classical breeding programs dedicated to the end are virtually impossible due to limited genetic variation, legal constraints on import and export of genetic material and limited academic knowledge and gene banks involved is such projects. 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 cultivated Cannabis plants with improved yield and more specifically with determinate growth habit and with significantly reduced requirement for short days period needed for induction of flowering. Breeding using genome editing allows a precise and significantly shorter breeding process in order to achieve these goals with a much higher success rate. Thus genome editing, has the potential to generate improved varieties faster and at a lower cost.
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).
Legal limitations and outdated breeding techniques significantly hamper the efforts of generating new and improved Cannabis varieties fit for intensive agriculture.
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.
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 Fl hybrid seeds. These hybrids are generated by crossing homozygous parental lines.
The next step for the Cannabis industry is the adoption and use of hybrid seeds for propagation, which is common practice in the conventional seed industry (from field crops to vegetables). In addition, breeding for basic agronomic traits that are completely lacking in currently available Cannabis varieties (with an emphasis on day length sensitivity and compact growth habit) will significantly increase grower's productivity. This will allow growing and supplying high quality raw material for the Cannabis industry.
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.
In general, plants flowering is triggered by seasonal changes in day length.
However, day-length sensitivity in crops limits their geographical range of cultivation, and thus modification of the photoperiod response is critical for their domestication.
The present invention provides for the first time Cannabis plants with improved domestication traits such as plant architecture and day length sensitivity. The current invention discloses the generation of non-transgenic Cannabis plants with improved yield traits, using the genome editing technology, e.g., the CRISPR/Cas9 highly precise tool. The generated mutations can be introduced into elite or locally adapted Cannabis lines rapidly, with relatively minimal effort and investment.
Genome editing is an efficient and useful tool for increasing crop productivity, and there is particular interest in advancing manipulation of domestication genes in Cannabis wild species, which often have undesirable characteristics.
Genome-editing technologies, such as the clustered regularly interspaced short palindromic repeats (CRISPR)¨CRISPR-associated protein-9 nuclease (Cas9) (CRISPR¨Cas9) provide opportunities to address these deficiencies, with the aims of increasing quality and yield, improve adaptation and expand geographical ranges of cultivation.
A major obstacle for CRISPR¨Cas9 plant genome editing is lack of efficient tissue culture and transformation methodologies. The present invention achieves these aims and surprisingly provides transformed and regenerated Cannabis plants with modified desirable domestication genes.
Precise editing of SELFPRUNING (SP) and SELF-PRUNING 5G (SP5G) in wild Cannabis species, as disclosed by the present invention, should serve as a first step towards generating commercially cultivable lines, without causing an associated linkage drag on other useful traits.
Genome engineering could thus be applied for de novo domestication of wild species to create climate-smart crops.
To that end, guide RNAs (gRNAs) were designed for each of the target genes identified in Cannabis to induce mutations in SP and SP5G through genome editing.
It was found that simultaneous mutation of SP and SP5G converted the indeterminate architecture of Cannabis into determinate growth with early termination of sympodial cycling, thus resulting in compact Cannabis plants with intensive and almost synchronous flowering. It should be emphasized that the desirable architectures and flower production traits are produced in just one generation.
As used herein the term "about" denotes 25% of the defined amount or measure or value.
As used herein the term "similar" denotes a correspondence or resemblance range of about 20%, particularly 15%, more particularly about 10% and even more particularly about 5%.
As used herein the term "corresponding" generally means similar, analogous, like, alike, akin, parallel, identical, resembling or comparable. In further aspects it means having or participating in the same relationship (such as type or species, kind, degree, position, correspondence, or function).
It further means related or accompanying. In some embodiments of the present invention it refers to plants of the same Cannabis species or strain or variety or to sibling plant, or one or more individuals having one or both parents in common.
A "plant" as used herein refers to any plant at any stage of development, particularly a seed plant.
The term "plant" includes the whole plant or any parts or derivatives thereof, such as plant cells, seeds, plant protoplasts, plant cell tissue culture from which tomato plants can be regenerated, plant callus or calli, meristematic cells, microspores, embryos, immature embryos, pollen, ovules, anthers, fruit, flowers, leaves, cotyledons, pistil, seeds, seed coat, roots, root tips and the like.
The term "plant cell" used herein refers to a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in a form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.
The term "plant cell culture" as used herein means cultures of plant units such as, for example, protoplasts, regenerable cells, cell culture, cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development, leaves, roots, root tips, anthers, meristematic cells, microspores, flowers, cotyledons, pistil, fruit, seeds, seed coat or any combination thereof.
The term "plant material" or "plant part" used herein refers to leaves, stems, roots, root tips, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, seed coat, cuttings, cell or tissue cultures, or any other part or product of a plant or a combination thereof.
A "plant organ" as used herein means a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower, flower bud, or embryo.
The term "Plant tissue" as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture, protoplasts, meristematic cells, calli and any group of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.
As used herein, the term "progeny" or "progenies" refers in a non limiting manner to offspring or descendant plants. According to certain embodiments, the term "progeny" or "progenies" refers to plants developed or grown or produced from the disclosed or deposited seeds as detailed inter alia. The grown plants preferably have the desired traits of the disclosed or deposited seeds, i.e.
loss of function mutation in at least one CsSP gene or at least one CsSP5G
gene.
The term "Cannabis" refers hereinafter to a genus of flowering plants in the family Cannabaceae.
Cannabis is an annual, dioecious, flowering herb that includes, but is not limited to three different species, Cannabis sativa, Cannabis indica and Cannabis ruderalis. The term also refers to hemp.
Cannabis plants produce a group of chemicals called cannabinoids.
Cannabinoids, terpenoids, and other compounds are secreted by glandular trichomes that occur most abundantly on the floral calyxes and bracts of female Cannabis plants.
The term 'SELF-PRUNING' or 'SP' in the context of the present invention refers to a gene which encodes a flowering repressor that modulates sympodial growth. It is herein shown that mutations in the SP orthologue cause an acceleration of sympodial cycling and shoot termination. It is further acknowledged that the SELF PRUNING (SP) gene controls the regularity of the vegetative-reproductive switch along the compound shoot of, for example, tomato and thus conditions the 'determinate (sp/sp) and 'indeterminate' (SP) growth habits of the plant. SP
is a developmental regulator which is homologous to CENTRORADIALIS (CEN) from Antirrhinum and TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT) from Arabidopsis.
The present invention discloses that SP is a member of a gene family in Cannabis composed of at least three genes. The newly revealed Cannabis SP genes comprise CsSP-1, CsSP-2 and CsSP-3, encoded by genomic sequence as set forth in SEQ. ID. NO: 1, 4 and 7, coding sequence as set forth in SEQ. ID. NO:2, 5 and 8, and amino acid sequence as set forth in SEQ. ID.
NO:3, 6 and 9, respectively. According to main aspects of the present invention, genome editing- targeted mutation in at least one of the aforementioned CsSP genes, which reduces the functional expression of the gene, affect the plant sympodial growth habit which plays a key role in determining plant architecture.
The term 'SELF-PRUNING 5G' or 'SP5G' in the context of the present invention refers to a gene encoding florigen paralog and flowering repressor responsible for loss of day-length-sensitive flowering. It is further acknowledged that SP5G expression is induced to high levels during long days in wild species. It is within the scope of the current invention that CRISPR/Cas9-engineered mutations in SP5G cause rapid flowering and enhance the compact determinate growth habit of Cannabis plants, resulting in a quick burst of flower production that turns to an early yield. The findings of the current invention suggest that variation in SP and/or SP5G
facilitate the production of cultivated Cannabis strains with improved demonstration traits. The inventors of the present invention use gene editing techniques to rapidly improve yield traits in crop such as Cannabis.
As used herein the term "genetic modification" refers hereinafter to genetic manipulation or modulation, which is the direct manipulation of an organism's genes using biotechnology. It also refers to a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species, targeted mutagenesis and genome editing technologies to produce improved organisms. According to main embodiments of the present invention, modified Cannabis plants with improved domestication traits are generated using genome editing mechanism. This technique enables to achieve in planta modification of specific genes that relate to and/or control the flowering time and plant architecture in the Cannabis plant.
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 Cas = CRISPR-associated genes Indel = insertion and/or deletion Cas9, Csn I = a CRISPR-associated protein NHEJ = Non-Homologous End Joining containing two nuclease domains, that is PAM = Protospacer-Adjacent Motif programmed by small RNAs to cleave DNA
RuvC = an endonuclease domain named for crRNA = CRISPR RNA an E, col i protein involved in DNA
repair dCAS9 = nuclease-deficient Cas9 sgRNA = single guide RNA
DSB = Double-Stranded Break tracrRNA, trRNA = trans-activating crRNA
gRNA = guide RNA TALEN = Transcription-Activator Like HDR = Homology-Directed Repair Effector Nuclease HNH = an endonuclease domain named ZFN = Zinc-Finger Nuclease for characteristic histidine and asparagine residues 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 Csn 1) is required for gene silencing. Cas9 participates in the processing of crRNAs, and is responsible for the destruction of the target DNA. Cas9's function in both of these steps relies on the presence of two nuclease domains, a RuvC-like nuclease domain located at the amino terminus and a HNH-like nuclease domain that resides in the mid-region of the protein. To achieve site-specific DNA
recognition and cleavage, Cas9 is complexed with both a crRNA and a separate trans-activating crRNA (tracrRNA or trRNA), that is partially complementary to the crRNA. The tracrRNA is required for crRNA maturation from a primary transcript encoding multiple pre-crRNAs. This occurs in the presence of RNase III and Cas9.
Without wishing to be bound by theory, it is herein acknowledged that during the destruction of target DNA, the HNH and RuvC-like nuclease domains cut both DNA strands, generating double-stranded breaks (DSBs) at sites defined by a 20-nucleotide target sequence within an associated crRNA transcript. The HNH domain cleaves the complementary strand, while the RuvC domain cleaves the noncomplementary strand.
It is further noted that the double-stranded endonuclease activity of Cas9 also requires that a short conserved sequence, (2-5 nts) known as protospacer-associated motif (PAM), follows immediately 3- of the crRNA complementary sequence.
According to further aspects of the invention, a two-component system may be used by the current invention, combining trRNA and crRNA into a single synthetic single guide RNA
(sgRNA) for guiding targeted gene alterations.
It is further within the scope that Cas9 nuclease variants include wild-type Cas9, Cas9D10A and nuclease-deficient Cas9 (dCas9).
Reference is now made to Fig. 1 schematically presenting an example of CRISPR/Cas9 mechanism of action as depicted by Xie, Kabin, and Yinong Yang. "RNA-guided genome editing in plants using a CRISPR¨Cas system." Molecular plant 6.6 (2013): 1975-1983.
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 an NGG motif (called protospacer-adjacent motif or PAM) that follows the base-pairing region in the complementary strand of the targeted DNA.
The term "meganucleases" as used herein refers hereinafter to endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA
sequences of 12 to 40 base pairs); as a result this site generally occurs only once in any given genome. Meganucleases are therefore considered to be the most specific naturally occurring restriction enzymes.
The term "protospacer adjacent motif" or "PAM' as used herein refers hereinafter to a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. PAM is a component of the invading virus or plasmid, but is not a component of the bacterial CRISPR locus. PAM is an essential targeting component which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR
locus from being targeted and destroyed by nuclease.
The term "Next-generation sequencing" or "NGS" as used herein refers hereinafter to massively, parallel, high- throughput or deep sequencing technology platforms that perform sequencing of millions of small fragments of DNA in parallel. Bioinformatics analyses are used to piece together these fragments by mapping the individual reads to the reference genome.
The term "gene knockdown" as used herein refers hereinafter to an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur through genetic modification, i.e. targeted genome editing or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript. The reduced expression can be at the level of RNA or at the level of protein. It is within the scope of the present invention that the term gene knockdown also refers to a loss of function mutation and /or gene knockout mutation in which an organism's genes is made inoperative or nonfunctional.
The term "gene silencing" as used herein refers hereinafter to the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation. In certain aspects of the invention, gene silencing is considered to have a similar meaning as gene knockdown. When genes are silenced, their expression is reduced. In contrast, when genes are knocked out, they are completely not expressed. Gene silencing may be considered a gene knockdown mechanism since the methods used to silence genes, such as RNAi, CRISPR, or siRNA, generally reduce the expression of a gene by at least 70% but do not completely eliminate it.
The term "loss of function mutation" as used herein refers to a type of mutation in which the altered gene product lacks the function of the wild-type gene. A synonyms of the term included within the scope of the present invention is null mutation.
The term "microRNAs" or "miRNAs" refers hereinafter to small non-coding RNAs that have been found in most of the eukaryotic organisms. They are involved in the regulation of gene expression at the post-transcriptional level in a sequence specific manner.
MiRNAs are produced from their precursors by Dicer-dependent small RNA biogenesis pathway. MiRNAs are candidates for studying gene function using different RNA-based gene silencing techniques. For example, artificial miRNAs (amiRNAs) targeting one or several genes of interest is a potential tool in functional genomics.
The term "in planta" means in the context of the present invention within the plant or plant cells.
More specifically, it means introducing CRISPR/Cas complex into plant material comprising a tissue culture of several cells, a whole plant, or into a single plant cell, without introducing a foreign gene or a mutated gene. It also used to describe conditions present in a non-laboratory environment (e.g. in vivo).
The term 'sympodial growth' as used herein refers to a type of bifurcating branching pattern where one branch develops more strongly than the other, resulting in the stronger branches forming the primary shoot and the weaker branches appearing laterally. A sympodium, also referred to as a sympode or pseudaxis, is the primary shoot, comprising the stronger branches, formed during sympodial growth. In some aspects of the present invention, sympodial growth occurs when the apical meristem is terminated and growth is continued by one or more lateral meristems, which repeat the process. The apical meristem may be consumed to make an inflorescence or other determinate structure, or it may be aborted.
It is further within the scope of the current invention that the shoot section between two successive inflorescences is called the 'sympodium', and the number of leaf nodes per sympodium is referred to as the 'sympodial index' (spi). The first termination event activates the 'sympodial cycle'. In sympodial plants, the apparent main shoot consists of a reiterated array of 'sympodial units'. A
mutant sp gene accelerates the termination of sympodial units but does not change the sympodial habit. The result is a progressive reduction in the number of vegetative nodes between inflorescences in a pattern that depends on light intensity and genetic background.
The term "earliness" refers hereinafter to early flowering and/or rapid transition from the vegetative to reproductive stages, or reduced 'time to initiation of flowering' and more generally to earlier completion of the life-cycle.
The term 'reduced flowering time' as used herein refers to time to production of first inflorescence. Such a trait can be evaluated or measured, for example, with reference to the number of leaves produced prior to appearance of the first inflorescence.
The term 'harvest index' can be herein defined as the total yield per plant weight.
The term 'day length' or 'day length sensitivity' as used in the context of the present invention generally refers to photoperiodism, which is the physiological reaction of organisms to the length of day or night. Photoperiodism can also be defined as the developmental responses of plants to the relative lengths of light and dark periods. Plants are classified under three groups according to the photoperiods: short-day plants, long-day plants, and day-neutral plants.
Photoperiodism affects flowering by inducing the shoot to produce floral buds instead of leaves and lateral buds. It is within the scope of the present invention that Cannabis is included within the short-day facultative plants. The Cannabis plants of the present invention are genetically modified so as to exhibit loss of day-length sensitivity, which is highly desirable agronomical trait enabling enhanced yield of the cultivated crop.
The term 'determinate' or 'determinate growth' as used herein refers to plant growth in which the main stem ends in an inflorescence or other reproductive structure (e.g. a bud) and stops continuing to elongate indefinitely with only branches from the main stem having further and similarly restricted growth. It also refers to growth characterized by sequential flowering from the central or uppermost bud to the lateral or basal buds. It further means naturally self-limited growth, resulting in a plant of a definite maximum size.
The term 'indeterminate' or 'indeterminate growth' as used herein refers to plant growth in which the main stem continues to elongate indefinitely without being limited by a terminal inflorescence or other reproductive structure. It also refers to growth characterized by sequential flowering from the lateral or basal buds to the central or uppermost buds.
The term "orthologue" as used herein refers hereinafter to one of two or more homologous gene sequences found in different species.
The term "functional variant" or "functional variant of a nucleic acid or amino acid sequence" as used herein, for example with reference to SEQ ID NOs: 1, 4 or 7 refers to a variant of a sequence or part of a sequence which retains the biological function of the full non-variant allele (e.g. CsSP or CsSP5G allele) and hence has the activity of SP or SP5G
expressed gene or protein. A functional variant also comprises a variant of the gene of interest encoding a polypeptide which has sequence alterations that do not affect function of the resulting protein, for example, in non-conserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example, in non-conserved residues, to the wild type nucleic acid or amino acid sequences of the alleles as shown herein, and is biologically active.
The term "variety" or "cultivar" used herein means a group of similar plants that by structural features and performance can be identified from other varieties within the same species.
The term "allele" used herein means any of one or more alternative or variant forms of a gene or a genetic unit at a particular locus, all of which alleles relate to one trait or characteristic at a specific locus. In a diploid cell of an organism, alleles of a given gene are located at a specific location, or locus (loci plural) on a chromosome. Alternative or variant forms of alleles may be the result of single nucleotide polymorphisms, insertions, inversions, translocations or deletions, or the consequence of gene regulation caused by, for example, by chemical or structural modification, transcription regulation or post-translational modification/regulation. An allele associated with a qualitative trait may comprise alternative or variant forms of various genetic units including those mat are identical or associated with a single gene or multiple genes or their products or even a gene disrupting or controlled by a genetic factor contributing to the phenotype represented by the locus.
According to further embodiments, the term "allele" designates any of one or more alternative forms of a gene at a particular locus. Heterozygous alleles are two different alleles at the same locus. Homozygous alleles are two identical alleles at a particular locus. A
wild type allele is a naturally occurring allele. In the context of the current invention, the term allele refers to the three identified SP genes in Cannabis, namely CsSP-1, CsSP-2 and CsSP-3 having the genomic nucleotide sequence as set forth in SEQ ID NOs: 1, 4 and 7, respectively. As well as to four identified SP5G genes in Cannabis, namely CsSP5G-1, CsSP5G-2, and CsSP5G-4 having the genomic nucleotide sequence as set forth in SEQ ID NOs: 10, 13, 18 and 19, 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.
In specific embodiments, the Cannabis plants of the present invention comprise homozygous configuration of at least one of the mutated Cssp genes (i.e. Cssp-1, Cssp-2 and Cssp-3) and/or the mutated Cssp5g genes (i.e. Cssp5g-1, Cssp5g-2, Cssp5g-3 and Cssp5g-4).
Conversely, as used herein, the term "heterozygous" means a genetic condition or configuration existing when two different or unlike alleles reside at a specific locus, but are positioned individually on corresponding pairs of homologous chromosomes in the cell of a diploid organism.
As used herein, the phrase "genetic marker" or "molecular marker" or "biomarker" refers to a feature in an individual's genome e.g., a nucleotide or a polynucleotide sequence that is associated with one or more loci or trait of interest In some embodiments, a genetic marker is polymorphic in a population of interest, or the locus occupied by the polymorphism, depending on context.
Genetic markers or molecular markers include, for example, single nucleotide polymorphisms (SNPs), indels (i.e. insertions deletions), simple sequence repeats (SSRs), restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAFDs), cleaved amplified polymorphic sequence (CAPS) markers, Diversity Arrays Technology (DArT) markers, and amplified fragment length polymorphisms (AFLPs) or combinations thereof, among many other examples such as the DNA sequence per se. Genetic markers can, for example, be used to locate genetic loci containing alleles on a chromosome that contribute to variability of phenotypic traits. The phrase "genetic marker" or "molecular marker" or "biomarker" can also refer to a polynucleotide sequence complementary or corresponding to a genomic sequence, such as a sequence of a nucleic acid used as a probe or primer.
As used herein, the term "germplasm" refers to the totality of the genotypes of a population or other group of individuals (e.g., a species). The term "germplasm" can also refer to plant material;
e.g., a group of plants that act as a repository for various alleles. Such germplasm genotypes or populations include plant materials of proven genetic superiority; e.g., for a given environment or geographical area, and plant materials of unknown or unproven genetic value;
that are not part of an established breeding population and that do not have a known relationship to a member of the established breeding population.
The terms "hybrid", "hybrid plant" and "hybrid progeny" used herein refers to an individual produced from genetically different parents (e.g., a genetically heterozygous or mostly heterozygous individual).
As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. The term further refers hereinafter to the amount of characters which match exactly between two different sequences. Hereby, gaps are not counted and the measurement is relational to the shorter of the two sequences.
It is further within the scope that the terms "similarity" and "identity"
additionally refer to local homology, identifying domains that are homologous or similar (in nucleotide and/or amino acid sequence). It is acknowledged that bioinformatics tools such as BLAST, SSEARCH, FASTA, and HMMER calculate local sequence alignments which identify the most similar region between two sequences. For domains that are found in different sequence contexts in different proteins, the alignment should be limited to the homologous domain, since the domain homology is providing the sequence similarity captured in the score. According to some aspects the term similarity or identity further includes a sequence motif, which is a nucleotide or amino-acid sequence pattern that is widespread and has, or is conjectured to have, a biological significance. Proteins may have a sequence motif and/or a structural motif, a motif formed by the three-dimensional arrangement of amino acids which may not be adjacent.
As used herein, the terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or RNA
molecules, and analogs of the DNA or RNA generated using nucleotide analogs.
It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term "gene", "allele" or "gene sequence" is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in combination with regulatory sequences. Thus, according to the various aspects of the invention, genomic DNA, cDNA or coding DNA may be used. In one embodiment, the nucleic acid is cDNA or coding DNA.
The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.
According to other aspects of the invention, a "modified" or a "mutant" plant is a plant that has been altered compared to the naturally occurring wild type (WT) plant.
Specifically, the endogenous nucleic acid sequences of each of the SP or SP5G homologs in Cannabis (nucleic acid sequences CsSP-1, CsSP-2 and CsSP-3; and/or CsSP5G-1, CsSP5G-2, CsSP5G-3 and CsSP5G-4) have been altered compared to wild type sequences using mutagenesis and/or genome editing methods as described herein. This causes inactivation of the endogenous SP
and/or SP5G genes and thus disables SP and/or SP5G function. Such plants have an altered phenotype and show improved domestication traits such as determinant plant architecture, synchronous and/or early flowering and loss of day length sensitivity compared to wild type plants.
Therefore, the improved domestication phenotype is conferred by the presence of at least one mutated endogenous Cssp and /or Cssp5g gene in the Cannabis plant genome which has been specifically targeted using genome editing technique.
According to further aspects of the present invention, the at least one improved domestication trait 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 CsSP-1, CsMSP-2 and CsSP-3; and CsSP5G-1, CsMSP5G-2, CsSP5G-3 and CsSP5G-4. Mutant sp and sp5g nucleic acid sequences use non-capitalization.
Cannabis plants of the invention are modified plants compared to wild type plants which comprise and express mutant Cssp and/or Cssp5g alleles.
It is further within the scope of the current invention that sp and/or sp5g mutations that down-regulate or disrupt functional expression of the wild-type SP and/or SP5G
sequence respectively, may be recessive, such that they are complemented by expression of a wild-type sequence.
It is further noted that a wild type Cannabis plant is a plant that does not have any mutant sp and/or sp5g alleles.
Main aspects of the invention involve targeted mutagenesis methods, specifically genome editing, and exclude embodiments that are solely based on generating plants by traditional breeding methods. In a further embodiment of the current invention, as explained herein, the improved domestication at least one trait is not due to the presence of a transgene.
The inventors have generated mutant Cannabis lines with mutations inactivating at least one CsSP
and/or CsSP5G homoeoallele which confer heritable improved domestication trait(s). In this way no functional CsSP and/or CsSP5G protein is made. Thus, the invention relates to these mutant Cannabis lines and related methods.
It is further within the scope of the present invention that breeding Cannabis cultivars with mutated sp allele enables the mechanical harvest of the plant. According to a further aspect of the present invention, loss of SP function results in compact Cannabis plants with reduced height, reduced number of sympodial units and determinate growth when compared with WT
Cannabis.
According to a main aspect of the present invention, modifying Cannabis shoot architecture by selection for mutations in florigen flowering pathway genes allowed major improvements in plant architecture and yield. In particular, a mutation in the antiflorigen SELFPRUNING (SP) gene (sp classic) provided compact 'determinate' growth that translated to a burst of flowers, thereby enabling largescale field production.
According to one embodiment of the present invention, SELFPRUNING (SP) homologues and related florigen family members such as SELF-PRUNING 5G (SP5G) have been identified in both genome and transcriptome in Cannabis.
The work inter alia described has important implications. The results have shown that CRISPR/Cas9 can be used to create heritable mutations in florigen pathway family members that result in desirable phenotypic effects.
To edit multiple domestication genes simultaneously and stack the resulting allelic variants, on option is that several gRNAs can be assembled to edit several genes into one construct, by using the Csy4 multi-gRNA system. The construct is then transformed via an appropriate vector into several wild-Cannabis accessions.
It is further within the scope of the current invention that Cannabis SP
genes, namely CsSP-1, CsSP-2 and CsSP-3 having genomic nucleotide sequence as set forth in SEQ. ID.
NO.:1, 4 and 7 respectively, were targeted using guide RNAs as set forth in SEQ ID NO:22-126, 127-211 and 212-283, respectively. Several mutated alleles have been identified. Notably, the plants with mutated sp alleles were more compact than the wild type plants lacking the mutated allele.
To identify other targets for plant architecture modification without negative effects on productivity, homologues of SELF-PRUNING 5G (SP5G) (Cs-SP5G), another florigen repressor, were identified in Cannabis. Cannabis SP5G genes, namely CsSP5G-1, CsSP5G-2, CsSP5G-3 and CsSP5G-4 having genomic nucleotide sequence as set forth in SEQ. ID. NO.:10, 13, 16 and 19 respectively, were targeted using guide RNAs as set forth in SEQ ID NO:284-516, 517-745, 746-828 and 829-916, respectively. Several mutated alleles have been identified.
It is herein acknowledged that SP5G represses flowering predominantly in primary and canonical axillary shoots.
According to one embodiment of the present invention, SP5G was shown to control primary and canonical axillary shoot flowering time and to contribute to controlling day-length sensitivity. In other words, reduced SP5G activity was shown to mitigate day-length sensitivity.
According to a further embodiment of the present invention, CRISPR¨Cas9-induced null sp5g mutations in Cannabis eliminate day-length sensitivity and causing faster primary and axillary shoot flowering.
The present invention discloses that the combination of both mutations sp5g and sp results in faster-flowering Cannabis plants with typical sp determinate growth. Such Cannabis plants could have agronomic value, particularly for the desirable trait of earliness of yield.
According to specific aspects of the present invention, Cs-sp5g/ sp double mutants are not simply additive but substantially more compact than mutant sp Cannabis plants owing to faster axillary shoot flowering and earlier termination of sympodial cycling.
According to further aspects of the present invention, compared with wild type and/or sp Cannabis plants, Cs-sp5g/ sp double mutant plants provide a more rapid flowering burst, and reach final harvest sooner.
It is further within the scope of the present invention that the harvest index (defined as the total yield per plant weight) of the Cs-sp5g/ sp double mutant plants is higher than that for wild type and/or sp mutant Cannabis plants.
According to a further specific aspect of the present invention, large-scale Cannabis production based on sp determinate growth may be achieved only in the absence of day-length sensitivity, i.e.
by loss of function mutation in at least one of Cssp5g-1, Cssp5g-2, Cssp5g-3 or Cssp5g-4 as set forth in SEQ ID NO.: 10, 13 16 and 19, respectively.
It is further within the scope of the present invention that targeting SP5G
homologs and/or other diurnally regulated CENTRORADIALIS/TERMINAL FLOWER 1/SELF-PRUNING (CETS) genes may allow immediate customization of day-length sensitivity in Cannabis elite germplasm to expand the geographical range of cultivation, and could serve as a first step toward engineering the domestication of wild Cannabis species with agricultural potential.
The loss of function mutation may be a deletion or insertion ("indels") with reference the wild type CsSP and/or CsSP5G allele sequence. The deletion may comprise 1-20 or more nucleotides, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18 or 20 nucleotides or more in one or more strand. The insertion may comprise 1-20 or more nucleotides, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18 or 20 or more nucleotides in one or more strand.
The plant of the invention includes plants wherein the plant is heterozygous for the each of the mutations. In a preferred embodiment however, the plant is homozygous for the mutations.
Progeny that is also homozygous can be generated from these plants according to methods known in the art.
It is further within the scope that variants of a particular CsSP and/or CsSP5G 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 CsSP and/or CsSP5G nucleotide sequence of the CsSP and/or CsSP5G allele as shown in SEQ ID
NO 1, 4 or 7; and/or SEQ ID NO 10, 13, 18 or 19, respectively. Sequence alignment programs to determine sequence identity are well known in the art.
Also, the various aspects of the invention encompass not only a CsSP and/or CsSP5G nucleic acid sequence or amino acid sequence, but also fragments thereof. By "fragment" is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence of the protein encoded thereby. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein, in this case improved domestication trait.
According to a further embodiment of the invention, the herein newly identified Cannabis SP
and/or SP5G locus (CsSP and/or CsSP5G) have been targeted using the double 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 Cannabis plants with at least one improved domestication trait, allows the modification of predetermined specific DNA sequences without introducing foreign DNA into the genome by GMO techniques. According to one embodiment of the present invention, this is achieved by combining the Cas nuclease (e.g. Cas9, Cpfl and the like) with a predefined guide RNA molecule (gRNA). The gRNA is complementary to a specific DNA sequence targeted for editing in the plant genome and which guides the Cas nuclease to a specific nucleotide sequence (for example see Fig. 1). The predefined gene specific gRNA's are cloned into the same plasmid as the Cas gene and this plasmid is inserted into plant cells. Insertion of the aforementioned plasmid DNA can be done, but not limited to, using different delivery systems, biological and/or mechanical, e.g. Agrobacterium infiltration, virus based plasmids for delivery of the genome editing molecules and mechanical insertion of DNA (PEG mediated DNA
transformation, biolistics, etc.).
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 I base pair deletion. As a result of this base pair deletion the gene coding sequence is disrupted and the translation of the encoded protein is compromised either by a premature stop codon or disruption of a functional or structural property of the protein.
Thus DNA is cut by the Cas9 protein and re-assembled by the cell's DNA repair mechanism.
It is further within the scope that improved domestication traits in Cannabis plants is herein produced by generating gRNA with homology to a specific site of predetermined genes in the Cannabis genome i.e. SP and/or SP5G genes, sub cloning this gRNA into a plasmid containing the Cas9 gene, and insertion of the plasmid into the Cannabis plant cells. In this way site specific mutations in the SP and/or SP5G genes are generated thus effectively creating non-active molecules, resulting in loss of day length sensitivity, reduced flowering time and determinant growth habit of the genome edited plant.
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 I
Production of Cannabis plants with improved domestication traits by targeted genome editing Production of Cannabis lines with mutated sp and/or sp5g gene may be achieved by at least one of the following breeding/cultivation schemes:
Scheme I:
= line stabilization by self pollination = Generation of F6 parental lines = Genome editing of parental lines = Crossing edited parental lines to generate an Fl hybrid plant Scheme 2:
= Identifying genes/alleles 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 about 6 self-crossing (6 generations) and done through a single seed descent (SSD) approach.
Fl hybrid seed production: Novel hybrids are produced by crosses between different Cannabis strains.
According to a further aspect of the current invention, shortening line stabilization is performed by Doubled Haploids (DH). More specifically, the CRISPR-Cas9 system is transformed into microspores to achieve DH homozygous parental lines. A doubled haploid (DH) is a genotype formed when haploid cells undergo chromosome doubling. Artificial production of doubled haploids is important in plant breeding. It is herein acknowledged that conventional inbreeding procedures take about six generations to achieve approximately complete homozygosity, whereas doubled haploidy achieves it in one generation.
It is within the scope of the current invention that genetic markers specific for Cannabis are developed and provided by the current invention:
= Sex markers- molecular markers are used for identification and selection of female vs male plants in the herein disclosed breeding program = Genotyping markers- germplasm used in the current invention is genotyped using molecular markers, in order to allow a more efficient breeding process and identification of the SP and/or SP5G editing event.
It is further within the scope of the current invention that allele and genetic variation is analysed for the Cannabis strains used.
Reference is now made to optional stages that have been used for the production of mutated SP
and/or SP5G Cannabis plants by genome editing:
Stage 1: Identifying Cannabis sativa (C. sativa), C. indica and C. ruderalis SP and SP5G
orthologues.
Three SP orthologues have herein been identified in Cannabis sativa (C.
sativa), C. indica and C.
ruderalis, namely CsSP-1, CsSP-2 and CsSP-3. These homologous genes have been sequenced and mapped. CsSP-1 has been mapped to CM011605.1:71328589-71330978 and has a genomic sequence as set forth in SEQ ID NO:1 (see Fig. 2A). The CsSP-1 gene has a coding sequence as set forth in SEQ ID NO:2 (see Fig. 2B) and it encodes an amino acid sequence as set forth in SEQ
ID NO:3 (see Fig. 2C).
CsSP-2 has been mapped to CM011605.1:25325478-25326672 and has a genomic sequence as set forth in SEQ ID NO:4 (see Fig. 3A). The CsSP-2 gene has a coding sequence as set forth in SEQ
ID NO:5 (see Fig. 3B) and it encodes an amino acid sequence as set forth in SEQ ID NO:6 (see Fig. 3C).
CsSP-3 has been mapped to CM011608.1:9602945-9603900 and has a genomic sequence as set forth in SEQ ID NO:7 (see Fig. 4A). The CsSP-3 gene has a coding sequence as set forth in SEQ
ID NO:8 (see Fig. 4B) and it encodes an amino acid sequence as set forth in SEQ ID NO:9 (see Fig. 4C).
Four SP5G orthologues have herein been identified in Cannabis sativa (C.
sativa), C. indica and C. ruderalis, namely CsSP5G-1, CsSP5G-2, CsSP5G-3 and CsSP5G-4. These homologous genes have been sequenced and mapped. CsSP5G-1 has been mapped to CM011610.1:5735300-and has a genomic sequence as set forth in SEQ ID NO:10 (see Fig. 5A). The CsSP-1 gene has a coding sequence as set forth in SEQ ID NO:11 (see Fig. 5B) and it encodes an amino acid sequence as set forth in SEQ ID NO:12 (see Fig. 5C).
CsSP5G-2 has been mapped to CM011610.1:6032638-6035504 and has a genomic sequence as set forth in SEQ ID NO:13 (see Fig. 6A). The CsSP5G-2 gene has a coding sequence as set forth in SEQ ID NO:14 (see Fig. 6B) and it encodes an amino acid sequence as set forth in SEQ ID
NO:15 (see Fig. 6C).
CsSP5G-3 has been mapped to CM011607.1:79899046-79900718 and has a genomic sequence as set forth in SEQ ID NO:16 (see Fig. 7A). The CsSP5G-3 gene has a coding sequence as set forth in SEQ ID NO:17 (see Fig. 7B) and it encodes an amino acid sequence as set forth in SEQ ID
NO:18 (see Fig. 7C).
CsSP5G-4 has been mapped to CM011614.1:9255475-9256908 and has a genomic sequence as set forth in SEQ ID NO:19 (see Fig. 8A). The CsSP5G-4 gene has a coding sequence as set forth in SEQ ID NO:20 (see Fig. 8B) and it encodes an amino acid sequence as set forth in SEQ ID
NO:21 (see Fig. 8C).
Stage 2: Designing and synthesizing gRNA molecules corresponding to the sequence targeted for editing, i.e. sequences of each of the genes CsSP-1, CsSP-2 and CsSP-3; and CsSP5G-1, CsSP5G-2, CsSP5G-3 and CsSP5G-4. 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 SP and/or SP5G
homologues of different Cannabis strains.
Reference is now made to Tables 1-3 presenting gRNA molecules targeted for silencing CsSP-1, CsSP-2 and CsSP-3, respectively; and Tables 4-7 presenting gRNA molecules targeted for silencing CsSP5G-1, CsSP5G-2, CsSP5G-3 and CsSP5G-4 respectively. The term 'PAM refers hereinafter to Protospacer Adjacent Motif, which is a 2-6 base pair DNA
sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR
bacterial adaptive immune system.
Table 1: gRNA sequences targeted for CsSP-1 Position on Strand Sequence PAM Efficiency SEQ. ID.
SEQ. ID. Score NO.
NO:1 831 1 TATATATATTAAGACTACGT AGG 69.79075 22 868 -1 ATAAGTGTGTAAGAGGCTCG TGG 66.70092 23 875 -1 TTATTATATAAGTGTGTAAG AGG 54.32121 24 964 -1 GCATGCATGCATGCATGCAT GGG 48.94366 25 965 -1 TGCATGCATGCATGCATGCA TGG 51.6943 26 1049 1 TTGAAGAAAAGAGCAGCCAC AGG 56.36067 27 1052 1 AAGAAAAGAGCAGCCACAGG AGG 61.86857 28 1054 -1 AAATGACCTTGGTCCTCCTG TGG 59.43912 29 1059 1 GAGCAGCCACAGGAGGACCA AGG 64.32062 30 1065 -1 TTTGCTACCCAAAATGACCT TGG 60.28351 31 1068 1 CAGGAGGACCAAGGTCATTT TGG 26.32254 32 1069 1 AGGAGGACCAAGGTCATTTT GGG 28.83447 33 1080 1 GGTCATTTTGGGTAGCAAAG AGG 69.55719 34 1109 1 TTGAAACGCTCTCTCGATGA AGG 54.24251 35 1112 1 AAACGCTCTCTCGATGAAGG TGG 60.45312 36 1129 -1 ACAGAAGAGAAGACAAACAG TGG 69.59222 37 1172 -1 GACCAAACATAGGAATACAT AGG 61.3781 38 1181 1 AACCTATGTATTCCTATGTT TGG 36.10233 39 1182 -1 GAAATGCCACGACCAAACAT AGG 59.3397 40 1187 1 TGTATTCCTATGTTTGGTCG TGG 47.61837 41 1224 -1 TTAAATGAATTAATATATGT AGG 44.76165 42 1324 -1 GGAACAACTGATGTGTCATT TGG 39.45477 43 1338 1 AATGACACATCAGTTGTTCC TGG 39.81732 44 1345 -1 AGGATAGTGACAGACATTCC AGG 51.58159 45 1365 -1 TTTGTTGTTGAAATAATTGC AGG 31.23807 46 1424 -1 GCAAAAGGCAAAATTTAAAA TGG 25.80444 47 1439 -1 TTTAATCAAACTTAAGCAAA AGG 41.06861 48 1478 1 TATTTTGTTAAATTATAAAT TGG 18.56233 49 1528 1 TAGTATATATATATTCTGAT TGG 48.76865 50 1572 1 GCAATAATAATTTAATGTAT AGG 42.88178 51 1631 1 TTAAAAAATCTTCTTCAAGT TGG 45.32853 52 1755 -1 TTATCTAGGGTTATAATAGT TGG 38.51395 53 1768 -1 GTATACACATATATTATCTA GGG 52.79043 54 1769 -1 TGTATACACATATATTATCT AGG 40.10661 55 1881 1 TCATTCAAAAGTAAAATAAT AGG 21.75194 56 1882 1 CATTCAAAAGTAAAATAATA GGG 35.40844 57 1890 1 AGTAAAATAATAGGGTAATT AGG 22.83575 58 1910 -1 TAGTAATATCAAAATTTTAA GGG 28.45209 59 1911 -1 TTAGTAATATCAAAATTTTA AGG 17.72295 60 1990 1 TATTGAGATTGTTAAATTTA AGG 6.261185 61 2063 -1 TTGGTAGATATTAAAATTTA GGG 23.20311 62 2064 -1 TTTGGTAGATATTAAAATTT AGG 24.70598 63 2082 -1 GAAAGTTCAGAGGCATGATT TGG 33.64684 64 2092 -1 TAACATGGAGGAAAGTTCAG AGG 66.62639 65 2104 -1 AAAAAAACTAATTAACATGG AGG 67.7548 66 2107 -1 AGTAAAAAAACTAATTAACA TGG 49.05741 67 2126 1 ATTAGTTTTTTTACTAAAAT TGG 20.24538 68 2154 -1 CTCTAAACTATTGAGATTGT TGG 27.96835 69 2214 1 ATAATATAATATATATATAT TGG 33.84373 70 2365 1 GACTAATTAATGATCATGTG TGG 67.15733 71 2382 -1 CTTAAGGGAATATATGCAAC TGG 42.83721 72 2397 -1 ATAATAATAAGTATACTTAA GGG 44.75504 73 2398 -1 TATAATAATAAGTATACTTA AGG 31.14549 74 2426 -1 TATATAATATATATATATAG GGG 51.83062 75 2427 -1 ATATATAATATATATATATA GGG 25.09287 76 2428 -1 TATATATAATATATATATAT AGG 21.06967 77 2527 -1 TGATCGATCGTTAGGGGGGA AGG 55.11339 78 2531 -1 ATCTTGATCGATCGTTAGGG GGG 69.00871 79 2532 -1 CATCTTGATCGATCGTTAGG GGG 62.62737 80 2533 -1 ACATCTTGATCGATCGTTAG GGG 62.65508 81 2534 -1 CACATCTTGATCGATCGTTA GGG 48.84918 82 2535 -1 ACACATCTTGATCGATCGTT AGG 40.46896 83 2692 1 ATATAAGAAATATGTATGAT CGG 52.65835 84 2703 1 ATGTATGATCGGAATTTTAT TGG 27.65155 85 2740 1 TCAATATTATATATAGTATT AGG 34.80095 86 2818 1 AACTTATATATGAATATTAT AGG 22.13664 87 2872 -1 TGTGATGACAAAAGTTTTTT GGG 25.03809 88 2873 -1 GTGTGATGACAAAAGTTTTT TGG 30.69267 89 2982 -1 ATTTGAGGGAGCATTTACAC TGG 63.0929 90 2996 -1 CCCTAGTGATCCTTATTTGA GGG 33.88568 91 2997 1 TGTAAATGCTCCCTCAAATA AGG 33.96309 92 2997 -1 GCCCTAGTGATCCTTATTTG AGG 41.63481 93 3006 1 TCCCTCAAATAAGGATCACT AGG 53.28568 94 3007 1 CCCTCAAATAAGGATCACTA GGG 61.56296 95 3012 1 AAATAAGGATCACTAGGGCC AGG 51.65425 96 3019 -1 ATGACTGATCCTGATGTTCC TGG 34.06368 97 3021 1 TCACTAGGGCCAGGAACATC AGG 43.87242 98 3044 -1 TGTTGTAAATAATATTAATT AGG 20.85671 99 3141 -1 CATGAGATCCTTTTTCACAC TGG 54.53186 100 3144 1 AATTATTACCAGTGTGAAAA AGG 40.03344 101 3167 -1 CCAAAAGTTGAGGTTCATGG TGG 71.52152 102 3170 -1 AAGCCAAAAGTTGAGGTTCA TGG 48.47928 103 3177 -1 TACTATCAAGCCAAAAGTTG AGG 51.74276 104 3178 1 CCACCATGAACCTCAACTTT TGG 32.68026 105 3195 1 TTTTGGCTTGATAGTAATTG TGG 54.13253 106 3199 1 GGCTTGATAGTAATTGTGGA AGG 53.09516 107 3224 -1 TCAAACAAGAAAGTCTACAA TGG 60.73706 108 3260 1 TGTAAGTCACTGTAAATTTT AGG 23.13386 109 3261 1 GTAAGTCACTGTAAATTTTA GGG 24.03811 110 3265 1 GTCACTGTAAATTTTAGGGT TGG 47.51408 111 3291 -1 TGGAAGAGTGATAGGAGATG TGG 64.74459 112 3299 -1 CTTATTATTGGAAGAGTGAT AGG 48.45991 113 3311 -1 TCAGATAATCCTCTTATTAT TGG 18.16136 114 3313 1 ATCACTCTTCCAATAATAAG AGG 52.38055 115 3342 -1 ATTTTTTTTTAAAAAATTAA TGG 17.24296 116 3444 -1 TGTGTCCTCTCTATAAATAA GGG 40.2693 117 3445 -1 TTGTGTCCTCTCTATAAATA AGG 27.63962 118 3450 1 TATATCCCTTATTTATAGAG AGG 51.01762 119 3488 1 GAGATGTTTAATATATTAAG TGG 50.52582 120 3489 1 AGATGTTTAATATATTAAGT GGG 41.77524 121 3490 1 GATGTTTAATATATTAAGTG GGG 62.38698 122 3534 1 AACTAAAATAATTTAAATGA AGG 46.81791 123 3549 1 AATGAAGGAATAGTATAAAA AGG 26.26061 124 3550 1 ATGAAGGAATAGTATAAAAA GGG 42.43128 125 3567 1 AAAGGGTCATCATCTTCAAA AGG 43.17344 126 Table 2: gRNA sequences targeted for CsSP-2 Position Strand Sequence PAM Efficiency SEQ. ID.
on SEQ. Score NO.
ID. NO:4 743 -1 TTTTTATCTTATCATGTTGT TGG 25.41093 127 773 -1 TGTAAGAGAACAGCTCTTAA GGG 32.52846 128 774 -1 TTGTAAGAGAACAGCTCTTA AGG 31.39567 129 812 -1 ACTAGTTTTAGTACGATGAT TGG 52.94793 130 907 1 AATAAGAGATTAACCGTGCG AGG 59.61949 131 909 -1 TAGTTTTCTTTTTCCTCGCA CGG 48.17976 132 967 1 TATTATATATATGTCATTAT TGG 18.73491 133 971 1 ATATATATGTCATTATTGGC AGG 41.76418 134 972 1 TATATATGTCATTATTGGCA GGG 54.92986 135 1048 1 ATTGAAGTATACAATAGCCA CGG 64.48335 136 1049 1 TTGAAGTATACAATAGCCAC GGG 53.86531 137 1050 1 TGAAGTATACAATAGCCACG GGG 75.49947 138 1054 -1 CAACAATCTGGGTCTCCCCG TGG 73.79643 139 1065 -1 TTTTCTGAAGACAACAATCT GGG 53.83692 140 1066 -1 ATTTTCTGAAGACAACAATC TGG 44.86974 141 1091 -1 TGAGAGACTGTTTTAACACA AGG 59.78212 142 1109 1 TTAAAACAGTCTCTCAAAGC TGG 53.49504 143 1140 -1 TTCATCTTATTCAAGCAAAG AGG 64.11009 144 1178 1 AATCTATGAATC CCC AC CAC CGG 66.90318 145 1178 -1 AAGCTCCAAAACCGGTGGTG GGG 60.93072 146 1179 -1 GAAGCTCCAAAACCGGTGGT GGG 54.2135 147 1180 -1 TGAAGCTCCAAAACCGGTGG TGG 46.33848 148 1183 -1 TTATGAAGCTCCAAAACCGG TGG 68.22724 149 1184 1 TGAATCCCCACCACCGGTTT TGG 29.72103 150 1186 -1 GAGTTATGAAGCTCCAAAAC CGG 48.10068 151 1221 -1 ATATATTTATAAACGAAAAC AGG 40.25381 152 1276 -1 TAACGAAATAAATATCTAAA TGG 33.5519 153 1329 -1 GTTTTTTTCTTTTGGTAGTG TGG 38.70555 154 1337 -1 TGCATTATGTTTTTTTCTTT TGG 14.19145 155 1351 1 AAAGAAAAAAACATAATGCA TGG 63.51026 156 1388 1 CACACATATATACGATAGTG AGG 59.34196 157 1409 1 GGACATGAAATACTCACCAA AGG 69.78964 158 1414 -1 GGTACAACTAATGTATCCTT TGG 46.18244 159 1428 1 AAGGATACATTAGTTGTACC TGG 57.10327 160 1435 -1 AGGATAGTCACAGACATTCC AGG 50.29588 161 1448 1 TGGAATGTCTGTGACTATCC TGG 45 .01183 162 1455 -1 ATAATACGTGTATTCTTTCC AGG 31.45682 163 1488 -1 GTGATCCTTTCCATTAATAT TGG 33.16337 164 1489 1 AATTAAATATCCAATATTAA TGG 20.04105 165 1494 1 AATATCCAATATTAATGGAA AGG 55 .69722 166 1532 1 ATATATAAATATACAAGAAG AGG 52.03505 167 1606 1 ATTATATTACAAACCAGTGA AGG 62.03101 168 1608 -1 ACTTAAAAGAACACCTTCAC TGG 43.10988 169 1622 1 GTGAAGGTGTTCTTTTAAGT AGG 40.61279 170 1623 1 TGAAGGTGTTCTTTTAAGTA GGG 44.06002 171 1632 1 TCTTTTAAGTAGGGATCACT AGG 52.1437 172 1633 1 CTTTTAAGTAGGGATCAC TA GGG 60.66675 173 1638 1 AAGTAGGGATCACTAGGGCT TGG 43.63704 174 1647 1 TCACTAGGGCTTGGAGCATC TGG 33.29868 175 1664 -1 CACTTATTTCTTGAAGATTA TGG 30.34701 176 1676 1 CATAATCTTCAAGAAATAAG TGG 51.71581 177 1684 1 TCAAGAAATAAGTGGAAAAA AGG 33.99747 178 1685 1 CAAGAAATAAGTGGAAAAAA GGG 41.53648 179 1815 -1 AAACCTCGAGTAGATATTGG TGG 58.65631 180 1818 -1 TCTAAACCTCGAGTAGATAT TGG 41.98098 181 1823 1 TCTCCACCAATATCTACTCG AGG 63.24041 182 1844 1 GGTTTAGACAAAATATTAGA AGG 43.87082 183 1869 -1 CCTGATAAGCAAGTTTGTAA TGG 46.15333 184 1880 1 CCATTACAAACTTGCTTATC AGG 22.76102 185 1881 1 CATTACAAACTTGCTTATCA GGG 46.00555 186 1913 1 ATGACAGTCATTTTTACACT AGG 56.38646 187 1939 -1 GGCAAGAGTAATAGGAGATG TGG 67.01531 188 1947 -1 TTAAGTGTGGCAAGAGTAAT AGG 35 .65879 189 1960 -1 AGCAATACAAACTTTAAGTG TGG 64.17131 190 1985 -1 TTTATATAATAATAATGTCA AGG 61.85855 191 2025 1 AATAGATCAGATATATGATA AGG 43.78777 192 2033 1 AGATATATGATAAGGATGAG AGG 64.39285 193 2104 1 GTAGTAGTAGAGATCGAGTT AGG 52.76822 194 2114 1 AGATCGAGTTAGGTGATCAA TGG 47.8583 195 2115 1 GATCGAGTTAGGTGATCAAT GGG 43.40038 196 2134 1 TGGGTATTTATAAGCCAGCT AGG 53.71914 197 2135 1 GGGTATTTATAAGCCAGCTA GGG 59.56206 198 2137 -1 AAATTCCAACTTGCCCTAGC TGG 36.84358 199 2143 1 ATAAGCCAGCTAGGGCAAGT TGG 60.49436 200 2187 1 TTATTTGAATAGCGTAGTAG TGG 54.61689 201 2197 1 AGCGTAGTAGTGGCTGCTCT TGG 43.97502 202 2198 1 GCGTAGTAGTGGCTGCTCTT GGG 44.41787 203 2208 1 GGCTGCTCTTGGGAAATGCC AGG 52.38622 204 2215 -1 AATAAAGCAAATATTAAACC TGG 60.29947 205 2253 1 GTAAAAAGTTAAAATACCAA AGG 65.71087 206 2258 -1 GTGATGTTTTCAAATTCCTT TGG 44.14554 207 2309 -1 CATACGTGTCGAACTAGGAA TGG 55.15684 208 2314 -1 ACAACCATACGTGTCGAACT AGG 63.97993 209 2321 1 CATTCCTAGTTCGACACGTA TGG 57.55443 210 2372 1 TTGTTGATGTTGAAAGAAGT TGG 64.04881 211 Table 3: gRNA sequences targeted for CsSP-3 Position Strand Sequence PAM Efficiency SEQ. ID.
on SEQ. Score NO.
ID. NO:7 979 1 AGAATATATATATATATTAG TGG 52.75818 212 983 1 TATATATATATATTAGTGGT AGG 56.18088 213 987 1 TATATATATTAGTGGTAGGT AGG 46.88074 214 1012 1 GAGCTAGCGTCTTCTAGCTG CGG 57.33903 215 1039 1 TCTCTGAGCGTTGAAATAAA CGG 31.00695 216 1049 1 TTGAAATAAACGGCAGCGAC AGG 51.04418 217 1059 1 CGGCAGCGACAGGAAGACCG AGG 71.16066 218 1065 -1 TTCGCTGCTCAAAACGACCT CGG 66.77634 219 1108 -1 GGTGAGTATACCAAGCTCAA AGG 64.31989 220 1109 1 TTGAAATGATCCTTTGAGCT TGG 55.54256 221 1128 1 TTGGTATACTCACCGAGTCT CGG 54.52127 222 1129 -1 CAAACAGAAATGCCGAGACT CGG 54.73946 223 1169 -1 AGCCAAATATAGGGATTCAC AGG 49.91997 224 1178 1 AACCTGTGAATCCCTATATT TGG 23.1867 225 1178 -1 AGAGCCCAAAGCCAAATATA GGG 42.41948 226 1179 -1 GAGAGCCCAAAGCCAAATAT AGG 31.63154 227 1184 1 TGAATCCCTATATTTGGCTT TGG 26.63952 1185 1 GAATCCCTATATTTGGCTTT GGG 29.53778 229 1210 -1 TATGTACACAGGAAGGGAAG TGG 62.37694 230 1216 -1 AATGGATATGTACACAGGAA GGG 56.73565 231 1217 -1 AAATGGATATGTACACAGGA AGG 56.69376 232 1221 -1 ATTAAAATGGATATGTACAC AGG 64.92133 233 1234 -1 TTATAAATATAAAATTAAAA TGG 25 .89586 234 1279 1 TATACATATATAATTAATAA TGG 34.02683 235 1309 -1 GGCACCACAGACGCTACATT TGG 42.45215 236 ATTACCAAATGTAGCGTCTG TGG 57.76274 237 AATGTAGCGTCTGTGGTGCC TGG 52.49826 238 1330 -1 AGGATTGTGACAGACATACC AGG 64.90611 239 1350 -1 TTCTTCTTTGAAACATGGAC AGG 56.04975 240 1355 -1 TTATTTTCTTCTTTGAAACA TGG 51.31281 241 1380 1 AGAAAATAATTATTACAATC AGG 48.41061 242 1390 1 TATTACAATCAGGATATCTT AGG 46.56003 243 1441 -1 TTTAATTAACAAACTATTTT AGG 18.17429 244 1464 -1 ATGTCAGTAAGATTATAATT TGG 33.42864 245 1479 1 ATTATAATCTTACTGACATT TGG 32.26823 246 1522 1 ACTATTTATTATACCAGTGA AGG 55 .21337 247 1524 -1 ACTTAAGGGAGCACCTTCAC TGG 49.73888 248 1538 -1 CCCAAGTGATCCTTACTTAA GGG 38.09188 249 1539 1 TGAAGGTGCTCCCTTAAGTA AGG 48.60231 250 1539 -1 GCCCAAGTGATCCTTACTTA AGG 40.75021 251 1548 1 TCCCTTAAGTAAGGATCACT TGG 45 .83122 252 1549 1 CCCTTAAGTAAGGATCACTT GGG 69.93595 253 1554 1 AAGTAAGGATCACTTGGGCC AGG 52.25638 254 1561 -1 ATGACAGATCCAGATGTTCC TGG 43.93102 255 1563 1 TCACTTGGGCCAGGAACATC TGG 40.0198 256 1586 -1 CTCTCTCTTTATTTTATTTT AGG 4.865075 1623 -1 AATTAATGTTGTTCTTTTTT TGG 15 .45049 258 1663 -1 AGATCTCTAAATGATGAACA AGG 65 .19671 259 1722 1 TTTTTTTATTAGTGTGAAGA AGG 48.17114 1748 -1 AAACCTAGGGTTGAGATTCA AGG 38.00239 261 1756 1 GCTCCTTGAATCTCAACC CT AGG 58.80331 262 1761 1 TTGAATCTCAACCCTAGGTT TGG 37.75685 263 1761 -1 AGTTGTCATCTCCAAACCTA GGG 52.43725 264 1762 -1 CAGTTGTCATCTCCAAACCT AGG 57.30496 265 1777 1 GGTTTGGAGATGACAACTGA AGG 63.04415 266 1802 -1 AACAAGAAGCAAGTCTGTAA TGG 45 .44633 267 1836 1 ATAAGAGACAGACATTTTTA TGG 28.08049 268 GAGACAGACATTTTTATGGA TGG 52.94092 269 1874 -1 CTTGTGGTTGGAAGAGTGAT AGG 54.20481 270 1886 -1 ATGTTAGAGCCTCTTGTGGT TGG 53.01781 271 1888 1 ATCACTCTTCCAACCACAAG AGG 57.38136 272 1890 -1 AAGAATGTTAGAGCCTCTTG TGG 65.83401 273 1914 -1 TATTACTATAAATATAATTA TGG 28.32586 274 1950 -1 ATATATTTTAAGTACTTAAT TGG 18.0389 275 2019 1 TTTATATATGTGTTTGTGTG TGG 49.7003 276 2060 1 AATTATTACTTAGTTCGTGA TGG 53.17869 277 2065 1 TTACTTAGTTCGTGATGGAA TGG 52.02412 278 2072 1 GTTCGTGATGGAATGGAACT TGG 45.80324 279 2087 -1 TGTAGACACTATAAATACAT GGG 59.04909 280 2088 -1 ATGTAGACACTATAAATACA TGG 56.82992 281 2110 1 TAGTGTCTACATGATATCTT TGG 36.6185 282 2124 -1 TAAAAGTAAAGTTACAATTA AGG 32.62771 283 Table 4: gRNA sequences targeted for CsSP5G-1 Position Strand Sequence PAM Efficiency SEQ. ID.
on SEQ. Score NO.
ID.
NO:10 43 1 ATATTAATATATAACAAGTT TGG 33.4577 284 127 1 AATAATTAATTAAGAATATA TGG 32.43589 285 138 1 AAGAATATATGGCTAGAGAT AGG 45.23675 286 139 1 AGAATATATGGCTAGAGATA GGG 53.98613 287 151 1 TAGAGATAGGGACCCTCTTG TGG 52.53234 288 152 -1 TTACTCTACCAACCACAAGA GGG 67.79474 289 153 -1 ATTACTCTACCAACCACAAG AGG 59.18615 290 155 1 GATAGGGACCCTCTTGTGGT TGG 56.67036 291 167 1 CTTGTGGTTGGTAGAGTAAT AGG 35.27607 292 178 1 TAGAGTAATAGGAGATGTTT TGG 32.95839 293 192 1 ATGTTTTGGATCCTTTTACA AGG 55.39173 294 192 -1 AGAGAGACTGACCTTGTAAA AGG 42.08228 295 218 1 GTCTCTCTTAGAGTGAGTTA TGG 47.06405 296 229 1 AGTGAGTTATGGTAATAGAG AGG 54.93598 297 239 1 GGTAATAGAGAGGTCAACAA TGG 58.93327 298 264 -1 GGTTGGTTAACAATTTGGGA AGG 61.46045 299 268 -1 ACGAGGTTGGTTAACAATTT GGG 32.45336 300 269 -1 CACGAGGTTGGTTAACAATT TGG 23.81735 301 281 -1 CACCAATATCAACACGAGGT TGG 61.80226 302 285 -1 TCACCACCAATATCAACACG AGG 75.84355 303 290 1 AACCAACCTCGTGTTGATAT TGG 44.46178 304 293 1 CAACCTCGTGTTGATATTGG TGG 58.81318 305 306 1 ATATTGGTGGTGATGACCTA AGG 60.13671 306 311 -1 CCAAAGTGTAGAAGGTCCTT AGG 42.09425 307 319 -1 TTAATTTACCAAAGTGTAGA AGG 48.09029 308 322 1 CCTAAGGACCTTCTACACTT TGG 50.47553 309 357 -1 TGAAATCATTATGAATATTG AGG 50.25272 310 436 1 CATATATTGAAAATTATTAC AGG 36.58192 311 442 1 TTGAAAATTATTACAGGTCA TGG 43.08159 312 445 1 AAAATTATTACAGGTCATGG TGG 65 .82262 313 451 1 ATTACAGGTCATGGTGGATC CGG 50.20677 314 459 -1 TTGCTAGGGCTAGGAGCATC CGG 49.91113 315 468 -1 AGATTGGGGTTGCTAGGGCT AGG 44.68519 316 473 -1 CCCTTAGATTGGGGTTGCTA GGG 47.67473 317 474 -1 TCCCTTAGATTGGGGTTGCT AGG 44.15699 318 482 -1 GC AAATACTCCCTTAGATTG GGG 58.46921 319 483 1 GCCCTAGCAACCCCAATCTA AGG 36.0562 320 483 -1 TGCAAATACTCCCTTAGATT GGG 30.97985 321 484 1 CCCTAGCAACCCCAATCTAA GGG 45 .48361 322 484 -1 ATGCAAATACTCCCTTAGAT TGG 38.79211 323 498 1 ATCTAAGGGAGTATTTGCAT TGG 59.37908 324 556 1 ATATTATTATTAAATAGATG AGG 50.44408 325 557 1 TATTATTATTAAATAGATGA GGG 52.51763 326 689 1 TTTAATTTTGTATAAAAC TT TGG 34.74999 327 808 -1 TTCATGCACACAACACATGT TGG 55 .60825 328 869 -1 CAAAAAGTAAAGACATATTT TGG 14.91886 329 881 1 CAAAATATGTCTTTACTTTT TGG 10.5253 330 916 1 CATTTTATAAAGATGTTAGT TGG 33.17467 331 917 1 ATTTTATAAAGATGTTAGTT GGG 40.20867 332 1105 1 TTTTAGTGTCAGTTTTGAAT TGG 29.61892 333 1123 -1 AC AAAATTCTGTAATTATTA GGG 26.7988 334 1124 -1 TACAAAATTCTGTAATTATT AGG 13.97206 335 1156 -1 AC AAATTAAAAC AAGCTTTA GGG 23.11439 336 1157 -1 AACAAATTAAAACAAGCTTT AGG 32.2923 337 1180 1 TTTAATTTGTTAAAGTGACT AGG 54.58344 338 1228 -1 AACATGTAAAAAGAATTTAA GGG 33.91047 339 1229 -1 AAACATGTAAAAAGAATTTA AGG 15 .92615 340 1258 -1 GCTAGCATATATGGAATTTG TGG 45 .56144 341 1267 -1 ATTTATATAGCTAGCATATA TGG 26.77656 342 1288 1 TAGCTATATAAATATAAATA TGG 35.H496 343 1293 1 ATATAAATATAAATATGGAA AGG 60.53312 344 1301 1 ATAAATATGGAAAGGATATA TGG 32.49959 345 1302 1 TAAATATGGAAAGGATATAT GGG 33.709 346 1351 1 AAAGCTGATGAGAAAGAATG TGG 66.11521 347 1356 1 TGATGAGAAAGAATGTGGTT TGG 50.94728 348 1357 1 GATGAGAAAGAATGTGGTTT GGG 37.02061 349 1358 1 ATGAGAAAGAATGTGGTTTG GGG 59.03998 350 1379 1 GGATGAATTTTGAATGATGA AGG 48.10583 351 1380 1 GATGAATTTTGAATGATGAA GGG 53.0293 352 1386 1 TTTTGAATGATGAAGGGATG AGG 52.46871 353 1398 1 AAGGGATGAGGCTGTGTGTG TGG 55.94352 354 1421 -1 GGGACATGCTATAGCTAGCA GGG 60.76795 355 1422 -1 GGGGACATGCTATAGCTAGC AGG 49.84652 356 1441 -1 TTTTAATGGTGGGACAAAAG GGG 54.93961 357 1442 -1 ATTTTAATGGTGGGACAAAA GGG 35.09294 358 1443 -1 CATTTTAATGGTGGGACAAA AGG 27.24033 359 1451 -1 GAGGTGGCCATTTTAATGGT GGG 62.03513 360 1452 -1 TGAGGTGGCCATTTTAATGG TGG 64.3868 361 1455 1 CTTTTGTCCCACCATTAAAA TGG 29.86891 362 1455 -1 GTGTGAGGTGGCCATTTTAA TGG 29.73425 363 1467 -1 AAAACCTTCTTAGTGTGAGG TGG 69.54075 364 1470 -1 GTGAAAACCTTCTTAGTGTG AGG 60.43941 365 1474 1 ATGGCCACCTCACACTAAGA AGG 56.86845 366 1543 1 ATATATACATACACATGTAT AGG 45.18352 367 1544 1 TATATACATACACATGTATA GGG 55.90543 368 1612 -1 CTATGTTCGAATTCAAATTC GGG 34.62224 369 1613 -1 TCTATGTTCGAATTCAAATT CGG 36.42467 370 1635 1 TTCGAACATAGACTCAGATT TGG 35.59295 371 1648 -1 AGGGTTCAGGGTCGAATTTA GGG 28.94531 372 1649 -1 CAGGGTTCAGGGTCGAATTT AGG 26.64298 373 1660 -1 AGTTCGTGTTTCAGGGTTCA GGG 43.58548 374 1661 -1 AAGTTCGTGTTTCAGGGTTC AGG 26.7905 375 1667 -1 GAGTCTAAGTTCGTGTTTCA GGG 35.03576 376 1668 -1 TGAGTCTAAGTTCGTGTTTC AGG 15.64056 377 1686 1 CACGAACTTAGACTCAGACC TGG 55.22454 378 1692 1 CTTAGACTCAGACCTGGACC TGG 54.41072 379 1693 -1 TTGGGTCAAGGTCCAGGTCC AGG 45.45826 380 1699 -1 CGGGGTTTGGGTCAAGGTCC AGG 43.16961 381 1705 -1 TCGGGTCGGGGTTTGGGTCA AGG 38.7222 382 1711 -1 CGGGGTTCGGGTCGGGGTTT GGG 29.12979 383 1712 -1 TCGGGGTTCGGGTCGGGGTT TGG 16.04517 384 1717 -1 TCGAGTCGGGGTTCGGGTCG GGG 28.40008 385 1718 -1 TTCGAGTCGGGGTTCGGGTC GGG 28.3022 386 1719 -1 GTTCGAGTCGGGGTTCGGGT CGG 44.74237 387 1723 -1 CGGGGTTCGAGTCGGGGTTC GGG 33.72492 388 1724 -1 TCGGGGTTCGAGTCGGGGTT CGG 29.847 389 1729 -1 TAGTTTCGGGGTTCGAGTCG GGG 54.61581 390 1730 -1 CTAGTTTCGGGGTTCGAGTC GGG 35.21533 391 1731 -1 TCTAGTTTCGGGGTTCGAGT CGG 47.1652 392 1741 -1 CCAGGTCCAGTCTAGTTTCG GGG 59.72421 393 1742 -1 TCCAGGTCCAGTCTAGTTTC GGG 29.76351 394 1743 -1 GTCCAGGTCCAGTCTAGTTT CGG 32.43822 395 1746 1 CTCGAAC CC CGAAACTAGAC TGG 41.77216 396 1752 1 CCCCGAAACTAGACTGGACC TGG 44.07102 397 1759 1 ACTAGACTGGACCTGGACTC TGG 56.19546 398 1759 -1 TAGGTCCTAGGCCAGAGTCC AGG 53.14281 399 1765 1 CTGGACCTGGACTCTGGC CT AGG 55.36929 400 1771 -1 CTAGACCCGAGCTAGGTCCT AGG 55.5272 401 1776 1 CTCTGGCCTAGGACCTAGCT CGG 44.89925 402 1777 1 TCTGGCCTAGGACCTAGCTC GGG 55.94427 403 1778 -1 CTGAACTCTAGACCCGAGCT AGG 64.71982 404 1790 1 CTAGCTCGGGTCTAGAGTTC AGG 38.28516 405 1800 1 TCTAGAGTTCAGGTCCAGTC CGG 44.68981 406 1801 1 CTAGAGTTCAGGTCCAGTCC GGG 51.19192 407 1802 1 TAGAGTTCAGGTCCAGTCCG GGG 64.76165 408 1803 -1 CCTGACCTCGGACCCCGGAC TGG 42.34928 409 1808 -1 CAGATCCTGACCTCGGACCC CGG 54.44243 410 1809 1 CAGGTCCAGTCCGGGGTCCG AGG 55.03576 411 1814 1 CCAGTCCGGGGTCCGAGGTC AGG 39.80181 412 1815 -1 ACGAACCCAGATCCTGACCT CGG 70.13695 413 1820 1 CGGGGTCCGAGGTCAGGATC TGG 27.07 414 1821 1 GGGGTCCGAGGTCAGGATCT GGG 45.99404 415 1833 1 CAGGATCTGGGTTCGTGTTC TGG 13.13476 416 1834 1 AGGATCTGGGTTCGTGTTCT GGG 32.34104 417 1835 1 GGATCTGGGTTCGTGTTCTG GGG 61.73636 418 1841 1 GGGTTCGTGTTCTGGGGTTC AGG 32.84982 419 1845 1 TCGTGTTCTGGGGTTCAGGT TGG 32.94333 420 1846 1 CGTGTTCTGGGGTTCAGGTT GGG 37.64639 421 1850 1 TTCTGGGGTTCAGGTTGGGT TGG 36.37666 422 1851 1 TCTGGGGTTCAGGTTGGGTT GGG 39.38005 423 1856 1 GGTTCAGGTTGGGTTGGGTC TGG 33.59531 424 1863 1 GTTGGGTTGGGTCTGGAGTC TGG 19.45666 425 1864 1 TTGGGTTGGGTCTGGAGTCT GGG 39.24034 426 1870 1 TGGGTCTGGAGTCTGGGTCT AGG 26.29809 427 1871 1 GGGTCTGGAGTCTGGGTCTA GGG 46.06491 428 1883 1 TGGGTCTAGGGTCCAGATTC AGG 36.11531 429 1884 -1 CCTGAAC CC GATCCTGAATC TGG 41.73702 430 1888 1 CTAGGGTCCAGATTCAGGAT CGG 53.25207 431 1889 1 TAGGGTCCAGATTCAGGATC GGG 49.77329 432 1895 1 CCAGATTCAGGATCGGGTTC AGG 32.80081 433 1901 1 TCAGGATCGGGTTCAGGTTA AGG 33.91225 434 1919 1 TAAGGTTTGAGTCTGAGTCC AGG 59.59865 435 1925 1 TTGAGTCTGAGTCCAGGTAT AGG 52.86993 436 1926 -1 TCCCGACCAGAACCTATACC TGG 43.98773 437 1931 1 CTGAGTCCAGGTATAGGTTC TGG 45.95561 438 1935 1 GTCCAGGTATAGGTTCTGGT CGG 54.99259 439 1936 1 TCCAGGTATAGGTTCTGGTC GGG 55.16665 440 1964 1 AGTTCGAGAGTTTGAATTCA AGG 34.8731 441 1974 1 TTTGAATTCAAGGTCCAATT TGG 30.63103 442 1977 -1 GAACTCATCCAACTCCAAAT TGG 38.88806 443 1980 1 TTCAAGGTCCAATTTGGAGT TGG 43.86757 444 1997 1 AGTTGGATGAGTTCATGTCA TGG 67.34083 445 2063 -1 TTTAAAATTTTAATAGTGTT TGG 34.83327 446 2179 1 ATTCATAATTTTTAAATTAG AGG 42.99532 447 2180 1 TTCATAATTTTTAAATTAGA GGG 36.49527 448 2196 -1 TTATTTTTATCTTACTTATA GGG 25.50447 449 2197 -1 ATTATTTTTATCTTACTTAT AGG 20.4776 450 2308 -1 TTTACTGTACCGAATATTCA CGG 41.6042 451 2310 1 TTGTAGTTACCGTGAATATT CGG 32.91515 452 2325 1 ATATTCGGTACAGTAAATTA AGG 39.9063 453 2329 1 TCGGTACAGTAAATTAAGGA TGG 57.49355 454 2410 -1 ATATATAAAAATATAAATTG TGG 59.46361 455 2441 1 TATATTATTAATCTAGATAA TGG 49.32964 456 2493 -1 ATAATTATACTATATATTAT AGG 26.08693 457 2523 1 TTATAATAATTATACATGTT TGG 35.08265 458 2538 1 ATGTTTGGCAATTTCAATTT AGG 24.64794 459 2542 1 TTGGCAATTTCAATTTAGGT TGG 51.99459 460 2558 1 AGGTTGGTGACTGATATTCC TGG 39.09227 461 2565 -1 AAGCTTGGCCCGGTAGTTCC AGG 41.23058 462 2567 1 ACTGATATTCCTGGAACTAC CGG 48.93325 463 2568 1 CTGATATTCCTGGAACTACC GGG 62.10752 464 2575 -1 CGGCTCACCGAAGCTTGGCC CGG 48.9322 465 2579 1 GGAACTACCGGGCCAAGCTT CGG 46.22153 466 2580 -1 ATGAACGGCTCACCGAAGCT TGG 59.46928 467 2595 -1 GTATTATTATGAAGTATGAA CGG 57.67305 468 2666 1 ACGCTGTAAACAAAATAGTG CGG 67.72415 469 2768 1 ATTAATTGTTTATTATGTGT AGG 49.28958 470 2776 1 TTTATTATGTGTAGGACAAG AGG 55.5504 471 2779 1 ATTATGTGTAGGACAAGAGG TGG 65.45521 472 2799 1 TGGTGTGCTACGAGAACCCG CGG 69.35789 473 2804 -1 GAATCCCCACCGTCGGCCGC GGG 51.25283 474 2805 -1 TGAATCCCCACCGTCGGCCG CGG 46.05954 475 2806 1 CTACGAGAACCCGCGGCCGA CGG 55.45967 476 2809 1 CGAGAACCCGCGGCCGACGG TGG 52.81179 477 2810 1 GAGAACCCGCGGCCGACGGT GGG 56.14245 478 2811 1 AGAACCCGCGGCCGACGGTG GGG 60.30596 479 2811 -1 TACCGATGAATCCCCACCGT CGG 68.50256 480 2820 1 GGCCGACGGTGGGGATTCAT CGG 48.29492 481 2841 1 GGTATGTATTTGTGTTGTTC CGG 42.62051 482 2848 1 ATTTGTGTTGTTCCGGCAAT TGG 33.46831 483 2849 1 TTTGTGTTGTTCCGGCAATT GGG 46.97766 484 2849 -1 CCGTTTGCCTTCCCAATTGC CGG 39.46262 485 2853 1 TGTTGTTCCGGCAATTGGGA AGG 55.97024 486 2860 1 CCGGCAATTGGGAAGGCAAA CGG 54.12282 487 2872 1 AAGGCAAACGGTGTTCGCGC CGG 41.65064 488 2873 1 AGGCAAACGGTGTTCGCGCC GGG 37.60685 489 2874 1 GGCAAACGGTGTTCGCGCCG GGG 56.96599 490 2877 1 AAACGGTGTTCGCGCCGGGG TGG 55.4399 491 2880 -1 TTGAAGTTCTGACGCCACCC CGG 59.80253 492 2905 -1 ATAAAGCTCAGCAAAGTCTT TGG 43.946 493 2924 1 TTTGCTGAGCTTTATAACCT TGG 44.20648 494 2930 -1 GGGCAGCAACAGGCAAACCA AGG 68.3844 495 2940 -1 TTGTAATAAAGGGCAGCAAC AGG 46.09672 496 2950 -1 CCTTTGGCAGTTGTAATAAA GGG 32.56095 497 2951 -1 CCCTTTGGCAGTTGTAATAA AGG 29.14878 498 2961 1 CCCTTTATTACAACTGCCAA AGG 54.00419 499 2962 1 CCTTTATTACAACTGCCAAA GGG 58.42829 500 2966 -1 CCCCAGATCCTGTCTCCCTT TGG 39.01106 501 2969 1 TACAACTGCCAAAGGGAGAC AGG 46.03039 502 2975 1 TGCCAAAGGGAGACAGGATC TGG 35.48964 503 2976 1 GCCAAAGGGAGACAGGATCT GGG 44.90032 504 2977 1 CCAAAGGGAGACAGGATCTG GGG 58.20173 505 2978 1 CAAAGGGAGACAGGATCTGG GGG 63.61687 506 2982 1 GGGAGACAGGATCTGGGGGA AGG 54.73323 507 2985 1 AGACAGGATCTGGGGGAAGG AGG 49.86207 508 2998 -1 ATATATATATGTCTATGTGG AGG 65.35548 509 3001 -1 TATATATATATATGTCTATG TGG 58.96155 510 3084 1 GATTCTTAATGATGATATCA TGG 42.04328 511 3110 -1 AAAGCTTATTATATTAATAA TGG 32.75644 512 3167 1 AAGATGAAGAAGAAGAAAAC AGG 48.09653 513 3253 1 ATTATTGTACTTAATTCAGC TGG 42.31373 514 3275 -1 TACATAATATAATATTAGCA AGG 60.27522 515 3312 1 ATGAGATACACACATATATA TGG 43.91874 516 Table 5: gRNA sequences targeted for CsSP5G-2 Position Strand Sequence PAM Specificity Efficiency SEQ.
ID.
on SEQ. Score Score NO.
ID.
NO:13 263 1 TATAATTAATTAAGAATATA TGG 22.23226 32.43589 517 274 1 AAGAATATATGGCTAGAGAT AGG 79.37203 45.23675 518 275 1 AGAATATATGGCTAGAGATA GGG 68.00411 53.98613 519 287 1 TAGAGATAGGGACCCTCTTG TGG 94.26857 52.53234 520 288 -1 TTACTCTACCAACCACAAGA GGG 86.20032 67.79474 521 289 -1 ATTACTCTACCAACCACAAG AGG 78.08364 59.18615 522 291 1 GATAGGGACCCTCTTGTGGT TGG 83.63088 56.67036 523 303 1 CTTGTGGTTGGTAGAGTAAT AGG 79.58025 35.27607 524 314 1 TAGAGTAATAGGAGATGTTT TGG 65.94111 32.95839 525 328 1 ATGTTTTGGATCCTTTTACA AGG 70.02417 55.39173 526 328 -1 AGAGAGACTGACCTTGTAAA AGG 61.64142 42.08228 527 354 1 GTCTCTCTTAGAGTGAGTTA TGG 77.15915 47.06405 528 364 1 GAGTGAGTTATGGTAATGAG AGG 88.2009 66.48722 529 375 1 GGTAATGAGAGGTCAACAGA TGG 69.50771 64.33275 530 400 -1 GGTTGGTTAACAATTTGGGA AGG 71.89396 61.46045 531 404 -1 ACGAGGTTGGTTAACAATTT GGG 74.7603 32.45336 532 405 -1 CACGAGGTTGGTTAACAATT TGG 47.58272 23.81735 533 417 -1 CACCAATATCAACACGAGGT TGG 87.81265 61.80226 534 421 -1 TCACCACCAATATCAACACG AGG 83.79878 75.84355 535 426 1 AACCAACCTCGTGTTGATAT TGG 89.69152 44.46178 536 429 1 CAACCTCGTGTTGATATTGG TGG 86.47779 58.81318 537 442 1 ATATTGGTGGTGATGACCTA AGG 85.41543 60.13671 538 447 -1 CCAAAGTGTAGAAGGTCCTT AGG 92.80423 42.09425 539 455 -1 TTAATTTACCAAAGTGTAGA AGG 72.5548 48.09029 540 458 1 CCTAAGGACCTTCTACACTT TGG 96.23278 50.47553 541 493 -1 TGAAATCATTATGAATATTG AGG 48.22121 50.25272 542 570 1 TCATATATTGAAATTATTAC AGG 52.69845 36.5369 543 576 1 ATTGAAATTATTACAGGTCA TGG 77.63024 44.4074 544 579 1 GAAATTATTACAGGTCATGG TGG 68.71918 67.377 545 585 1 ATTACAGGTCATGGTGGATC CGG 91.58358 50.20677 546 593 -1 TTGCTAGGGCTAGGAGCATC CGG 91.68151 49.91113 547 602 -1 AGATTGGGGTTGCTAGGGCT AGG 88.79083 44.68519 548 607 -1 CCCTTAGATTGGGGTTGCTA GGG 95.67441 47.67473 549 608 -1 TCCCTTAGATTGGGGTTGCT AGG 93.93984 44.15699 550 616 -1 GCAAATACTCCCTTAGATTG GGG 90.51159 58.46921 551 617 1 GCCCTAGCAACCCCAATCTA AGG 96.11133 36.0562 552 617 -1 TGCAAATACTCCCTTAGATT GGG 80.38169 30.97985 553 618 1 CCCTAGCAACCCCAATCTAA GGG 91.08172 45.48361 554 618 -1 ATGCAAATACTCCCTTAGAT TGG 83.72207 38.79211 555 632 1 ATCTAAGGGAGTATTTGCAT TGG 72.37072 59.37908 556 691 1 TATTATTATTATAATAGATG AGG 33.94489 49.0571 557 692 1 ATTATTATTATAATAGATGA GGG 46.17269 54.14818 558 824 1 TTTAATTTTGTATAAAAATT TGG 30.69736 25.68348 559 941 -1 TTCATGCACACAACACATGT TGG 73.37183 55.60825 560 1002 -1 CAAAAGATAAAGACATATTT TGG 40.5511 14.24454 561 1014 1 CAAAATATGTCTTTATCTTT TGG 49.25714 18.66841 562 1049 1 CATTTTATAAAGATGTTAGT TGG 62.22119 33.17467 563 1050 1 ATTTTATAAAGATGTTAGTT GGG 55.65628 40.20867 564 1235 1 TTTTAGTGTCAGTTTTGAAT TGG 56.17701 29.61892 565 1253 -1 ACAAAATTCTGTAATTATTA GGG 40.47765 26.7988 566 1254 -1 TACAAAATTCTGTAATTATT AGG 42.0428 13.97206 567 1286 -1 ACAAATTAAAACAAGCTTTA GGG 61.75581 23.11439 568 1287 -1 AACAAATTAAAACAAGCTTT AGG 57.55848 32.2923 569 1310 1 TTTAATTTGTTAAAGTGACT AGG 43.2 54.58344 1358 -1 AAACATGTAAAAGAATTTAA GGG 28.72101 30.13118 571 1359 -1 TAAACATGTAAAAGAATTTA AGG 36.55236 17.03894 572 1386 -1 TAGCTAGCATATATGGATTG TGG 85.80949 58.5557 573 1393 -1 ATTTATATAGCTAGCATATA TGG 67.09149 25.17453 574 1414 1 TAGCTATATAAATATAAATA TGG 34.98771 35.14496 575 1419 1 ATATAAATATAAATATGGAA AGG 38.5034 60.53312 576 1427 1 ATAAATATGGAAAGGATATA TGG 50.85775 32.49959 577 1428 1 TAAATATGGAAAGGATATAT GGG 55.43276 33.709 578 1476 1 AAAGCTGATGAGAAAGAATG TGG 59.04283 66.42004 579 1481 1 TGATGAGAAAGAATGTGGTT TGG 56.44644 50.94728 580 1482 1 GATGAGAAAGAATGTGGTTT GGG 45.55022 37.02061 581 1483 1 ATGAGAAAGAATGTGGTTTG GGG 32.76114 59.03998 582 1504 1 GGATGAATTTTGAATGATGA AGG 57.96939 48.10583 583 1505 1 GATGAATTTTGAATGATGAA GGG 51.67398 53.0293 584 1511 1 TTTTGAATGATGAAGGGATG AGG 74.87655 52.46871 585 1523 1 AAGGGATGAGGCTGTGTGTG TGG 91.76706 55.94352 586 1545 -1 GGGACATGCTATAGCTAGCA GGG 93.3302 60.76795 587 1546 -1 GGGGACATGCTATAGCTAGC AGG 96.3887 49.84652 588 1565 -1 ATTTTAATGGGGGACAAAAG GGG 75.18965 50.95202 589 1566 -1 CATTTTAATGGGGGACAAAA GGG 70.07484 28.24863 590 1567 -1 CC ATTTTAATGGGGGACAAA AGG 72.90401 29.61281 1575 -1 TGAGGTGGCCATTTTAATGG GGG 88.0117 64.57289 592 1576 -1 GTGAGGTGGCCATTTTAATG GGG 88.89714 54.06851 593 1577 -1 TGTGAGGTGGCCATTTTAAT GGG 86.9284 20.4018 594 1578 1 CCTTTTGTCCCCCATTAAAA TGG 84.04315 22.66327 595 1578 -1 GTGTGAGGTGGCCATTTTAA TGG 90.68493 23.47984 596 1590 -1 AAAACCTTCTTAGTGTGAGG TGG 86.37634 69.54075 597 1593 -1 GTGAAAACCTTCTTAGTGTG AGG 80.43362 60.43941 598 1597 1 ATGGCCACCTCACACTAAGA AGG 96.3153 56.86845 599 1665 1 ATATATACATACACATGTAT AGG 43.76861 45.18352 600 1666 1 TATATACATACACATGTATA GGG 22.29849 55.90543 601 1733 -1 CTATGTTCGAATTCAAATTC GGG 59.04622 34.62224 602 1734 -1 TCTATGTTCGAATTCAAATT CGG 55.60998 36.42467 603 1756 1 TTCGAACATAGACTCAGATT TGG 67.83184 35.59295 604 1769 -1 CAGGGTTCAGGGTCGAATTA GGG 96.49558 35.95472 605 1770 -1 TCAGGGTTCAGGGTCGAATT AGG 93.28847 30.0118 606 1780 -1 AGTTCGTGTTTCAGGGTTC A GGG 91.81519 43.58548 1781 -1 AAGTTCGTGTTTCAGGGTTC AGG 94.75493 26.7905 608 1787 -1 GAGTCTAAGTTCGTGTTTC A GGG 82.9438 35.03576 609 1788 -1 TGAGTCTAAGTTCGTGTTTC AGG 86.51258 15.64056 610 1806 1 CACGAACTTAGACTCAGACC TGG 97.32676 55.22454 611 1811 1 ACTTAGACTCAGACCTGGAC TGG 94.91925 50.92057 612 1813 -1 TTTGGGTCAAGGTCCAGTCC AGG 94.63584 46.64457 613 1824 -1 TCGGGTCGGGGTTTGGGTCA AGG 85.77517 38.7222 614 1830 -1 CGGGGTTCGGGTCGGGGTTT GGG 93.19713 29.12979 615 1831 -1 TCGGGGTTCGGGTCGGGGTT TGG 90.00364 16.04517 616 1836 -1 TCGAGTCGGGGTTCGGGTCG GGG 94.47451 28.40008 617 1837 -1 TTCGAGTCGGGGTTCGGGTC GGG 89.12695 28.3022 618 1838 -1 GTTCGAGTCGGGGTTCGGGT CGG 93.33815 44.74237 619 1842 -1 CGGGGTTCGAGTCGGGGTTC GGG 97.78823 33.72492 620 1843 -1 TCGGGGTTCGAGTCGGGGTT CGG 95.88259 29.847 621 1848 -1 TAGTTTCGGGGTTCGAGTCG GGG 89.32043 54.61581 622 1849 -1 CTAGTTTCGGGGTTCGAGTC GGG 95.12488 35.21533 623 1850 -1 TCTAGTTTCGGGGTTCGAGT CGG 89.32442 47.1652 624 1860 -1 CC AGGTCCAGTCTAGTTTCG GGG 97.39694 59.72421 1861 -1 TCCAGGTCCAGTCTAGTTTC GGG 95.56199 29.76351 626 1862 -1 GTCCAGGTCCAGTCTAGTTT CGG 93.75616 32.43822 627 1865 1 CTCGAACCCCGAAACTAGAC TGG 95.01385 41.77216 628 1871 1 CC CC GAAACTAGACTGGACC TGG 99.6993 44.07102 629 1878 1 ACTAGACTGGACCTGGACTC TGG 98.4311 56.19546 630 1878 -1 TAGGTCCTAGGCCAGAGTCC AGG 98.14848 53.14281 631 1884 1 CTGGACCTGGACTCTGGCCT AGG 98.33955 55.36929 632 1890 -1 CTAGACCCGAGCTAGGTCCT AGG 99.08664 55.22238 633 1895 1 CTCTGGCCTAGGACCTAGCT CGG 97.78611 44.89925 634 1896 1 TCTGGCCTAGGACCTAGCTC GGG 98.32979 55.94427 635 1897 -1 GCCTGACCTAGACCCGAGCT AGG 99.73282 62.86618 636 1902 1 CTAGGACCTAGCTCGGGTCT AGG 99.47133 48.45849 637 1907 1 ACCTAGCTCGGGTCTAGGTC AGG 98.95846 46.63816 638 1914 1 TCGGGTCTAGGTCAGGCGTC CGG 98.55442 40.11313 639 1915 1 CGGGTCTAGGTCAGGCGTCC GGG 99.96756 43.65285 640 1922 1 AGGTCAGGCGTCCGGGTCCG AGG 99.22245 53.42575 641 1922 -1 CC CAGATCTGACCTCGGACC CGG 99.30512 55.96585 1928 -1 CACGAACCCAGATCTGACCT CGG 96.77532 68.87557 643 1932 1 TCCGGGTCCGAGGTCAGATC TGG 99.24263 31.05262 644 1933 1 CCGGGTCCGAGGTCAGATCT GGG 97.78879 47.515 645 1945 1 TCAGATCTGGGTTCGTGTTC TGG 98.00119 23.41796 646 1946 1 CAGATCTGGGTTCGTGTTCT GGG 90.6576 34.46177 647 1947 1 AGATCTGGGTTCGTGTTCTG GGG 72.74378 61.20728 648 1953 1 GGGTTCGTGTTCTGGGGTTC AGG 96.0542 32.84982 649 1957 1 TCGTGTTCTGGGGTTCAGGT TGG 96.79548 32.94333 650 1958 1 CGTGTTCTGGGGTTCAGGTT GGG 97.29827 37.64639 651 1962 1 TTCTGGGGTTCAGGTTGGGT TGG 95.13759 36.37666 652 1963 1 TCTGGGGTTCAGGTTGGGTT GGG 88.17509 39.38005 653 1968 1 GGTTCAGGTTGGGTTGGGTC TGG 80.71071 33.59531 654 1975 1 GTTGGGTTGGGTCTGGAGTC TGG 89.11478 19.45666 655 1976 1 TTGGGTTGGGTCTGGAGTCT GGG 91.69313 39.24034 656 1982 1 TGGGTCTGGAGTCTGGGTCT AGG 93.64853 26.29809 657 1983 1 GGGTCTGGAGTCTGGGTCTA GGG 95.12459 46.06491 658 1995 1 TGGGTCTAGGGTCCAGATTC AGG 81.74099 36.11531 659 1996 -1 CCTGAACCCGATCCTGAATC TGG 95.331 41.73702 660 2000 1 CTAGGGTCCAGATTCAGGAT CGG 92.37173 53.25207 661 2001 1 TAGGGTCCAGATTCAGGATC GGG 89.64241 49.77329 662 2007 1 CC AGATTCAGGATCGGGTTC AGG 95.93196 32.80081 2013 1 TCAGGATCGGGTTCAGGTTA AGG 87.67606 33.91225 664 2031 1 TAAGGTTTGAGTCTGAGTCC AGG 94.41411 59.59865 665 2037 1 TTGAGTCTGAGTCCAGGTAT AGG 90.1772 52.86993 666 2038 -1 TCCCGACCAGAACCTATACC TGG 87.92881 43.98773 667 2043 1 CTGAGTCCAGGTATAGGTTC TGG 92.36672 45.95561 668 2047 1 GTCCAGGTATAGGTTCTGGT CGG 94.39855 54.99259 669 2048 1 TCCAGGTATAGGTTCTGGTC GGG 94.37527 55.16665 670 2075 1 GAGTTCAGAGTTTGAATTCA AGG 63.71772 35.78178 671 2085 1 TTTGAATTCAAGGTCCAATT TGG 68.36156 30.63103 672 2088 -1 GAACTCATCCAACTCCAAAT TGG 63.82882 38.88806 673 2091 1 TTCAAGGTCCAATTTGGAGT TGG 83.895 43.86757 674 2108 1 AGTTGGATGAGTTCATGTC A TGG 82.12315 67.34083 2174 -1 TTTAAAATTTTAATAGTGTT TGG 46.71727 34.83327 676 2291 1 TTCATAATTTTTAAAATTAG AGG 22.81635 37.38885 677 2292 1 TCATAATTTTTAAAATTAGA GGG 36.07909 41.07827 678 2308 -1 TATTTTTATCTTTACTTATA GGG 47.52393 23.50322 679 2309 -1 TTATTTTTATCTTTACTTAT AGG 46.63947 16.34323 680 2421 -1 TTTACTGTACCGAATATTCA CGG 79.17329 41.6042 681 2423 1 TTGTAGTTACCGTGAATATT CGG 84.39107 32.91515 682 2438 1 ATATTCGGTACAGTAAATTA AGG 78.81327 39.9063 683 2442 1 TCGGTACAGTAAATTAAGGA TGG 89.21293 57.49355 684 2523 -1 ATATATAAAAATATAAATTG TGG 25.27378 59.76844 685 2552 1 TATATTATTAATCTAGATAA TGG 50.26283 44.83698 686 2604 -1 TTATAATTATACTAATATAT AGG 35.57578 34.45299 687 2632 1 TTATAATAATTATACATGTT TGG 44.45843 35.08265 688 2648 1 TGTTTGGCAATTTCAATTTT AGG 48.67666 19.55566 689 2652 1 TGGCAATTTCAATTTTAGGT TGG 62.81544 45.19466 690 2668 1 AGGTTGGTGACTGATATTCC TGG 90.67651 39.09227 691 2675 -1 AAGCTTGGCCCGGTAGTTCC AGG 100 41.23058 692 2677 1 ACTGATATTCCTGGAACTAC CGG 83.0177 48.93325 693 2678 1 CTGATATTCCTGGAACTACC GGG 96.95467 62.10752 694 2685 -1 CGGCTCACCGAAGCTTGGCC CGG 98.17534 48.9322 695 2689 1 GGAACTACCGGGCCAAGCTT CGG 98.10529 46.22153 696 2690 -1 ATGAACGGCTCACCGAAGCT TGG 97.02801 59.46928 697 2705 -1 GTATTATTATGAAGTATGAA CGG 54.7198 57.67305 698 2776 1 ACGCTGTAAACAAAATAGTG CGG 81.23666 66.20102 699 2870 1 ATTAATTGTTTATTATGTGT AGG 37.44309 49.28958 700 2878 1 TTTATTATGTGTAGGACAAG AGG 75.38758 55.5504 701 2881 1 ATTATGTGTAGGACAAGAGG TGG 76.37859 65.45521 702 2901 1 TGGTGTGCTACGAGAACCCG CGG 98.37073 69.35789 703 2905 1 GTGCTACGAGAACCCGCGGC CGG 100 49.67584 704 2906 1 TGCTACGAGAACCCGCGGCC GGG 99.48738 51.94565 705 2906 -1 ATGAATCCCCACCCGGCCGC GGG 99.3641 52.12455 706 2907 -1 GATGAATCCCCACCCGGCCG CGG 99.86331 48.71998 707 2909 1 TACGAGAACCCGCGGCCGGG TGG 99.98179 55.19917 708 2910 1 ACGAGAACCCGCGGCCGGGT GGG 100 48.89766 709 2911 1 CGAGAACCCGCGGCCGGGTG GGG 99.8583 44.45536 710 2913 -1 CATAC CGATGAATCC CC ACC CGG 91.97498 55.96558 2920 1 GCGGCCGGGTGGGGATTCAT CGG 96.23188 49.33169 712 2941 1 GGTATGTATTTGTGTTGTTC CGG 49.44229 42.62051 713 2948 1 ATTTGTGTTGTTCCGGCAAT TGG 93.12907 33.46831 714 2949 1 TTTGTGTTGTTCCGGCAATT GGG 95.02167 46.97766 715 2949 -1 CCGTTTGCCTTCCCAATTGC CGG 95.00883 39.46262 716 2953 1 TGTTGTTCCGGCAATTGGGA AGG 88.65091 55.97024 717 2960 1 CCGGCAATTGGGAAGGCAAA CGG 93.92485 54.12282 718 2972 1 AAGGCAAACGGTGTTCGCGC CGG 99.47463 41.65064 719 2973 1 AGGCAAACGGTGTTCGCGCC GGG 99.96331 37.60685 720 2974 1 GGCAAACGGTGTTCGCGCCG GGG 99.57812 56.96599 721 2977 1 AAACGGTGTTCGCGCCGGGG TGG 99.29854 55.4399 722 2980 -1 TTGAAGTTCTGACGCCACCC CGG 97.94034 59.80253 723 3005 -1 ATAAAGCTCAGCAAAGTCTT TGG 76.47984 43.946 724 3024 1 TTTGCTGAGCTTTATAACCT TGG 70.03213 44.20648 725 3030 -1 GGGCAGCAACAGGCAAACCA AGG 96.32764 68.3844 726 3040 -1 TTGTAATAAAGGGCAGCAAC AGG 93.04706 46.09672 727 3050 -1 CCTTTGGCAGTTGTAATAAA GGG 84.67114 32.56095 728 3051 -1 CC CTTTGGCAGTTGTAATAA AGG 86.81624 29.14878 3061 1 CC CTTTATTACAACTGCCAA AGG 95.16301 54.00419 3062 1 CCTTTATTACAACTGCCAAA GGG 87.58701 58.42829 731 3066 -1 CC CC AGATCCTGTCTCCC TT TGG 97.97572 39.01106 3069 1 TACAACTGCCAAAGGGAGAC AGG 93.01226 46.03039 733 3075 1 TGCCAAAGGGAGACAGGATC TGG 98.02303 35.48964 734 3076 1 GCCAAAGGGAGACAGGATCT GGG 98.25717 44.90032 735 3077 1 CC AAAGGGAGAC AGGATCTG GGG 94.1697 58.20173 736 3078 1 CAAAGGGAGACAGGATCTGG GGG 96.85202 63.61687 737 3082 1 GGGAGACAGGATCTGGGGGA AGG 98.34396 54.73323 738 3085 1 AGACAGGATCTGGGGGAAGG AGG 95.84931 49.86207 739 3098 -1 ATATATATATGTCTATGTGG AGG 57.02276 65.35548 740 3101 -1 TATATATATATATGTCTATG TGG 42.14695 58.96155 741 3185 1 GATTCTTAATGATGATATCA TGG 35.38161 43.13298 742 3213 -1 AAAGCTTATTATATTAATAA TGG 35.17605 32.75644 743 3250 1 TATATATATATAGAATAAGA TGG 45.50324 48.36613 744 3261 1 AGAATAAGATGGAAGAAAAC AGG 48.6601 44.82802 745 Table 6: gRNA sequences targeted for CsSP5G-3 Position Strand Sequence PAM Efficiency SEQ. ID.
on SEQ. Score NO.
ID.
NO:16 1007 -1 AACTTGGCTGTGGTGGAAGG AGG 62.58111 746 1010 -1 AGGAACTTGGCTGTGGTGGA AGG 53.35214 747 1014 -1 CAAAAGGAACTTGGCTGTGG TGG 59.7649 748 1017 -1 TGCCAAAAGGAACTTGGCTG TGG 49.9405 749 1023 -1 TTCAACTGCCAAAAGGAACT TGG 48.07818 750 1026 1 CACCACAGCCAAGTTCCTTT TGG 32.23959 751 1030 -1 CGTTTACTTCAACTGCCAAA AGG 54.51517 752 1052 1 TTGAAGTAAACGACAGCAAC AGG 41.20281 753 1062 1 CGACAGCAACAGGCAATCCA AGG 59.31913 754 1068 -1 TTTGCTGAGATCTACAACCT TGG 39.26965 755 1112 1 TTGAAGTTATGTCGCCACCC AGG 64.35662 756 1115 -1 AGACAGTGTATGCACCTGGG TGG 69.83127 757 1118 -1 GGCAGACAGTGTATGCACCT GGG 69.43409 758 1119 -1 AGGCAGACAGTGTATGCACC TGG 46.20011 759 1139 -1 TTCTGTTTCGACAGTTAGGA AGG 47.13328 760 1143 -1 TTTGTTCTGTTTCGACAGTT AGG 49.53212 761 1181 1 TAGCGATGTATTCCCACCGT CGG 68.69506 762 1182 -1 GAGAGCCCTAGACCGACGGT GGG 72.28535 763 1183 -1 CGAGAGCCCTAGACCGACGG TGG 62.48353 764 1186 -1 CTACGAGAGCCCTAGACCGA CGG 67.63038 765 1187 1 TGTATTCCCACCGTCGGTCT AGG 37.13047 766 1188 1 GTATTCCCACCGTCGGTCTA GGG 52.6053 767 1224 -1 GTGTATATACGCATATATGT AGG 53.19418 768 1367 1 TTAACTTATCGTTAACTTAT TGG 21.66841 769 1452 1 AGTGTATTAATATGTACCAA AGG 64.2117 770 1457 -1 GCAACTACGGGAACAGCCTT TGG 43.45249 771 1469 -1 ACTGATATTCCCGCAACTAC GGG 56.57771 772 1470 1 AAAGGCTGTTCCCGTAGTTG CGG 54.3468 773 1470 -1 GACTGATATTCCCGCAACTA CGG 47.03522 774 1471 1 AAGGCTGTTCCCGTAGTTGC GGG 43.75953 775 1494 -1 AGTGTATAACTTTTTCAGGT TGG 45.74911 776 1498 -1 TTTAAGTGTATAACTTTTTC AGG 12.25519 777 1553 1 ATAAAATTAATAATTATTAG TGG 40.90627 778 1569 1 TTAGTGGCACACACTATTGA TGG 50.84478 779 1583 -1 AATATTACTAACGAATGACA AGG 71.2185 780 1633 -1 TGTACTTAGTAATATCATTT CGG 39.17215 781 1658 -1 ATCTTAAAGAATATTTGCAT TGG 51.43028 782 1673 1 TGCAAATATTCTTTAAGATT AGG 33.25661 783 1682 1 TCTTTAAGATTAGGTTCGCT AGG 47.68808 784 1683 1 CTTTAAGATTAGGTTCGCTA GGG 53.86326 785 1688 1 AGATTAGGTTCGCTAGGGTT AGG 44.74391 786 1713 -1 ACTGTTAATGTTGCAGGTTA TGG 38.16734 787 1719 -1 ATTAATACTGTTAATGTTGC AGG 25.17433 788 1839 1 CGTATACTTAATTTTATGCT AGG 51.56297 789 1980 1 TATTTGTGCAATTATAAGTT AGG 41.35824 790 1992 -1 GTCAAAGACCCACAACAATA AGG 47.19197 791 1994 1 TAAGTTAGGCCTTATTGTTG TGG 52.99944 792 1995 1 AAGTTAGGCCTTATTGTTGT GGG 35.64647 793 2051 1 TTAACAGTGTTAATTTTAAA CGG 27.14498 794 2196 -1 TTGGTTTATGAAAAATTAGT GGG 43.4587 795 2197 -1 CTTGGTTTATGAAAAATTAG TGG 50.90688 796 2215 -1 TGCCTATATAACTAAATTCT TGG 33.54393 797 2224 1 AACCAAGAATTTAGTTATAT AGG 34.54892 798 2274 -1 TCTCAGGACTTTCTACACTT TGG 48.01676 799 2290 -1 AGATTGGTGGAGATGATCTC AGG 51.3529 800 2303 -1 TCACCTAGGGTTGAGATTGG TGG 50.45115 801 2306 -1 AACTCACCTAGGGTTGAGAT TGG 52.86728 802 2311 1 TCTCCACCAATCTCAACCCT AGG 61.53779 803 2316 -1 TCAAGTTGCTAACTCACCTA GGG 50.65517 804 2317 -1 CTCAAGTTGCTAACTCACCT AGG 56.33574 805 2332 1 GGTGAGTTAGCAACTTGAGA AGG 55.71603 806 2339 1 TAGCAACTTGAGAAGGTCTA AGG 50.2432 807 2357 -1 GGGATTAGGGCTATTAAC AA TGG 57.57407 808 2370 -1 AGTATCATATAGTGGGATTA GGG 45.56512 809 2371 -1 AAGTATCATATAGTGGGATT AGG 31.11638 810 2377 -1 CTCTTAAAGTATCATATAGT GGG 47.53587 811 2378 -1 TCTCTTAAAGTATCATATAG TGG 49.25586 812 2407 1 AGAGAGACAGTTTTTGTGAA AGG 50.90759 813 2408 1 GAGAGACAGTTTTTGTGAAA GGG 39.7937 814 2421 -1 GAGAGTGATAAGTGATGTGT TGG 59.68802 815 2443 -1 ATAGGGATCCTCTTGTGGTT GGG 49.32786 816 2444 -1 AATAGGGATCCTCTTGTGGT TGG 40.13389 817 2446 1 ATCACTCTCCCAACCACAAG AGG 54.01665 818 2448 -1 GGCTAATAGGGATCCTCTTG TGG 57.16178 819 2460 -1 ATATATATAAATGGCTAATA GGG 36.81696 820 2461 -1 TATATATATAAATGGCTAAT AGG 23.14055 821 2469 -1 ATAGTTGTTATATATATAAA TGG 34.2573 822 2520 1 AGAGAGATAGAGAGAAGAAG AGG 53.49955 823 2521 1 GAGAGATAGAGAGAAGAAGA GGG 63.65805 824 2537 1 AAGAGGGTTTGATGAGTTTT TGG 25.7462 825 2550 1 GAGTTTTTGGTTGTATAATT TGG 28.78721 826 2553 1 TTTTTGGTTGTATAATTTGG TGG 50.48919 827 2574 1 GGCTGACATTCAACAATTTA TGG 15.68184 828 Table 7: gRNA sequences targeted for CsSP5G-4 Position on SEQ. Strand Sequence PAM Efficiency SEQ.
ID. NO:19 Score ID. NO.
732 -1 TAAAGTTATTGGGAGTTGTG TGG 66.8211 829 742 -1 CCGACTACTGTAAAGTTATT GGG 28.42176 830 743 -1 GCCGACTACTGTAAAGTTAT TGG 36.02326 831 753 1 CCCAATAACTTTACAGTAGT CGG 40.53914 832 765 -1 TTATATAATTCTTATAGCAA TGG 48.27692 833 819 -1 AAAGGTTCTAATATATATTG TGG 47.44654 834 837 -1 TTAGCTTTTGTAACATCAAA AGG 33.39633 835 893 -1 GGCTGACATTCAACAATTTA TGG 15.68184 836 914 -1 GTTTTGGTTATATAATTTGG TGG 57.11172 837 917 -1 TGAGTTTTGGTTATATAATT TGG 28.40639 838 930 -1 GAAGAGGGTTTGATGAGTTT TGG 34.58611 839 945 -1 TGTAGAGAGAGATCAGAAGA GGG 60.53333 840 946 -1 ATGTAGAGAGAGATCAGAAG AGG 54.06301 841 990 1 ATAGTTGTTATATATATAAA TGG 33.58298 842 998 1 TATATATATAAATGGCTAAT AGG 23.14055 843 999 1 ATATATATAAATGGCTAATA GGG 36.81696 844 1011 1 GGCTAATAGGGATCCTCTTG TGG 57.16178 845 1013 -1 ATCACTCTCCCAACCACAAG AGG 54.01665 846 1015 1 AATAGGGATCCTCTTGTGGT TGG 40.13389 847 1016 1 ATAGGGATCCTCTTGTGGTT GGG 49.32786 848 1038 1 GAGAGTGATAAGTGATGTGT TGG 59.68802 849 1051 -1 GAGACACAGTTTTTGTGAAA GGG 37.91168 850 1052 -1 AGAGACACAGTTTTTGTGAA AGG 52.69581 851 1081 1 TCTCTTAAAGTATCATATAG TGG 51.27177 852 1082 1 CTCTTAAAGTATCATATAGT GGG 49.44647 853 1088 1 AAGTATCATATAGTGGGAAT AGG 36.1639 854 1089 1 AGTATCATATAGTGGGAATA GGG 49.45047 855 1102 1 GGGAATAGGGCTATTAACAA TGG 57.4132 856 1120 -1 TAGCAACTTGAGAAGGTCTA AGG 50.2432 857 1127 -1 GGTGAGTTAGCAACTTGAGA AGG 55.71603 858 1142 1 CTCAAGTTGCTAACTCACCT AGG 56.33574 859 1143 1 TCAAGTTGCTAACTCACCTA GGG 50.65517 860 1148 -1 TCTCCACCAATCTCAACCCT AGG 61.53779 861 1153 1 AACTCACCTAGGGTTGAGAT TGG 52.86728 862 1156 1 TCACCTAGGGTTGAGATTGG TGG 50.45115 863 1169 1 AGATTGGTGGAGATGATCTC AGG 51.3529 864 1185 1 TCTCAGGACTTTCTACACTT TGG 48.01676 865 1268 1 ATTAATACTGTTAATGTTGC AGG 25.17433 866 1274 1 ACTGTTAATGTTGCAGGTTA TGG 38.16734 867 1291 -1 TCGCTAGGGTTAGGAGCATC AGG 47.478 868 1300 -1 AGATTAGGTTCGCTAGGGTT AGG 44.74391 869 1305 -1 CTTTAAGATTAGGTTCGC TA GGG 53.86326 870 1306 -1 TCTTTAAGATTAGGTTCGCT AGG 47.68808 871 1315 -1 TGCAAATATTCTTTAAGATT AGG 33.25661 872 1330 1 ATCTTAAAGAATATTTGCAT TGG 51.43028 873 1402 1 AACTTTTAGATATATTACTT AGG 42.90897 874 1412 1 TATATTACTTAGGAATCACA AGG 71.76671 875 1426 -1 TAGTGGCACACACTTATTGA TGG 44.48106 876 1443 -1 TAAAAATTAATAATTATTAG TGG 38.53536 877 1503 1 TTAAAGTGTATAATTTTGTC AGG 40.17797 878 1507 1 AGTGTATAATTTTGTCAGGT TGG 38.69861 879 1530 -1 AAGCCTGTTCCCGTAGTTGC GGG 43.75953 880 1531 1 GACTGATATTCCCGCAAC TA CGG 47.03522 881 1531 -1 AAAGCCTGTTCCCGTAGTTG CGG 53.51908 882 1532 1 ACTGATATTCCCGCAACTAC GGG 56.57771 883 1538 1 ATTCCCGCAACTACGGGAAC AGG 40.62686 884 1544 1 GCAACTACGGGAACAGGCTT TGG 40.90731 885 1634 -1 TTAACTTACCATTAACTTAT TGG 23.66032 886 1637 1 AAATAACACCAATAAGTTAA TGG 36.79107 887 1740 1 TGTGTGTGATGATTTAATGA TGG 55.89947 888 1741 1 GTGTGTGATGATTTAATGAT GGG 53.71776 889 1759 1 ATGGGCGTACGCATATATGT AGG 58.24576 890 1795 -1 GAATTCCCACCGTCGGTCTT GGG 43.57541 891 1796 -1 TGAATTCCCACCGTCGGTCT TGG 31.13209 892 1797 1 CTACGAGAGCCCAAGACCGA CGG 67.05847 893 1800 1 CGAGAGC CC AAGACCGACGG TGG 58.98627 894 1801 1 GAGAGCCCAAGACCGACGGT GGG 70.56006 895 1802 -1 AAGCGATGAATTCCCACCGT CGG 68.48212 896 1840 1 TTTGTTCTGTTTCGACAGTT AGG 49.53212 897 1844 1 TTCTGTTTCGACAGTTAGGA AGG 47.13328 898 1864 1 AGGCAGACAGTGTATGCACC CGG 53.95754 899 1865 1 GGCAGACAGTGTATGCACCC GGG 67.05624 900 1868 1 AGACAGTGTATGCACCCGGG TGG 71.26007 901 1871 -1 TTGAAGTTATGTCGCCAC CC GGG 64.36734 902 1872 -1 GTTGAAGTTATGTCGCCACC CGG 57.40751 903 1915 1 TTTGCTGAAATCTACAAC CT TGG 36.83098 904 1921 -1 CGGCAGCAACAGGCAATCCA AGG 56.65812 905 1931 -1 TTGAAGTAAACGGCAGCAAC AGG 42.77031 906 1941 -1 CTTTTGGCAGTTGAAGTAAA CGG 46.14269 907 1953 1 CGTTTACTTCAACTGCCAAA AGG 54.51517 908 1957 -1 C AC CACAGCCAAGTTCCTTT TGG 32.23959 909 1960 1 TTCAACTGCCAAAAGGAACT TGG 48.07818 910 1966 1 TGCCAAAAGGAACTTGGCTG TGG 49.9405 911 1969 1 CAAAAGGAACTTGGCTGTGG TGG 59.7649 912 1973 1 AGGAACTTGGCTGTGGTGGA AGG 53.35214 913 1976 1 AACTTGGCTGTGGTGGAAGG AGG 62.58111 914 2046 -1 GACATATATAGATAGATAGA TGG 51.68794 915 2351 1 TGTATCATCAATAATATATA TGG 32.79628 916 Reference is made to Table 8 presenting a summary of the sequences within the scope of the current invention.
Table 8: Summary of sequences within the scope of the present invention Sequence type CsSP-1 CsSP-2 CsSP-3 CsSP5G-1 CsSP5G-2 CsSP5G-3 CsSP5G-4 Genomic SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
sequences NO:1 NO:4 NO:7 NO:10 NO:13 NO:16 NO:19 (Fig. 2A) (Fig. 3A) (Fig. 4A) (Fig. 5A) (Fig. 6A) (Fig.
7A) (Fig. 8A) Coding sequences SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
(CDS) NO:2 NO:5 NO:8 NO:11 NO:14 NO:17 NO:20 (Fig. 2B) (Fig. 3B) (Fig. 4B) (Fig. 5B) (Fig. 6B) (Fig. 7B) (Fig. 8B) Amino acid SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
sequences NO:3 NO:6 NO:9 NO:12 NO:15 NO:18 NO:21 (Fig. 2C) (Fig. 3C) (Fig. 4C) (Fig. 5C) (Fig. 6C) (Fig. 7C) (Fig. 8C) gRNA sequences SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
NO:22- NO:127- NO:212- NO:284- NO:517- NO:746- NO:829-SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
NO:126 NO:211 NO:283 NO:516 NO:745 NO:828 NO:916 (Table 1) (Table 2) (Table 3) (Table 4) (Table 5) (Table 6) (Table 7) The above gRNA molecules have been cloned into suitable vectors and their sequence has been verified. In addition different Cas9 versions have been analyzed for optimal compatibility between the Cas9 protein activity and the gRNA molecule in the Cannabis plant.
The efficiency of the designed gRNA molecules have been validated by transiently transforming Cannabis tissue culture. A plasmid carrying a gRNA sequence together with the Cas9 gene has been transformed into Cannabis protoplasts. The protoplast cells have been grown for a short period of time and then were analyzed for existence of genome editing events.
The positive constructs have been subjected to the herein established stable transformation protocol into Cannabis plant tissue for producing genome edited Cannabis plants in SP and/or SP5G genes.
Stage 3: Transforming Cannabis plants using Agrobacterium or biolistics (gene gun) methods. For Agrobacterium and bioloistics, a DNA plasmid carrying (Cas9 + gene specific gRNA) can be used.
A vector containing a selection marker, Cas9 gene and relevant gene specific gRNA's is constructed. For biolistics, Ribonucleoprotein (RNP) complexes carrying (Cas9 protein + gene specific gRNA) are used. RNP complexes are created by mixing the Cas9 protein with relevant gene specific gRNA' s.
According to some embodiments of the present invention, transformation of various Cannabis tissues was performed using particle bombardment of:
= 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. 9 photographically presenting GUS staining after transient transformation of the following Cannabis tissues (A) axillary buds (B) leaf (C) calli, and (D) cotyledons.
Fig. 9 demonstrates that various Cannabis tissues have been successfully transiently transformed using biolistics system. Transformation has been performed into calli, leaves, axillary buds and cotyledons of Cannabis.
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. 10A-C 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. 11 showing PCR detection of Cas9 DNA in shoots of transformed Cannabis plants. DNA extracted from shoots of plants transformed with Cas9 using biolistics. This figure shows that three weeks post transformation, Cas9 DNA was detected in shoots of transformed plants.
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. 12 presenting results of in vitro analysis of CRISPR/Cas9 cleavage activity. Fig. 12A schematically shows the genomic area targeted for editing (PAM is marked in red) and amplified by the reverse and forward designed primers Fig. 12B
photographically presents a gel showing successful digestion of the resulted PCR amplicon containing the gene specific gRNA sequence, by RNP complex containing Cas9. The analysis included the following steps:
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 sp and sp5g related phenotypes as described above. It is within the scope that different gRNA promoters were tested in order to maximize editing efficiency.
References:
Sebastian Soyk, Niels A Muller, Soon Ju Park, Inga Schmalenbach, Ke Jiang, Ryosuke Hayama, Lei Zhang, Joyce Van Eck, Jose M Jimenez-Gomez & Zachary B Lippman "Variation in the flowering gene SELF PRUNING 5G promotes day-neutrality and early yield in tomato" Nature Genetics, 2017 49, 162-168.
Tingdong Li, Xinping Yang, Yuan Yu, Xiaomin Si, Xiawan Zhai, Huawei Zhang, Wenxia Dong, Caixia Gao and Cao Xu "Domestication of wild tomato is accelerated by genome editing" Nature Biotechnology, 2018 36, 1160-1163.
Agustin Zsogon, TomaS Cermak, Emmanuel Rezende Naves, Marcela Morato Notini, Kai H Edel, Stefan Weinl, Luciano Freschi, Daniel F Voytas, Jorg Kudla and Lazar Eustaquio Pereira Peres "De novo domestication of wild tomato using genome editing" Nature Biotechnology, 2018 36, 1211-1216.
Zachary H. Lemmon, Nathan T. Reem, Justin Dalrymple, Sebastian Soyk, Kerry E.
Swartwood, Daniel Rodriguez-Leal, Joyce Van Eck and Zachary B. Lippman "Rapid improvement of domestication traits in an orphan crop by genome editing" Nature Plants, 2018 4, 766-770.
Xie, K. and Yang Y. "RNA-guided genome editing in plants using a CRISPR¨Cas system."
Molecular plant, 2013 6 (6), 1975-1983.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said functional variant has at least 75% sequence identity to said CsSP
or said CsSP5G nucleotide sequence.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation is introduced 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 said mutation is introduced using targeted genome modification.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation 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 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, Castl0d, Cas12, Cas13, Cas14, CasX, CasF, CasG, CasH, Csyl, Csy2, Csy3, Cse 1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc 1, Csc2, Csa5, Csnl, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz 1, Csx15, Csfl, Csf2, Csf3, Csf4, and Cu 1966and 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 mutated CsSP or CsSP5G gene is a CRISPR/Cas9-induced heritable mutated allele.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation is a missense mutation, nonsense mutation, insertion, deletion, indel, substitution or duplication.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the insertion or the deletion produces a gene comprising a frameshift.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant is homozygous for said at list one CsSP
mutated gene.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant is homozygous for said at list one CsSP5G
mutated gene.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant is a Cssp Cssp5g double mutant.
It is another object of the present invention to disclose the modified Cannabis plant 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, 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 said mutation 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 said mutation is generated in planta.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation 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:22-SEQ ID NO:916 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:22-916 and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation in said CsSP-1 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:22-SEQ ID NO:126 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ
ID NO:22-126 and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation in said CsSP-2 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:127-SEQ ID NO:211 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ
ID NO:127-211 and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation in said CsSP-3 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:212-SEQ ID NO:283 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ
ID NO:212-283 and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation in said CsSP5G-1 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:284-SEQ ID NO:516 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ
ID NO: 284-516 and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation in said CsSP5G-2 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:517-SEQ ID NO:745 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ
ID NO:517-745 and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation in said CsSP5G-3 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:746-SEQ ID NO:828 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ
ID NO: 746-828 and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said mutation in said CsSP5G-4 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:829-SEQ ID NO:916 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ
ID NO: 829-916 and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said gRNA sequence comprises a 3' NGG Protospacer Adjacent Motif (PAM).
It is another object of the present invention to disclose the modified 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 of the genome editing molecules or mechanical insertion such as polyethylene glycol (PEG) mediated DNA transformation, electroporation or gene gun biolistics.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant has decreased expression levels of at least one of said CsSP
genes.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the sequence of said expressed CsSP gene is selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:9 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 said plant has decreased expression levels of at least one of said CsSP5G
genes.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the sequence of said expressed CsSP5G gene is selected from the group consisting of: SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:21 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 said plant is semi-determinant.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant has determinant growth habit.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant flowers earlier than a corresponding wild type cannabis plant.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant exhibits improved earliness as compared to a corresponding wild type cannabis plant.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant exhibits suppressed sympodial shoot termination as compared to a corresponding wild type cannabis plant.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant exhibits similar sympodial shoot termination as compared to a corresponding wild type cannabis plant.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said plant exhibits suppressed or reduced day-length sensitivity as compared to a corresponding wild type cannabis plant.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said Cannabis plant is selected from the group of species that includes, but is not limited to, Cannabis sativa (C. sativa), C. indica, C. ruderalis and any hybrid or cultivated variety of the genus Cannabis.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said domestication trait is selected from the group consisting of reduced flowering time, earliness, synchronous flowering, reduced day-length sensitivity, determinant or semi-determinant architecture, early termination of sympodial cycling, earlier axillary shoot flowering, compact growth habit, reduced height, reduced number of sympodial units, adaptation to mechanical harvest, higher harvest index and any combination thereof.
It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein said Cssp Cssp5g double mutant is characterized by having a more than additive effect on a trait selected from the group consisting of compactness, earlier axillary shoot flowering, earlier termination of sympodial cycling, harvest index and any combination thereof as compared to wild type and/or sp mutant Cannabis plants.
It is another object of the present invention to disclose a modified Cannabis plant, plant part or plant cell as defined in any of the above, wherein said plant does not comprise a transgene.
It is another object of the present invention to disclose a plant part, plant cell or plant seed of a 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 said plant genotype is obtainable by deposit under accession number with NCIMB Aberdeen AB21 9YA, Scotland, UK or with ATCC.
It is another object of the present invention to disclose a method for producing a modified Cannabis plant exhibiting at least one improved domestication trait compared with wild type Cannabis, said method comprises steps of genetically modifying at least one Cannabis SELF
PRUNING (SP) (CsSP) gene and/or at least one Cannabis SELF PRUNING 5G (SP5G) (CsSP5G) gene.
It is another object of the present invention to disclose a method for producing a modified Cannabis plant exhibiting at least one improved domestication trait compared with wild type Cannabis by targeted genome modification, said method comprises steps of genetically introducing a loss of function mutation in at least one Cannabis SELF PRUNING (SP) (CsSP) gene and/or at least one Cannabis SELF PRUNING 5G (SP5G) (CsSP5G) gene.
It is another object of the present invention to disclose a method of improving at least one domestication trait compared with wild type Cannabis, comprising steps of producing a modified Cannabis plant, seed or plant part thereof, that is homozygous for at least one mutated CsSP5G
gene in an sp background and enabling growth of said Cannabis plant, seed or plant part thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said method comprises steps of: (a) identifying at least one Cannabis SP (CsSP) and/or at least one Cannabis SP5G (CsSP5G) allele; (b) synthetizing at least one guide RNA (gRNA) comprising a nucleotide sequence complementary to said at least one identified CsSP and/or CsSP5G allele; (c) transforming Cannabis plant cells with a construct comprising (a) Cas nucleotide sequence operably linked to said at least one gRNA, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and said at least one gRNA; (d) screening the genome of said transformed plant cells for induced targeted loss of function mutation in at least one of said CsSP
and/or CsSP5G allele; (e) regenerating Cannabis plants carrying said loss of function mutation in at least one of said CsSP and/or CsSP5G allele; and (f) screening said regenerated plants for a Cannabis plant with improved domestication trait.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said step of screening the genome of said transformed plant cells for induced targeted loss of function mutation further comprises steps of obtaining a nucleic acid sample of said transformed plant and performing a nucleic acid amplification and optionally restriction enzyme digestion to detect a mutation in said at least one of said CsSP and/or CsSP5G allele.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said SELF PRUNING (SP) Cannabis gene is selected from the group consisting of CsSP-1 having a genomic nucleotide sequence as set forth in SEQ ID NO:1 or a functional variant thereof, CsSP-2 having a genomic nucleotide sequence as set forth in SEQ ID
NO:4 or a functional variant thereof, CsSP-3 having a genomic nucleotide sequence as set forth in SEQ ID NO:7 or a functional variant thereof and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said SELF PRUNING 5G (SP5G) Cannabis gene is selected from the group consisting of CsSP5G-1 having a genomic nucleotide sequence as set forth in SEQ ID NO:10 or a functional variant thereof, CsSP5G-2 having a genomic nucleotide sequence as set forth in SEQ ID NO:13 or a functional variant thereof, CsSP5G-3 having a genomic nucleotide sequence as set forth in SEQ ID NO:16 or a functional variant thereof, CsSP5G-4 having a genomic nucleotide sequence as set forth in SEQ ID NO:19 or a functional variant thereof and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said functional variant has at least 75% sequence identity to said CsSP or said CsSP5G
nucleotide sequence.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation is introduced 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 method as defined in any of the above, wherein said mutation is introduced using targeted genome modification.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation 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 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, Castl0d, Cas12, Cas13, Cas14, CasX, CasF, CasG, CasH, Csyl, Csy2, Csy3, Cse 1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc 1, Csc2, Csa5, Csn 1, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Cszl, Csx15, Csfl, Csf2, Csf3, Csf4, and Cu1966and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein the mutated CsSP or CsSP5G gene is a CRISPR/Cas9- induced heritable mutated allele.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation is a missense mutation, nonsense mutation, insertion, deletion, indel, substitution or duplication.
It is another object of the present invention to disclose the method as defined in any of the above, wherein the insertion or the deletion produces a gene comprising a frameshift.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said plant is homozygous for said at list one CsSP mutated gene.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said plant is homozygous for said at list one CsSP5G mutated gene.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said plant is a Cssp Cssp5g double mutant.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation is in the coding region of said allele, a mutation in the regulatory region of said allele, or an epigenetic factor.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation is a silencing mutation, a knockdown mutation, a knockout mutation, a loss of function mutation or any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation is generated in planta.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation 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:22-SEQ
ID NO:916 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:22-916 and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation in said CsSP-1 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:22-SEQ ID NO:126 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:22-126 and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation in said CsSP-2 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:127-SEQ ID NO:211 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID
NO:127-211 and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation in said CsSP-3 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:212-SEQ ID NO:283 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID
NO:212-283 and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation in said CsSP5G-1 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:284-SEQ ID NO:516 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:
284-516 and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation in said CsSP5G-2 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:517-SEQ ID NO:745 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID
NO:517-745 and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation in said CsSP5G-3 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:746-SEQ ID NO:828 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:
746-828 and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said mutation in said CsSP5G-4 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:829-SEQ ID NO:916 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:
829-916 and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said gRNA sequence comprises a 3' NGG Protospacer Adjacent Motif (PAM).
It is another object of the present invention to disclose the method as defined in any of the above, wherein said construct is introduced into the plant cells via Agrobacterium infiltration, virus based plasmids for delivery of the genome editing molecules or mechanical insertion such as polyethylene glycol (PEG) mediated DNA transformation, electroporation or gene gun biolistics.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said plant has decreased expression levels of at least one of said CsSP genes.
It is another object of the present invention to disclose the method as defined in any of the above, wherein the sequence of said expressed CsSP gene is selected from the group consisting of: SEQ
ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:9 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 said plant has decreased expression levels of at least one of said CsSP5G genes.
It is another object of the present invention to disclose the method as defined in any of the above, wherein the sequence of said expressed CsSP5G gene is selected from the group consisting of:
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID
NO:18, SEQ ID NO:20 and SEQ ID NO:21 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 said plant is semi-determinant.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said plant has determinant growth habit.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said plant flowers earlier than a corresponding wild type cannabis plant.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said plant exhibits improved earliness as compared to a corresponding wild type cannabis plant.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said plant exhibits suppressed sympodial shoot termination as compared to a corresponding wild type cannabis plant.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said plant exhibits similar sympodial shoot termination as compared to a corresponding wild type cannabis plant.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said plant exhibits suppressed or reduced day-length sensitivity as compared to a corresponding wild type cannabis plant.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said Cannabis plant is selected from the group of species that includes, but is not limited to, Cannabis sativa (C. sativa), C. indica, C. ruderalis and any hybrid or cultivated variety of the genus Cannabis.
It is another object of the present invention to disclose a modified Cannabis plant, plant part or plant cell produced by the method as defined in any of the above, wherein said plant does not comprise a transgene.
It is another object of the present invention to disclose a plant part, plant cell or plant seed of a plant produced by the method 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 produced 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 said plant genotype is obtainable by deposit under accession number with NCIMB
Aberdeen AB21 9YA, Scotland, UK or with ATCC.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said at least one domestication trait is selected from the group consisting of reduced flowering time, earliness, synchronous flowering, reduced day-length sensitivity, determinant or semi-determinant architecture, early termination of sympodial cycling, earlier axillary shoot flowering, compact growth habit, reduced height, reduced number of sympodial units, adaptation to mechanical harvest, higher harvest index and any combination thereof.
It is another object of the present invention to disclose the method as defined in any of the above, wherein said Cssp Cssp5g double mutant is characterized by having a more than additive effect on a trait selected from the group consisting of compactness, earlier axillary shoot flowering, earlier termination of sympodial cycling, harvest index and any combination thereof as compared to wild type and/or sp mutant Cannabis plants.
It is another object of the present invention to disclose an isolated nucleotide sequence having at least 75% sequence identity to a CsSP genomic nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4 and SEQ ID NO:7.
It is another object of the present invention to disclose an isolated nucleotide sequence having at least 75% sequence identity to a CsSP5G genomic nucleotide sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16 and SEQ ID NO:19.
It is another object of the present invention to disclose an isolated nucleotide sequence having at least 75% sequence identity to a CsSP nucleotide coding sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.
It is another object of the present invention to disclose an isolated nucleotide sequence having at least 75% sequence identity to a CsSP5G nucleotide coding sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17 and SEQ ID NO:20.
It is another object of the present invention to disclose an isolated amino acid sequence having at least 75% sequence similarity to a CsSP amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:9.
It is another object of the present invention to disclose an isolated amino acid sequence having at least 75% sequence similarity to a CsSP5G amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18 and SEQ ID NO:21.
It is another object of the present invention to disclose an isolated nucleotide sequence having at least 75% sequence identity to a CsSP-targeted gRNA nucleotide sequence as set forth in SEQ ID
NO:22-283.
It is another object of the present invention to disclose an isolated nucleotide sequence having at least 75% sequence identity to a CsSP5G-targeted gRNA nucleotide sequence as set forth in SEQ
ID NO:284-916.
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:22-126 and any combination thereof for targeted genome modification of Cannabis SP-1 (CsSP-1) 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:127-211 and any combination thereof for targeted genome modification of Cannabis SP-2 (CsSP-2) 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:212-283 and any combination thereof for targeted genome modification of Cannabis SP-3 (CsSP-3) 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:284-516 and any combination thereof for targeted genome modification of Cannabis SP5G-1 (CsSP5G-1) 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:517-745 and any combination thereof for targeted genome modification of Cannabis SP5G-2 (CsSP5G-2) 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:746-828 and any combination thereof for targeted genome modification of Cannabis SP5G-3 (CsSP5G-3) 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:829-916 and any combination thereof for targeted genome modification of Cannabis SP5G-4 (CsSP5G-4) allele.
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.
Fig. 1 is schematically presenting CRISPR/Cas9 mode of action as depicted by Xie, Kabin, and Yinong Yang. "RNA-guided genome editing in plants using a CRISPR¨Cas system."
Molecular plant 6.6 (2013): 1975-1983;
Fig. 2A-C is presenting Cannabis SP-1 gene (CsSP-1) (A) nucleotide genomic sequence as set forth in SEQ ID NO:1, (B) nucleotide coding sequence (CDS) set forth in SEQ ID
NO:2, and (C) amino acid sequence as set forth in SEQ ID NO:3;
Fig. 3A-C is presenting Cannabis SP-2 gene (CsSP-2) (A) nucleotide genomic sequence as set forth in SEQ ID NO:4, (B) nucleotide coding sequence (CDS) set forth in SEQ ID
NO:5, and (C) amino acid sequence as set forth in SEQ ID NO:6;
Fig. 4A-C is presenting Cannabis SP-3 gene (CsSP-3) (A) nucleotide genomic sequence as set forth in SEQ ID NO:7, (B) nucleotide coding sequence (CDS) set forth in SEQ ID
NO:8, and (C) amino acid sequence as set forth in SEQ ID NO:9;
Fig. 5A-C is presenting Cannabis SP5G-1 gene (CsSP5G-1) (A) nucleotide genomic sequence as set forth in SEQ ID NO:10, (B) nucleotide coding sequence (CDS) set forth in SEQ ID NO:11, and (C) amino acid sequence as set forth in SEQ ID NO:12;
Fig. 6A-C is presenting Cannabis SP5G-2 gene (CsSP5G-2) (A) nucleotide genomic sequence as set forth in SEQ ID NO:13, (B) nucleotide coding sequence (CDS) set forth in SEQ ID NO:14, and (C) amino acid sequence as set forth in SEQ ID NO:15;
Fig. 7A-C is presenting Cannabis SP5G-3 gene (CsSP5G-3) (A) nucleotide genomic sequence as set forth in SEQ ID NO:16, (B) nucleotide coding sequence (CDS) set forth in SEQ ID NO:17, and (C) amino acid sequence as set forth in SEQ ID NO:18;
Fig. 8A-C is presenting Cannabis SP5G-4 gene (CsSP5G-3) (A) nucleotide genomic sequence as set forth in SEQ ID NO:19, (B) nucleotide coding sequence (CDS) set forth in SEQ ID NO:20, and (C) amino acid sequence as set forth in SEQ ID NO:21;
Figs 9A-D is photographically presenting staining of Cannabis plants after transient GUS
transformation of (A) axillary buds (B) leaf (C) calli, and (D) cotyledons;
Figs 10A-C is presenting regenerated transformed Cannabis tissue;
Fig. 11 is photographically presenting PCR detection of Cas9 DNA in shoots of Cannabis plants transformed using biolistics; and Figs 12A-B is illustrating in vitro cleavage activity of CRISPR/Cas9; (A) a scheme of genomic area targeted for editing, and (B) a gel showing digestion of PCR amplicon containing RNP
complex of Cas9 and gene specific gRNA.
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 at least one improved domestication trait compared with wild type Cannabis, wherein said modified plant comprises at least one mutated Cannabis SELF PRUNING (SP) (CsSP) gene and/or at least one mutated Cannabis SELF PRUNING 5G (SP5G) (CsSP5G) gene. The present invention further provides methods for producing the aforementioned modified Cannabis plant using genome editing or other genome modification techniques.
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.
To date, there are no Cannabis varieties with improved domestication traits on the market.
Classical breeding programs dedicated to the end are virtually impossible due to limited genetic variation, legal constraints on import and export of genetic material and limited academic knowledge and gene banks involved is such projects. 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 cultivated Cannabis plants with improved yield and more specifically with determinate growth habit and with significantly reduced requirement for short days period needed for induction of flowering. Breeding using genome editing allows a precise and significantly shorter breeding process in order to achieve these goals with a much higher success rate. Thus genome editing, has the potential to generate improved varieties faster and at a lower cost.
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).
Legal limitations and outdated breeding techniques significantly hamper the efforts of generating new and improved Cannabis varieties fit for intensive agriculture.
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.
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 Fl hybrid seeds. These hybrids are generated by crossing homozygous parental lines.
The next step for the Cannabis industry is the adoption and use of hybrid seeds for propagation, which is common practice in the conventional seed industry (from field crops to vegetables). In addition, breeding for basic agronomic traits that are completely lacking in currently available Cannabis varieties (with an emphasis on day length sensitivity and compact growth habit) will significantly increase grower's productivity. This will allow growing and supplying high quality raw material for the Cannabis industry.
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.
In general, plants flowering is triggered by seasonal changes in day length.
However, day-length sensitivity in crops limits their geographical range of cultivation, and thus modification of the photoperiod response is critical for their domestication.
The present invention provides for the first time Cannabis plants with improved domestication traits such as plant architecture and day length sensitivity. The current invention discloses the generation of non-transgenic Cannabis plants with improved yield traits, using the genome editing technology, e.g., the CRISPR/Cas9 highly precise tool. The generated mutations can be introduced into elite or locally adapted Cannabis lines rapidly, with relatively minimal effort and investment.
Genome editing is an efficient and useful tool for increasing crop productivity, and there is particular interest in advancing manipulation of domestication genes in Cannabis wild species, which often have undesirable characteristics.
Genome-editing technologies, such as the clustered regularly interspaced short palindromic repeats (CRISPR)¨CRISPR-associated protein-9 nuclease (Cas9) (CRISPR¨Cas9) provide opportunities to address these deficiencies, with the aims of increasing quality and yield, improve adaptation and expand geographical ranges of cultivation.
A major obstacle for CRISPR¨Cas9 plant genome editing is lack of efficient tissue culture and transformation methodologies. The present invention achieves these aims and surprisingly provides transformed and regenerated Cannabis plants with modified desirable domestication genes.
Precise editing of SELFPRUNING (SP) and SELF-PRUNING 5G (SP5G) in wild Cannabis species, as disclosed by the present invention, should serve as a first step towards generating commercially cultivable lines, without causing an associated linkage drag on other useful traits.
Genome engineering could thus be applied for de novo domestication of wild species to create climate-smart crops.
To that end, guide RNAs (gRNAs) were designed for each of the target genes identified in Cannabis to induce mutations in SP and SP5G through genome editing.
It was found that simultaneous mutation of SP and SP5G converted the indeterminate architecture of Cannabis into determinate growth with early termination of sympodial cycling, thus resulting in compact Cannabis plants with intensive and almost synchronous flowering. It should be emphasized that the desirable architectures and flower production traits are produced in just one generation.
As used herein the term "about" denotes 25% of the defined amount or measure or value.
As used herein the term "similar" denotes a correspondence or resemblance range of about 20%, particularly 15%, more particularly about 10% and even more particularly about 5%.
As used herein the term "corresponding" generally means similar, analogous, like, alike, akin, parallel, identical, resembling or comparable. In further aspects it means having or participating in the same relationship (such as type or species, kind, degree, position, correspondence, or function).
It further means related or accompanying. In some embodiments of the present invention it refers to plants of the same Cannabis species or strain or variety or to sibling plant, or one or more individuals having one or both parents in common.
A "plant" as used herein refers to any plant at any stage of development, particularly a seed plant.
The term "plant" includes the whole plant or any parts or derivatives thereof, such as plant cells, seeds, plant protoplasts, plant cell tissue culture from which tomato plants can be regenerated, plant callus or calli, meristematic cells, microspores, embryos, immature embryos, pollen, ovules, anthers, fruit, flowers, leaves, cotyledons, pistil, seeds, seed coat, roots, root tips and the like.
The term "plant cell" used herein refers to a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in a form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.
The term "plant cell culture" as used herein means cultures of plant units such as, for example, protoplasts, regenerable cells, cell culture, cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development, leaves, roots, root tips, anthers, meristematic cells, microspores, flowers, cotyledons, pistil, fruit, seeds, seed coat or any combination thereof.
The term "plant material" or "plant part" used herein refers to leaves, stems, roots, root tips, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, seed coat, cuttings, cell or tissue cultures, or any other part or product of a plant or a combination thereof.
A "plant organ" as used herein means a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower, flower bud, or embryo.
The term "Plant tissue" as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture, protoplasts, meristematic cells, calli and any group of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.
As used herein, the term "progeny" or "progenies" refers in a non limiting manner to offspring or descendant plants. According to certain embodiments, the term "progeny" or "progenies" refers to plants developed or grown or produced from the disclosed or deposited seeds as detailed inter alia. The grown plants preferably have the desired traits of the disclosed or deposited seeds, i.e.
loss of function mutation in at least one CsSP gene or at least one CsSP5G
gene.
The term "Cannabis" refers hereinafter to a genus of flowering plants in the family Cannabaceae.
Cannabis is an annual, dioecious, flowering herb that includes, but is not limited to three different species, Cannabis sativa, Cannabis indica and Cannabis ruderalis. The term also refers to hemp.
Cannabis plants produce a group of chemicals called cannabinoids.
Cannabinoids, terpenoids, and other compounds are secreted by glandular trichomes that occur most abundantly on the floral calyxes and bracts of female Cannabis plants.
The term 'SELF-PRUNING' or 'SP' in the context of the present invention refers to a gene which encodes a flowering repressor that modulates sympodial growth. It is herein shown that mutations in the SP orthologue cause an acceleration of sympodial cycling and shoot termination. It is further acknowledged that the SELF PRUNING (SP) gene controls the regularity of the vegetative-reproductive switch along the compound shoot of, for example, tomato and thus conditions the 'determinate (sp/sp) and 'indeterminate' (SP) growth habits of the plant. SP
is a developmental regulator which is homologous to CENTRORADIALIS (CEN) from Antirrhinum and TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT) from Arabidopsis.
The present invention discloses that SP is a member of a gene family in Cannabis composed of at least three genes. The newly revealed Cannabis SP genes comprise CsSP-1, CsSP-2 and CsSP-3, encoded by genomic sequence as set forth in SEQ. ID. NO: 1, 4 and 7, coding sequence as set forth in SEQ. ID. NO:2, 5 and 8, and amino acid sequence as set forth in SEQ. ID.
NO:3, 6 and 9, respectively. According to main aspects of the present invention, genome editing- targeted mutation in at least one of the aforementioned CsSP genes, which reduces the functional expression of the gene, affect the plant sympodial growth habit which plays a key role in determining plant architecture.
The term 'SELF-PRUNING 5G' or 'SP5G' in the context of the present invention refers to a gene encoding florigen paralog and flowering repressor responsible for loss of day-length-sensitive flowering. It is further acknowledged that SP5G expression is induced to high levels during long days in wild species. It is within the scope of the current invention that CRISPR/Cas9-engineered mutations in SP5G cause rapid flowering and enhance the compact determinate growth habit of Cannabis plants, resulting in a quick burst of flower production that turns to an early yield. The findings of the current invention suggest that variation in SP and/or SP5G
facilitate the production of cultivated Cannabis strains with improved demonstration traits. The inventors of the present invention use gene editing techniques to rapidly improve yield traits in crop such as Cannabis.
As used herein the term "genetic modification" refers hereinafter to genetic manipulation or modulation, which is the direct manipulation of an organism's genes using biotechnology. It also refers to a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species, targeted mutagenesis and genome editing technologies to produce improved organisms. According to main embodiments of the present invention, modified Cannabis plants with improved domestication traits are generated using genome editing mechanism. This technique enables to achieve in planta modification of specific genes that relate to and/or control the flowering time and plant architecture in the Cannabis plant.
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 Cas = CRISPR-associated genes Indel = insertion and/or deletion Cas9, Csn I = a CRISPR-associated protein NHEJ = Non-Homologous End Joining containing two nuclease domains, that is PAM = Protospacer-Adjacent Motif programmed by small RNAs to cleave DNA
RuvC = an endonuclease domain named for crRNA = CRISPR RNA an E, col i protein involved in DNA
repair dCAS9 = nuclease-deficient Cas9 sgRNA = single guide RNA
DSB = Double-Stranded Break tracrRNA, trRNA = trans-activating crRNA
gRNA = guide RNA TALEN = Transcription-Activator Like HDR = Homology-Directed Repair Effector Nuclease HNH = an endonuclease domain named ZFN = Zinc-Finger Nuclease for characteristic histidine and asparagine residues 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 Csn 1) is required for gene silencing. Cas9 participates in the processing of crRNAs, and is responsible for the destruction of the target DNA. Cas9's function in both of these steps relies on the presence of two nuclease domains, a RuvC-like nuclease domain located at the amino terminus and a HNH-like nuclease domain that resides in the mid-region of the protein. To achieve site-specific DNA
recognition and cleavage, Cas9 is complexed with both a crRNA and a separate trans-activating crRNA (tracrRNA or trRNA), that is partially complementary to the crRNA. The tracrRNA is required for crRNA maturation from a primary transcript encoding multiple pre-crRNAs. This occurs in the presence of RNase III and Cas9.
Without wishing to be bound by theory, it is herein acknowledged that during the destruction of target DNA, the HNH and RuvC-like nuclease domains cut both DNA strands, generating double-stranded breaks (DSBs) at sites defined by a 20-nucleotide target sequence within an associated crRNA transcript. The HNH domain cleaves the complementary strand, while the RuvC domain cleaves the noncomplementary strand.
It is further noted that the double-stranded endonuclease activity of Cas9 also requires that a short conserved sequence, (2-5 nts) known as protospacer-associated motif (PAM), follows immediately 3- of the crRNA complementary sequence.
According to further aspects of the invention, a two-component system may be used by the current invention, combining trRNA and crRNA into a single synthetic single guide RNA
(sgRNA) for guiding targeted gene alterations.
It is further within the scope that Cas9 nuclease variants include wild-type Cas9, Cas9D10A and nuclease-deficient Cas9 (dCas9).
Reference is now made to Fig. 1 schematically presenting an example of CRISPR/Cas9 mechanism of action as depicted by Xie, Kabin, and Yinong Yang. "RNA-guided genome editing in plants using a CRISPR¨Cas system." Molecular plant 6.6 (2013): 1975-1983.
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 an NGG motif (called protospacer-adjacent motif or PAM) that follows the base-pairing region in the complementary strand of the targeted DNA.
The term "meganucleases" as used herein refers hereinafter to endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA
sequences of 12 to 40 base pairs); as a result this site generally occurs only once in any given genome. Meganucleases are therefore considered to be the most specific naturally occurring restriction enzymes.
The term "protospacer adjacent motif" or "PAM' as used herein refers hereinafter to a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. PAM is a component of the invading virus or plasmid, but is not a component of the bacterial CRISPR locus. PAM is an essential targeting component which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR
locus from being targeted and destroyed by nuclease.
The term "Next-generation sequencing" or "NGS" as used herein refers hereinafter to massively, parallel, high- throughput or deep sequencing technology platforms that perform sequencing of millions of small fragments of DNA in parallel. Bioinformatics analyses are used to piece together these fragments by mapping the individual reads to the reference genome.
The term "gene knockdown" as used herein refers hereinafter to an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur through genetic modification, i.e. targeted genome editing or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript. The reduced expression can be at the level of RNA or at the level of protein. It is within the scope of the present invention that the term gene knockdown also refers to a loss of function mutation and /or gene knockout mutation in which an organism's genes is made inoperative or nonfunctional.
The term "gene silencing" as used herein refers hereinafter to the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation. In certain aspects of the invention, gene silencing is considered to have a similar meaning as gene knockdown. When genes are silenced, their expression is reduced. In contrast, when genes are knocked out, they are completely not expressed. Gene silencing may be considered a gene knockdown mechanism since the methods used to silence genes, such as RNAi, CRISPR, or siRNA, generally reduce the expression of a gene by at least 70% but do not completely eliminate it.
The term "loss of function mutation" as used herein refers to a type of mutation in which the altered gene product lacks the function of the wild-type gene. A synonyms of the term included within the scope of the present invention is null mutation.
The term "microRNAs" or "miRNAs" refers hereinafter to small non-coding RNAs that have been found in most of the eukaryotic organisms. They are involved in the regulation of gene expression at the post-transcriptional level in a sequence specific manner.
MiRNAs are produced from their precursors by Dicer-dependent small RNA biogenesis pathway. MiRNAs are candidates for studying gene function using different RNA-based gene silencing techniques. For example, artificial miRNAs (amiRNAs) targeting one or several genes of interest is a potential tool in functional genomics.
The term "in planta" means in the context of the present invention within the plant or plant cells.
More specifically, it means introducing CRISPR/Cas complex into plant material comprising a tissue culture of several cells, a whole plant, or into a single plant cell, without introducing a foreign gene or a mutated gene. It also used to describe conditions present in a non-laboratory environment (e.g. in vivo).
The term 'sympodial growth' as used herein refers to a type of bifurcating branching pattern where one branch develops more strongly than the other, resulting in the stronger branches forming the primary shoot and the weaker branches appearing laterally. A sympodium, also referred to as a sympode or pseudaxis, is the primary shoot, comprising the stronger branches, formed during sympodial growth. In some aspects of the present invention, sympodial growth occurs when the apical meristem is terminated and growth is continued by one or more lateral meristems, which repeat the process. The apical meristem may be consumed to make an inflorescence or other determinate structure, or it may be aborted.
It is further within the scope of the current invention that the shoot section between two successive inflorescences is called the 'sympodium', and the number of leaf nodes per sympodium is referred to as the 'sympodial index' (spi). The first termination event activates the 'sympodial cycle'. In sympodial plants, the apparent main shoot consists of a reiterated array of 'sympodial units'. A
mutant sp gene accelerates the termination of sympodial units but does not change the sympodial habit. The result is a progressive reduction in the number of vegetative nodes between inflorescences in a pattern that depends on light intensity and genetic background.
The term "earliness" refers hereinafter to early flowering and/or rapid transition from the vegetative to reproductive stages, or reduced 'time to initiation of flowering' and more generally to earlier completion of the life-cycle.
The term 'reduced flowering time' as used herein refers to time to production of first inflorescence. Such a trait can be evaluated or measured, for example, with reference to the number of leaves produced prior to appearance of the first inflorescence.
The term 'harvest index' can be herein defined as the total yield per plant weight.
The term 'day length' or 'day length sensitivity' as used in the context of the present invention generally refers to photoperiodism, which is the physiological reaction of organisms to the length of day or night. Photoperiodism can also be defined as the developmental responses of plants to the relative lengths of light and dark periods. Plants are classified under three groups according to the photoperiods: short-day plants, long-day plants, and day-neutral plants.
Photoperiodism affects flowering by inducing the shoot to produce floral buds instead of leaves and lateral buds. It is within the scope of the present invention that Cannabis is included within the short-day facultative plants. The Cannabis plants of the present invention are genetically modified so as to exhibit loss of day-length sensitivity, which is highly desirable agronomical trait enabling enhanced yield of the cultivated crop.
The term 'determinate' or 'determinate growth' as used herein refers to plant growth in which the main stem ends in an inflorescence or other reproductive structure (e.g. a bud) and stops continuing to elongate indefinitely with only branches from the main stem having further and similarly restricted growth. It also refers to growth characterized by sequential flowering from the central or uppermost bud to the lateral or basal buds. It further means naturally self-limited growth, resulting in a plant of a definite maximum size.
The term 'indeterminate' or 'indeterminate growth' as used herein refers to plant growth in which the main stem continues to elongate indefinitely without being limited by a terminal inflorescence or other reproductive structure. It also refers to growth characterized by sequential flowering from the lateral or basal buds to the central or uppermost buds.
The term "orthologue" as used herein refers hereinafter to one of two or more homologous gene sequences found in different species.
The term "functional variant" or "functional variant of a nucleic acid or amino acid sequence" as used herein, for example with reference to SEQ ID NOs: 1, 4 or 7 refers to a variant of a sequence or part of a sequence which retains the biological function of the full non-variant allele (e.g. CsSP or CsSP5G allele) and hence has the activity of SP or SP5G
expressed gene or protein. A functional variant also comprises a variant of the gene of interest encoding a polypeptide which has sequence alterations that do not affect function of the resulting protein, for example, in non-conserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example, in non-conserved residues, to the wild type nucleic acid or amino acid sequences of the alleles as shown herein, and is biologically active.
The term "variety" or "cultivar" used herein means a group of similar plants that by structural features and performance can be identified from other varieties within the same species.
The term "allele" used herein means any of one or more alternative or variant forms of a gene or a genetic unit at a particular locus, all of which alleles relate to one trait or characteristic at a specific locus. In a diploid cell of an organism, alleles of a given gene are located at a specific location, or locus (loci plural) on a chromosome. Alternative or variant forms of alleles may be the result of single nucleotide polymorphisms, insertions, inversions, translocations or deletions, or the consequence of gene regulation caused by, for example, by chemical or structural modification, transcription regulation or post-translational modification/regulation. An allele associated with a qualitative trait may comprise alternative or variant forms of various genetic units including those mat are identical or associated with a single gene or multiple genes or their products or even a gene disrupting or controlled by a genetic factor contributing to the phenotype represented by the locus.
According to further embodiments, the term "allele" designates any of one or more alternative forms of a gene at a particular locus. Heterozygous alleles are two different alleles at the same locus. Homozygous alleles are two identical alleles at a particular locus. A
wild type allele is a naturally occurring allele. In the context of the current invention, the term allele refers to the three identified SP genes in Cannabis, namely CsSP-1, CsSP-2 and CsSP-3 having the genomic nucleotide sequence as set forth in SEQ ID NOs: 1, 4 and 7, respectively. As well as to four identified SP5G genes in Cannabis, namely CsSP5G-1, CsSP5G-2, and CsSP5G-4 having the genomic nucleotide sequence as set forth in SEQ ID NOs: 10, 13, 18 and 19, 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.
In specific embodiments, the Cannabis plants of the present invention comprise homozygous configuration of at least one of the mutated Cssp genes (i.e. Cssp-1, Cssp-2 and Cssp-3) and/or the mutated Cssp5g genes (i.e. Cssp5g-1, Cssp5g-2, Cssp5g-3 and Cssp5g-4).
Conversely, as used herein, the term "heterozygous" means a genetic condition or configuration existing when two different or unlike alleles reside at a specific locus, but are positioned individually on corresponding pairs of homologous chromosomes in the cell of a diploid organism.
As used herein, the phrase "genetic marker" or "molecular marker" or "biomarker" refers to a feature in an individual's genome e.g., a nucleotide or a polynucleotide sequence that is associated with one or more loci or trait of interest In some embodiments, a genetic marker is polymorphic in a population of interest, or the locus occupied by the polymorphism, depending on context.
Genetic markers or molecular markers include, for example, single nucleotide polymorphisms (SNPs), indels (i.e. insertions deletions), simple sequence repeats (SSRs), restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAFDs), cleaved amplified polymorphic sequence (CAPS) markers, Diversity Arrays Technology (DArT) markers, and amplified fragment length polymorphisms (AFLPs) or combinations thereof, among many other examples such as the DNA sequence per se. Genetic markers can, for example, be used to locate genetic loci containing alleles on a chromosome that contribute to variability of phenotypic traits. The phrase "genetic marker" or "molecular marker" or "biomarker" can also refer to a polynucleotide sequence complementary or corresponding to a genomic sequence, such as a sequence of a nucleic acid used as a probe or primer.
As used herein, the term "germplasm" refers to the totality of the genotypes of a population or other group of individuals (e.g., a species). The term "germplasm" can also refer to plant material;
e.g., a group of plants that act as a repository for various alleles. Such germplasm genotypes or populations include plant materials of proven genetic superiority; e.g., for a given environment or geographical area, and plant materials of unknown or unproven genetic value;
that are not part of an established breeding population and that do not have a known relationship to a member of the established breeding population.
The terms "hybrid", "hybrid plant" and "hybrid progeny" used herein refers to an individual produced from genetically different parents (e.g., a genetically heterozygous or mostly heterozygous individual).
As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. The term further refers hereinafter to the amount of characters which match exactly between two different sequences. Hereby, gaps are not counted and the measurement is relational to the shorter of the two sequences.
It is further within the scope that the terms "similarity" and "identity"
additionally refer to local homology, identifying domains that are homologous or similar (in nucleotide and/or amino acid sequence). It is acknowledged that bioinformatics tools such as BLAST, SSEARCH, FASTA, and HMMER calculate local sequence alignments which identify the most similar region between two sequences. For domains that are found in different sequence contexts in different proteins, the alignment should be limited to the homologous domain, since the domain homology is providing the sequence similarity captured in the score. According to some aspects the term similarity or identity further includes a sequence motif, which is a nucleotide or amino-acid sequence pattern that is widespread and has, or is conjectured to have, a biological significance. Proteins may have a sequence motif and/or a structural motif, a motif formed by the three-dimensional arrangement of amino acids which may not be adjacent.
As used herein, the terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or RNA
molecules, and analogs of the DNA or RNA generated using nucleotide analogs.
It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term "gene", "allele" or "gene sequence" is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in combination with regulatory sequences. Thus, according to the various aspects of the invention, genomic DNA, cDNA or coding DNA may be used. In one embodiment, the nucleic acid is cDNA or coding DNA.
The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.
According to other aspects of the invention, a "modified" or a "mutant" plant is a plant that has been altered compared to the naturally occurring wild type (WT) plant.
Specifically, the endogenous nucleic acid sequences of each of the SP or SP5G homologs in Cannabis (nucleic acid sequences CsSP-1, CsSP-2 and CsSP-3; and/or CsSP5G-1, CsSP5G-2, CsSP5G-3 and CsSP5G-4) have been altered compared to wild type sequences using mutagenesis and/or genome editing methods as described herein. This causes inactivation of the endogenous SP
and/or SP5G genes and thus disables SP and/or SP5G function. Such plants have an altered phenotype and show improved domestication traits such as determinant plant architecture, synchronous and/or early flowering and loss of day length sensitivity compared to wild type plants.
Therefore, the improved domestication phenotype is conferred by the presence of at least one mutated endogenous Cssp and /or Cssp5g gene in the Cannabis plant genome which has been specifically targeted using genome editing technique.
According to further aspects of the present invention, the at least one improved domestication trait 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 CsSP-1, CsMSP-2 and CsSP-3; and CsSP5G-1, CsMSP5G-2, CsSP5G-3 and CsSP5G-4. Mutant sp and sp5g nucleic acid sequences use non-capitalization.
Cannabis plants of the invention are modified plants compared to wild type plants which comprise and express mutant Cssp and/or Cssp5g alleles.
It is further within the scope of the current invention that sp and/or sp5g mutations that down-regulate or disrupt functional expression of the wild-type SP and/or SP5G
sequence respectively, may be recessive, such that they are complemented by expression of a wild-type sequence.
It is further noted that a wild type Cannabis plant is a plant that does not have any mutant sp and/or sp5g alleles.
Main aspects of the invention involve targeted mutagenesis methods, specifically genome editing, and exclude embodiments that are solely based on generating plants by traditional breeding methods. In a further embodiment of the current invention, as explained herein, the improved domestication at least one trait is not due to the presence of a transgene.
The inventors have generated mutant Cannabis lines with mutations inactivating at least one CsSP
and/or CsSP5G homoeoallele which confer heritable improved domestication trait(s). In this way no functional CsSP and/or CsSP5G protein is made. Thus, the invention relates to these mutant Cannabis lines and related methods.
It is further within the scope of the present invention that breeding Cannabis cultivars with mutated sp allele enables the mechanical harvest of the plant. According to a further aspect of the present invention, loss of SP function results in compact Cannabis plants with reduced height, reduced number of sympodial units and determinate growth when compared with WT
Cannabis.
According to a main aspect of the present invention, modifying Cannabis shoot architecture by selection for mutations in florigen flowering pathway genes allowed major improvements in plant architecture and yield. In particular, a mutation in the antiflorigen SELFPRUNING (SP) gene (sp classic) provided compact 'determinate' growth that translated to a burst of flowers, thereby enabling largescale field production.
According to one embodiment of the present invention, SELFPRUNING (SP) homologues and related florigen family members such as SELF-PRUNING 5G (SP5G) have been identified in both genome and transcriptome in Cannabis.
The work inter alia described has important implications. The results have shown that CRISPR/Cas9 can be used to create heritable mutations in florigen pathway family members that result in desirable phenotypic effects.
To edit multiple domestication genes simultaneously and stack the resulting allelic variants, on option is that several gRNAs can be assembled to edit several genes into one construct, by using the Csy4 multi-gRNA system. The construct is then transformed via an appropriate vector into several wild-Cannabis accessions.
It is further within the scope of the current invention that Cannabis SP
genes, namely CsSP-1, CsSP-2 and CsSP-3 having genomic nucleotide sequence as set forth in SEQ. ID.
NO.:1, 4 and 7 respectively, were targeted using guide RNAs as set forth in SEQ ID NO:22-126, 127-211 and 212-283, respectively. Several mutated alleles have been identified. Notably, the plants with mutated sp alleles were more compact than the wild type plants lacking the mutated allele.
To identify other targets for plant architecture modification without negative effects on productivity, homologues of SELF-PRUNING 5G (SP5G) (Cs-SP5G), another florigen repressor, were identified in Cannabis. Cannabis SP5G genes, namely CsSP5G-1, CsSP5G-2, CsSP5G-3 and CsSP5G-4 having genomic nucleotide sequence as set forth in SEQ. ID. NO.:10, 13, 16 and 19 respectively, were targeted using guide RNAs as set forth in SEQ ID NO:284-516, 517-745, 746-828 and 829-916, respectively. Several mutated alleles have been identified.
It is herein acknowledged that SP5G represses flowering predominantly in primary and canonical axillary shoots.
According to one embodiment of the present invention, SP5G was shown to control primary and canonical axillary shoot flowering time and to contribute to controlling day-length sensitivity. In other words, reduced SP5G activity was shown to mitigate day-length sensitivity.
According to a further embodiment of the present invention, CRISPR¨Cas9-induced null sp5g mutations in Cannabis eliminate day-length sensitivity and causing faster primary and axillary shoot flowering.
The present invention discloses that the combination of both mutations sp5g and sp results in faster-flowering Cannabis plants with typical sp determinate growth. Such Cannabis plants could have agronomic value, particularly for the desirable trait of earliness of yield.
According to specific aspects of the present invention, Cs-sp5g/ sp double mutants are not simply additive but substantially more compact than mutant sp Cannabis plants owing to faster axillary shoot flowering and earlier termination of sympodial cycling.
According to further aspects of the present invention, compared with wild type and/or sp Cannabis plants, Cs-sp5g/ sp double mutant plants provide a more rapid flowering burst, and reach final harvest sooner.
It is further within the scope of the present invention that the harvest index (defined as the total yield per plant weight) of the Cs-sp5g/ sp double mutant plants is higher than that for wild type and/or sp mutant Cannabis plants.
According to a further specific aspect of the present invention, large-scale Cannabis production based on sp determinate growth may be achieved only in the absence of day-length sensitivity, i.e.
by loss of function mutation in at least one of Cssp5g-1, Cssp5g-2, Cssp5g-3 or Cssp5g-4 as set forth in SEQ ID NO.: 10, 13 16 and 19, respectively.
It is further within the scope of the present invention that targeting SP5G
homologs and/or other diurnally regulated CENTRORADIALIS/TERMINAL FLOWER 1/SELF-PRUNING (CETS) genes may allow immediate customization of day-length sensitivity in Cannabis elite germplasm to expand the geographical range of cultivation, and could serve as a first step toward engineering the domestication of wild Cannabis species with agricultural potential.
The loss of function mutation may be a deletion or insertion ("indels") with reference the wild type CsSP and/or CsSP5G allele sequence. The deletion may comprise 1-20 or more nucleotides, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18 or 20 nucleotides or more in one or more strand. The insertion may comprise 1-20 or more nucleotides, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18 or 20 or more nucleotides in one or more strand.
The plant of the invention includes plants wherein the plant is heterozygous for the each of the mutations. In a preferred embodiment however, the plant is homozygous for the mutations.
Progeny that is also homozygous can be generated from these plants according to methods known in the art.
It is further within the scope that variants of a particular CsSP and/or CsSP5G 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 CsSP and/or CsSP5G nucleotide sequence of the CsSP and/or CsSP5G allele as shown in SEQ ID
NO 1, 4 or 7; and/or SEQ ID NO 10, 13, 18 or 19, respectively. Sequence alignment programs to determine sequence identity are well known in the art.
Also, the various aspects of the invention encompass not only a CsSP and/or CsSP5G nucleic acid sequence or amino acid sequence, but also fragments thereof. By "fragment" is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence of the protein encoded thereby. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein, in this case improved domestication trait.
According to a further embodiment of the invention, the herein newly identified Cannabis SP
and/or SP5G locus (CsSP and/or CsSP5G) have been targeted using the double 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 Cannabis plants with at least one improved domestication trait, allows the modification of predetermined specific DNA sequences without introducing foreign DNA into the genome by GMO techniques. According to one embodiment of the present invention, this is achieved by combining the Cas nuclease (e.g. Cas9, Cpfl and the like) with a predefined guide RNA molecule (gRNA). The gRNA is complementary to a specific DNA sequence targeted for editing in the plant genome and which guides the Cas nuclease to a specific nucleotide sequence (for example see Fig. 1). The predefined gene specific gRNA's are cloned into the same plasmid as the Cas gene and this plasmid is inserted into plant cells. Insertion of the aforementioned plasmid DNA can be done, but not limited to, using different delivery systems, biological and/or mechanical, e.g. Agrobacterium infiltration, virus based plasmids for delivery of the genome editing molecules and mechanical insertion of DNA (PEG mediated DNA
transformation, biolistics, etc.).
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 I base pair deletion. As a result of this base pair deletion the gene coding sequence is disrupted and the translation of the encoded protein is compromised either by a premature stop codon or disruption of a functional or structural property of the protein.
Thus DNA is cut by the Cas9 protein and re-assembled by the cell's DNA repair mechanism.
It is further within the scope that improved domestication traits in Cannabis plants is herein produced by generating gRNA with homology to a specific site of predetermined genes in the Cannabis genome i.e. SP and/or SP5G genes, sub cloning this gRNA into a plasmid containing the Cas9 gene, and insertion of the plasmid into the Cannabis plant cells. In this way site specific mutations in the SP and/or SP5G genes are generated thus effectively creating non-active molecules, resulting in loss of day length sensitivity, reduced flowering time and determinant growth habit of the genome edited plant.
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 I
Production of Cannabis plants with improved domestication traits by targeted genome editing Production of Cannabis lines with mutated sp and/or sp5g gene may be achieved by at least one of the following breeding/cultivation schemes:
Scheme I:
= line stabilization by self pollination = Generation of F6 parental lines = Genome editing of parental lines = Crossing edited parental lines to generate an Fl hybrid plant Scheme 2:
= Identifying genes/alleles 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 about 6 self-crossing (6 generations) and done through a single seed descent (SSD) approach.
Fl hybrid seed production: Novel hybrids are produced by crosses between different Cannabis strains.
According to a further aspect of the current invention, shortening line stabilization is performed by Doubled Haploids (DH). More specifically, the CRISPR-Cas9 system is transformed into microspores to achieve DH homozygous parental lines. A doubled haploid (DH) is a genotype formed when haploid cells undergo chromosome doubling. Artificial production of doubled haploids is important in plant breeding. It is herein acknowledged that conventional inbreeding procedures take about six generations to achieve approximately complete homozygosity, whereas doubled haploidy achieves it in one generation.
It is within the scope of the current invention that genetic markers specific for Cannabis are developed and provided by the current invention:
= Sex markers- molecular markers are used for identification and selection of female vs male plants in the herein disclosed breeding program = Genotyping markers- germplasm used in the current invention is genotyped using molecular markers, in order to allow a more efficient breeding process and identification of the SP and/or SP5G editing event.
It is further within the scope of the current invention that allele and genetic variation is analysed for the Cannabis strains used.
Reference is now made to optional stages that have been used for the production of mutated SP
and/or SP5G Cannabis plants by genome editing:
Stage 1: Identifying Cannabis sativa (C. sativa), C. indica and C. ruderalis SP and SP5G
orthologues.
Three SP orthologues have herein been identified in Cannabis sativa (C.
sativa), C. indica and C.
ruderalis, namely CsSP-1, CsSP-2 and CsSP-3. These homologous genes have been sequenced and mapped. CsSP-1 has been mapped to CM011605.1:71328589-71330978 and has a genomic sequence as set forth in SEQ ID NO:1 (see Fig. 2A). The CsSP-1 gene has a coding sequence as set forth in SEQ ID NO:2 (see Fig. 2B) and it encodes an amino acid sequence as set forth in SEQ
ID NO:3 (see Fig. 2C).
CsSP-2 has been mapped to CM011605.1:25325478-25326672 and has a genomic sequence as set forth in SEQ ID NO:4 (see Fig. 3A). The CsSP-2 gene has a coding sequence as set forth in SEQ
ID NO:5 (see Fig. 3B) and it encodes an amino acid sequence as set forth in SEQ ID NO:6 (see Fig. 3C).
CsSP-3 has been mapped to CM011608.1:9602945-9603900 and has a genomic sequence as set forth in SEQ ID NO:7 (see Fig. 4A). The CsSP-3 gene has a coding sequence as set forth in SEQ
ID NO:8 (see Fig. 4B) and it encodes an amino acid sequence as set forth in SEQ ID NO:9 (see Fig. 4C).
Four SP5G orthologues have herein been identified in Cannabis sativa (C.
sativa), C. indica and C. ruderalis, namely CsSP5G-1, CsSP5G-2, CsSP5G-3 and CsSP5G-4. These homologous genes have been sequenced and mapped. CsSP5G-1 has been mapped to CM011610.1:5735300-and has a genomic sequence as set forth in SEQ ID NO:10 (see Fig. 5A). The CsSP-1 gene has a coding sequence as set forth in SEQ ID NO:11 (see Fig. 5B) and it encodes an amino acid sequence as set forth in SEQ ID NO:12 (see Fig. 5C).
CsSP5G-2 has been mapped to CM011610.1:6032638-6035504 and has a genomic sequence as set forth in SEQ ID NO:13 (see Fig. 6A). The CsSP5G-2 gene has a coding sequence as set forth in SEQ ID NO:14 (see Fig. 6B) and it encodes an amino acid sequence as set forth in SEQ ID
NO:15 (see Fig. 6C).
CsSP5G-3 has been mapped to CM011607.1:79899046-79900718 and has a genomic sequence as set forth in SEQ ID NO:16 (see Fig. 7A). The CsSP5G-3 gene has a coding sequence as set forth in SEQ ID NO:17 (see Fig. 7B) and it encodes an amino acid sequence as set forth in SEQ ID
NO:18 (see Fig. 7C).
CsSP5G-4 has been mapped to CM011614.1:9255475-9256908 and has a genomic sequence as set forth in SEQ ID NO:19 (see Fig. 8A). The CsSP5G-4 gene has a coding sequence as set forth in SEQ ID NO:20 (see Fig. 8B) and it encodes an amino acid sequence as set forth in SEQ ID
NO:21 (see Fig. 8C).
Stage 2: Designing and synthesizing gRNA molecules corresponding to the sequence targeted for editing, i.e. sequences of each of the genes CsSP-1, CsSP-2 and CsSP-3; and CsSP5G-1, CsSP5G-2, CsSP5G-3 and CsSP5G-4. 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 SP and/or SP5G
homologues of different Cannabis strains.
Reference is now made to Tables 1-3 presenting gRNA molecules targeted for silencing CsSP-1, CsSP-2 and CsSP-3, respectively; and Tables 4-7 presenting gRNA molecules targeted for silencing CsSP5G-1, CsSP5G-2, CsSP5G-3 and CsSP5G-4 respectively. The term 'PAM refers hereinafter to Protospacer Adjacent Motif, which is a 2-6 base pair DNA
sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR
bacterial adaptive immune system.
Table 1: gRNA sequences targeted for CsSP-1 Position on Strand Sequence PAM Efficiency SEQ. ID.
SEQ. ID. Score NO.
NO:1 831 1 TATATATATTAAGACTACGT AGG 69.79075 22 868 -1 ATAAGTGTGTAAGAGGCTCG TGG 66.70092 23 875 -1 TTATTATATAAGTGTGTAAG AGG 54.32121 24 964 -1 GCATGCATGCATGCATGCAT GGG 48.94366 25 965 -1 TGCATGCATGCATGCATGCA TGG 51.6943 26 1049 1 TTGAAGAAAAGAGCAGCCAC AGG 56.36067 27 1052 1 AAGAAAAGAGCAGCCACAGG AGG 61.86857 28 1054 -1 AAATGACCTTGGTCCTCCTG TGG 59.43912 29 1059 1 GAGCAGCCACAGGAGGACCA AGG 64.32062 30 1065 -1 TTTGCTACCCAAAATGACCT TGG 60.28351 31 1068 1 CAGGAGGACCAAGGTCATTT TGG 26.32254 32 1069 1 AGGAGGACCAAGGTCATTTT GGG 28.83447 33 1080 1 GGTCATTTTGGGTAGCAAAG AGG 69.55719 34 1109 1 TTGAAACGCTCTCTCGATGA AGG 54.24251 35 1112 1 AAACGCTCTCTCGATGAAGG TGG 60.45312 36 1129 -1 ACAGAAGAGAAGACAAACAG TGG 69.59222 37 1172 -1 GACCAAACATAGGAATACAT AGG 61.3781 38 1181 1 AACCTATGTATTCCTATGTT TGG 36.10233 39 1182 -1 GAAATGCCACGACCAAACAT AGG 59.3397 40 1187 1 TGTATTCCTATGTTTGGTCG TGG 47.61837 41 1224 -1 TTAAATGAATTAATATATGT AGG 44.76165 42 1324 -1 GGAACAACTGATGTGTCATT TGG 39.45477 43 1338 1 AATGACACATCAGTTGTTCC TGG 39.81732 44 1345 -1 AGGATAGTGACAGACATTCC AGG 51.58159 45 1365 -1 TTTGTTGTTGAAATAATTGC AGG 31.23807 46 1424 -1 GCAAAAGGCAAAATTTAAAA TGG 25.80444 47 1439 -1 TTTAATCAAACTTAAGCAAA AGG 41.06861 48 1478 1 TATTTTGTTAAATTATAAAT TGG 18.56233 49 1528 1 TAGTATATATATATTCTGAT TGG 48.76865 50 1572 1 GCAATAATAATTTAATGTAT AGG 42.88178 51 1631 1 TTAAAAAATCTTCTTCAAGT TGG 45.32853 52 1755 -1 TTATCTAGGGTTATAATAGT TGG 38.51395 53 1768 -1 GTATACACATATATTATCTA GGG 52.79043 54 1769 -1 TGTATACACATATATTATCT AGG 40.10661 55 1881 1 TCATTCAAAAGTAAAATAAT AGG 21.75194 56 1882 1 CATTCAAAAGTAAAATAATA GGG 35.40844 57 1890 1 AGTAAAATAATAGGGTAATT AGG 22.83575 58 1910 -1 TAGTAATATCAAAATTTTAA GGG 28.45209 59 1911 -1 TTAGTAATATCAAAATTTTA AGG 17.72295 60 1990 1 TATTGAGATTGTTAAATTTA AGG 6.261185 61 2063 -1 TTGGTAGATATTAAAATTTA GGG 23.20311 62 2064 -1 TTTGGTAGATATTAAAATTT AGG 24.70598 63 2082 -1 GAAAGTTCAGAGGCATGATT TGG 33.64684 64 2092 -1 TAACATGGAGGAAAGTTCAG AGG 66.62639 65 2104 -1 AAAAAAACTAATTAACATGG AGG 67.7548 66 2107 -1 AGTAAAAAAACTAATTAACA TGG 49.05741 67 2126 1 ATTAGTTTTTTTACTAAAAT TGG 20.24538 68 2154 -1 CTCTAAACTATTGAGATTGT TGG 27.96835 69 2214 1 ATAATATAATATATATATAT TGG 33.84373 70 2365 1 GACTAATTAATGATCATGTG TGG 67.15733 71 2382 -1 CTTAAGGGAATATATGCAAC TGG 42.83721 72 2397 -1 ATAATAATAAGTATACTTAA GGG 44.75504 73 2398 -1 TATAATAATAAGTATACTTA AGG 31.14549 74 2426 -1 TATATAATATATATATATAG GGG 51.83062 75 2427 -1 ATATATAATATATATATATA GGG 25.09287 76 2428 -1 TATATATAATATATATATAT AGG 21.06967 77 2527 -1 TGATCGATCGTTAGGGGGGA AGG 55.11339 78 2531 -1 ATCTTGATCGATCGTTAGGG GGG 69.00871 79 2532 -1 CATCTTGATCGATCGTTAGG GGG 62.62737 80 2533 -1 ACATCTTGATCGATCGTTAG GGG 62.65508 81 2534 -1 CACATCTTGATCGATCGTTA GGG 48.84918 82 2535 -1 ACACATCTTGATCGATCGTT AGG 40.46896 83 2692 1 ATATAAGAAATATGTATGAT CGG 52.65835 84 2703 1 ATGTATGATCGGAATTTTAT TGG 27.65155 85 2740 1 TCAATATTATATATAGTATT AGG 34.80095 86 2818 1 AACTTATATATGAATATTAT AGG 22.13664 87 2872 -1 TGTGATGACAAAAGTTTTTT GGG 25.03809 88 2873 -1 GTGTGATGACAAAAGTTTTT TGG 30.69267 89 2982 -1 ATTTGAGGGAGCATTTACAC TGG 63.0929 90 2996 -1 CCCTAGTGATCCTTATTTGA GGG 33.88568 91 2997 1 TGTAAATGCTCCCTCAAATA AGG 33.96309 92 2997 -1 GCCCTAGTGATCCTTATTTG AGG 41.63481 93 3006 1 TCCCTCAAATAAGGATCACT AGG 53.28568 94 3007 1 CCCTCAAATAAGGATCACTA GGG 61.56296 95 3012 1 AAATAAGGATCACTAGGGCC AGG 51.65425 96 3019 -1 ATGACTGATCCTGATGTTCC TGG 34.06368 97 3021 1 TCACTAGGGCCAGGAACATC AGG 43.87242 98 3044 -1 TGTTGTAAATAATATTAATT AGG 20.85671 99 3141 -1 CATGAGATCCTTTTTCACAC TGG 54.53186 100 3144 1 AATTATTACCAGTGTGAAAA AGG 40.03344 101 3167 -1 CCAAAAGTTGAGGTTCATGG TGG 71.52152 102 3170 -1 AAGCCAAAAGTTGAGGTTCA TGG 48.47928 103 3177 -1 TACTATCAAGCCAAAAGTTG AGG 51.74276 104 3178 1 CCACCATGAACCTCAACTTT TGG 32.68026 105 3195 1 TTTTGGCTTGATAGTAATTG TGG 54.13253 106 3199 1 GGCTTGATAGTAATTGTGGA AGG 53.09516 107 3224 -1 TCAAACAAGAAAGTCTACAA TGG 60.73706 108 3260 1 TGTAAGTCACTGTAAATTTT AGG 23.13386 109 3261 1 GTAAGTCACTGTAAATTTTA GGG 24.03811 110 3265 1 GTCACTGTAAATTTTAGGGT TGG 47.51408 111 3291 -1 TGGAAGAGTGATAGGAGATG TGG 64.74459 112 3299 -1 CTTATTATTGGAAGAGTGAT AGG 48.45991 113 3311 -1 TCAGATAATCCTCTTATTAT TGG 18.16136 114 3313 1 ATCACTCTTCCAATAATAAG AGG 52.38055 115 3342 -1 ATTTTTTTTTAAAAAATTAA TGG 17.24296 116 3444 -1 TGTGTCCTCTCTATAAATAA GGG 40.2693 117 3445 -1 TTGTGTCCTCTCTATAAATA AGG 27.63962 118 3450 1 TATATCCCTTATTTATAGAG AGG 51.01762 119 3488 1 GAGATGTTTAATATATTAAG TGG 50.52582 120 3489 1 AGATGTTTAATATATTAAGT GGG 41.77524 121 3490 1 GATGTTTAATATATTAAGTG GGG 62.38698 122 3534 1 AACTAAAATAATTTAAATGA AGG 46.81791 123 3549 1 AATGAAGGAATAGTATAAAA AGG 26.26061 124 3550 1 ATGAAGGAATAGTATAAAAA GGG 42.43128 125 3567 1 AAAGGGTCATCATCTTCAAA AGG 43.17344 126 Table 2: gRNA sequences targeted for CsSP-2 Position Strand Sequence PAM Efficiency SEQ. ID.
on SEQ. Score NO.
ID. NO:4 743 -1 TTTTTATCTTATCATGTTGT TGG 25.41093 127 773 -1 TGTAAGAGAACAGCTCTTAA GGG 32.52846 128 774 -1 TTGTAAGAGAACAGCTCTTA AGG 31.39567 129 812 -1 ACTAGTTTTAGTACGATGAT TGG 52.94793 130 907 1 AATAAGAGATTAACCGTGCG AGG 59.61949 131 909 -1 TAGTTTTCTTTTTCCTCGCA CGG 48.17976 132 967 1 TATTATATATATGTCATTAT TGG 18.73491 133 971 1 ATATATATGTCATTATTGGC AGG 41.76418 134 972 1 TATATATGTCATTATTGGCA GGG 54.92986 135 1048 1 ATTGAAGTATACAATAGCCA CGG 64.48335 136 1049 1 TTGAAGTATACAATAGCCAC GGG 53.86531 137 1050 1 TGAAGTATACAATAGCCACG GGG 75.49947 138 1054 -1 CAACAATCTGGGTCTCCCCG TGG 73.79643 139 1065 -1 TTTTCTGAAGACAACAATCT GGG 53.83692 140 1066 -1 ATTTTCTGAAGACAACAATC TGG 44.86974 141 1091 -1 TGAGAGACTGTTTTAACACA AGG 59.78212 142 1109 1 TTAAAACAGTCTCTCAAAGC TGG 53.49504 143 1140 -1 TTCATCTTATTCAAGCAAAG AGG 64.11009 144 1178 1 AATCTATGAATC CCC AC CAC CGG 66.90318 145 1178 -1 AAGCTCCAAAACCGGTGGTG GGG 60.93072 146 1179 -1 GAAGCTCCAAAACCGGTGGT GGG 54.2135 147 1180 -1 TGAAGCTCCAAAACCGGTGG TGG 46.33848 148 1183 -1 TTATGAAGCTCCAAAACCGG TGG 68.22724 149 1184 1 TGAATCCCCACCACCGGTTT TGG 29.72103 150 1186 -1 GAGTTATGAAGCTCCAAAAC CGG 48.10068 151 1221 -1 ATATATTTATAAACGAAAAC AGG 40.25381 152 1276 -1 TAACGAAATAAATATCTAAA TGG 33.5519 153 1329 -1 GTTTTTTTCTTTTGGTAGTG TGG 38.70555 154 1337 -1 TGCATTATGTTTTTTTCTTT TGG 14.19145 155 1351 1 AAAGAAAAAAACATAATGCA TGG 63.51026 156 1388 1 CACACATATATACGATAGTG AGG 59.34196 157 1409 1 GGACATGAAATACTCACCAA AGG 69.78964 158 1414 -1 GGTACAACTAATGTATCCTT TGG 46.18244 159 1428 1 AAGGATACATTAGTTGTACC TGG 57.10327 160 1435 -1 AGGATAGTCACAGACATTCC AGG 50.29588 161 1448 1 TGGAATGTCTGTGACTATCC TGG 45 .01183 162 1455 -1 ATAATACGTGTATTCTTTCC AGG 31.45682 163 1488 -1 GTGATCCTTTCCATTAATAT TGG 33.16337 164 1489 1 AATTAAATATCCAATATTAA TGG 20.04105 165 1494 1 AATATCCAATATTAATGGAA AGG 55 .69722 166 1532 1 ATATATAAATATACAAGAAG AGG 52.03505 167 1606 1 ATTATATTACAAACCAGTGA AGG 62.03101 168 1608 -1 ACTTAAAAGAACACCTTCAC TGG 43.10988 169 1622 1 GTGAAGGTGTTCTTTTAAGT AGG 40.61279 170 1623 1 TGAAGGTGTTCTTTTAAGTA GGG 44.06002 171 1632 1 TCTTTTAAGTAGGGATCACT AGG 52.1437 172 1633 1 CTTTTAAGTAGGGATCAC TA GGG 60.66675 173 1638 1 AAGTAGGGATCACTAGGGCT TGG 43.63704 174 1647 1 TCACTAGGGCTTGGAGCATC TGG 33.29868 175 1664 -1 CACTTATTTCTTGAAGATTA TGG 30.34701 176 1676 1 CATAATCTTCAAGAAATAAG TGG 51.71581 177 1684 1 TCAAGAAATAAGTGGAAAAA AGG 33.99747 178 1685 1 CAAGAAATAAGTGGAAAAAA GGG 41.53648 179 1815 -1 AAACCTCGAGTAGATATTGG TGG 58.65631 180 1818 -1 TCTAAACCTCGAGTAGATAT TGG 41.98098 181 1823 1 TCTCCACCAATATCTACTCG AGG 63.24041 182 1844 1 GGTTTAGACAAAATATTAGA AGG 43.87082 183 1869 -1 CCTGATAAGCAAGTTTGTAA TGG 46.15333 184 1880 1 CCATTACAAACTTGCTTATC AGG 22.76102 185 1881 1 CATTACAAACTTGCTTATCA GGG 46.00555 186 1913 1 ATGACAGTCATTTTTACACT AGG 56.38646 187 1939 -1 GGCAAGAGTAATAGGAGATG TGG 67.01531 188 1947 -1 TTAAGTGTGGCAAGAGTAAT AGG 35 .65879 189 1960 -1 AGCAATACAAACTTTAAGTG TGG 64.17131 190 1985 -1 TTTATATAATAATAATGTCA AGG 61.85855 191 2025 1 AATAGATCAGATATATGATA AGG 43.78777 192 2033 1 AGATATATGATAAGGATGAG AGG 64.39285 193 2104 1 GTAGTAGTAGAGATCGAGTT AGG 52.76822 194 2114 1 AGATCGAGTTAGGTGATCAA TGG 47.8583 195 2115 1 GATCGAGTTAGGTGATCAAT GGG 43.40038 196 2134 1 TGGGTATTTATAAGCCAGCT AGG 53.71914 197 2135 1 GGGTATTTATAAGCCAGCTA GGG 59.56206 198 2137 -1 AAATTCCAACTTGCCCTAGC TGG 36.84358 199 2143 1 ATAAGCCAGCTAGGGCAAGT TGG 60.49436 200 2187 1 TTATTTGAATAGCGTAGTAG TGG 54.61689 201 2197 1 AGCGTAGTAGTGGCTGCTCT TGG 43.97502 202 2198 1 GCGTAGTAGTGGCTGCTCTT GGG 44.41787 203 2208 1 GGCTGCTCTTGGGAAATGCC AGG 52.38622 204 2215 -1 AATAAAGCAAATATTAAACC TGG 60.29947 205 2253 1 GTAAAAAGTTAAAATACCAA AGG 65.71087 206 2258 -1 GTGATGTTTTCAAATTCCTT TGG 44.14554 207 2309 -1 CATACGTGTCGAACTAGGAA TGG 55.15684 208 2314 -1 ACAACCATACGTGTCGAACT AGG 63.97993 209 2321 1 CATTCCTAGTTCGACACGTA TGG 57.55443 210 2372 1 TTGTTGATGTTGAAAGAAGT TGG 64.04881 211 Table 3: gRNA sequences targeted for CsSP-3 Position Strand Sequence PAM Efficiency SEQ. ID.
on SEQ. Score NO.
ID. NO:7 979 1 AGAATATATATATATATTAG TGG 52.75818 212 983 1 TATATATATATATTAGTGGT AGG 56.18088 213 987 1 TATATATATTAGTGGTAGGT AGG 46.88074 214 1012 1 GAGCTAGCGTCTTCTAGCTG CGG 57.33903 215 1039 1 TCTCTGAGCGTTGAAATAAA CGG 31.00695 216 1049 1 TTGAAATAAACGGCAGCGAC AGG 51.04418 217 1059 1 CGGCAGCGACAGGAAGACCG AGG 71.16066 218 1065 -1 TTCGCTGCTCAAAACGACCT CGG 66.77634 219 1108 -1 GGTGAGTATACCAAGCTCAA AGG 64.31989 220 1109 1 TTGAAATGATCCTTTGAGCT TGG 55.54256 221 1128 1 TTGGTATACTCACCGAGTCT CGG 54.52127 222 1129 -1 CAAACAGAAATGCCGAGACT CGG 54.73946 223 1169 -1 AGCCAAATATAGGGATTCAC AGG 49.91997 224 1178 1 AACCTGTGAATCCCTATATT TGG 23.1867 225 1178 -1 AGAGCCCAAAGCCAAATATA GGG 42.41948 226 1179 -1 GAGAGCCCAAAGCCAAATAT AGG 31.63154 227 1184 1 TGAATCCCTATATTTGGCTT TGG 26.63952 1185 1 GAATCCCTATATTTGGCTTT GGG 29.53778 229 1210 -1 TATGTACACAGGAAGGGAAG TGG 62.37694 230 1216 -1 AATGGATATGTACACAGGAA GGG 56.73565 231 1217 -1 AAATGGATATGTACACAGGA AGG 56.69376 232 1221 -1 ATTAAAATGGATATGTACAC AGG 64.92133 233 1234 -1 TTATAAATATAAAATTAAAA TGG 25 .89586 234 1279 1 TATACATATATAATTAATAA TGG 34.02683 235 1309 -1 GGCACCACAGACGCTACATT TGG 42.45215 236 ATTACCAAATGTAGCGTCTG TGG 57.76274 237 AATGTAGCGTCTGTGGTGCC TGG 52.49826 238 1330 -1 AGGATTGTGACAGACATACC AGG 64.90611 239 1350 -1 TTCTTCTTTGAAACATGGAC AGG 56.04975 240 1355 -1 TTATTTTCTTCTTTGAAACA TGG 51.31281 241 1380 1 AGAAAATAATTATTACAATC AGG 48.41061 242 1390 1 TATTACAATCAGGATATCTT AGG 46.56003 243 1441 -1 TTTAATTAACAAACTATTTT AGG 18.17429 244 1464 -1 ATGTCAGTAAGATTATAATT TGG 33.42864 245 1479 1 ATTATAATCTTACTGACATT TGG 32.26823 246 1522 1 ACTATTTATTATACCAGTGA AGG 55 .21337 247 1524 -1 ACTTAAGGGAGCACCTTCAC TGG 49.73888 248 1538 -1 CCCAAGTGATCCTTACTTAA GGG 38.09188 249 1539 1 TGAAGGTGCTCCCTTAAGTA AGG 48.60231 250 1539 -1 GCCCAAGTGATCCTTACTTA AGG 40.75021 251 1548 1 TCCCTTAAGTAAGGATCACT TGG 45 .83122 252 1549 1 CCCTTAAGTAAGGATCACTT GGG 69.93595 253 1554 1 AAGTAAGGATCACTTGGGCC AGG 52.25638 254 1561 -1 ATGACAGATCCAGATGTTCC TGG 43.93102 255 1563 1 TCACTTGGGCCAGGAACATC TGG 40.0198 256 1586 -1 CTCTCTCTTTATTTTATTTT AGG 4.865075 1623 -1 AATTAATGTTGTTCTTTTTT TGG 15 .45049 258 1663 -1 AGATCTCTAAATGATGAACA AGG 65 .19671 259 1722 1 TTTTTTTATTAGTGTGAAGA AGG 48.17114 1748 -1 AAACCTAGGGTTGAGATTCA AGG 38.00239 261 1756 1 GCTCCTTGAATCTCAACC CT AGG 58.80331 262 1761 1 TTGAATCTCAACCCTAGGTT TGG 37.75685 263 1761 -1 AGTTGTCATCTCCAAACCTA GGG 52.43725 264 1762 -1 CAGTTGTCATCTCCAAACCT AGG 57.30496 265 1777 1 GGTTTGGAGATGACAACTGA AGG 63.04415 266 1802 -1 AACAAGAAGCAAGTCTGTAA TGG 45 .44633 267 1836 1 ATAAGAGACAGACATTTTTA TGG 28.08049 268 GAGACAGACATTTTTATGGA TGG 52.94092 269 1874 -1 CTTGTGGTTGGAAGAGTGAT AGG 54.20481 270 1886 -1 ATGTTAGAGCCTCTTGTGGT TGG 53.01781 271 1888 1 ATCACTCTTCCAACCACAAG AGG 57.38136 272 1890 -1 AAGAATGTTAGAGCCTCTTG TGG 65.83401 273 1914 -1 TATTACTATAAATATAATTA TGG 28.32586 274 1950 -1 ATATATTTTAAGTACTTAAT TGG 18.0389 275 2019 1 TTTATATATGTGTTTGTGTG TGG 49.7003 276 2060 1 AATTATTACTTAGTTCGTGA TGG 53.17869 277 2065 1 TTACTTAGTTCGTGATGGAA TGG 52.02412 278 2072 1 GTTCGTGATGGAATGGAACT TGG 45.80324 279 2087 -1 TGTAGACACTATAAATACAT GGG 59.04909 280 2088 -1 ATGTAGACACTATAAATACA TGG 56.82992 281 2110 1 TAGTGTCTACATGATATCTT TGG 36.6185 282 2124 -1 TAAAAGTAAAGTTACAATTA AGG 32.62771 283 Table 4: gRNA sequences targeted for CsSP5G-1 Position Strand Sequence PAM Efficiency SEQ. ID.
on SEQ. Score NO.
ID.
NO:10 43 1 ATATTAATATATAACAAGTT TGG 33.4577 284 127 1 AATAATTAATTAAGAATATA TGG 32.43589 285 138 1 AAGAATATATGGCTAGAGAT AGG 45.23675 286 139 1 AGAATATATGGCTAGAGATA GGG 53.98613 287 151 1 TAGAGATAGGGACCCTCTTG TGG 52.53234 288 152 -1 TTACTCTACCAACCACAAGA GGG 67.79474 289 153 -1 ATTACTCTACCAACCACAAG AGG 59.18615 290 155 1 GATAGGGACCCTCTTGTGGT TGG 56.67036 291 167 1 CTTGTGGTTGGTAGAGTAAT AGG 35.27607 292 178 1 TAGAGTAATAGGAGATGTTT TGG 32.95839 293 192 1 ATGTTTTGGATCCTTTTACA AGG 55.39173 294 192 -1 AGAGAGACTGACCTTGTAAA AGG 42.08228 295 218 1 GTCTCTCTTAGAGTGAGTTA TGG 47.06405 296 229 1 AGTGAGTTATGGTAATAGAG AGG 54.93598 297 239 1 GGTAATAGAGAGGTCAACAA TGG 58.93327 298 264 -1 GGTTGGTTAACAATTTGGGA AGG 61.46045 299 268 -1 ACGAGGTTGGTTAACAATTT GGG 32.45336 300 269 -1 CACGAGGTTGGTTAACAATT TGG 23.81735 301 281 -1 CACCAATATCAACACGAGGT TGG 61.80226 302 285 -1 TCACCACCAATATCAACACG AGG 75.84355 303 290 1 AACCAACCTCGTGTTGATAT TGG 44.46178 304 293 1 CAACCTCGTGTTGATATTGG TGG 58.81318 305 306 1 ATATTGGTGGTGATGACCTA AGG 60.13671 306 311 -1 CCAAAGTGTAGAAGGTCCTT AGG 42.09425 307 319 -1 TTAATTTACCAAAGTGTAGA AGG 48.09029 308 322 1 CCTAAGGACCTTCTACACTT TGG 50.47553 309 357 -1 TGAAATCATTATGAATATTG AGG 50.25272 310 436 1 CATATATTGAAAATTATTAC AGG 36.58192 311 442 1 TTGAAAATTATTACAGGTCA TGG 43.08159 312 445 1 AAAATTATTACAGGTCATGG TGG 65 .82262 313 451 1 ATTACAGGTCATGGTGGATC CGG 50.20677 314 459 -1 TTGCTAGGGCTAGGAGCATC CGG 49.91113 315 468 -1 AGATTGGGGTTGCTAGGGCT AGG 44.68519 316 473 -1 CCCTTAGATTGGGGTTGCTA GGG 47.67473 317 474 -1 TCCCTTAGATTGGGGTTGCT AGG 44.15699 318 482 -1 GC AAATACTCCCTTAGATTG GGG 58.46921 319 483 1 GCCCTAGCAACCCCAATCTA AGG 36.0562 320 483 -1 TGCAAATACTCCCTTAGATT GGG 30.97985 321 484 1 CCCTAGCAACCCCAATCTAA GGG 45 .48361 322 484 -1 ATGCAAATACTCCCTTAGAT TGG 38.79211 323 498 1 ATCTAAGGGAGTATTTGCAT TGG 59.37908 324 556 1 ATATTATTATTAAATAGATG AGG 50.44408 325 557 1 TATTATTATTAAATAGATGA GGG 52.51763 326 689 1 TTTAATTTTGTATAAAAC TT TGG 34.74999 327 808 -1 TTCATGCACACAACACATGT TGG 55 .60825 328 869 -1 CAAAAAGTAAAGACATATTT TGG 14.91886 329 881 1 CAAAATATGTCTTTACTTTT TGG 10.5253 330 916 1 CATTTTATAAAGATGTTAGT TGG 33.17467 331 917 1 ATTTTATAAAGATGTTAGTT GGG 40.20867 332 1105 1 TTTTAGTGTCAGTTTTGAAT TGG 29.61892 333 1123 -1 AC AAAATTCTGTAATTATTA GGG 26.7988 334 1124 -1 TACAAAATTCTGTAATTATT AGG 13.97206 335 1156 -1 AC AAATTAAAAC AAGCTTTA GGG 23.11439 336 1157 -1 AACAAATTAAAACAAGCTTT AGG 32.2923 337 1180 1 TTTAATTTGTTAAAGTGACT AGG 54.58344 338 1228 -1 AACATGTAAAAAGAATTTAA GGG 33.91047 339 1229 -1 AAACATGTAAAAAGAATTTA AGG 15 .92615 340 1258 -1 GCTAGCATATATGGAATTTG TGG 45 .56144 341 1267 -1 ATTTATATAGCTAGCATATA TGG 26.77656 342 1288 1 TAGCTATATAAATATAAATA TGG 35.H496 343 1293 1 ATATAAATATAAATATGGAA AGG 60.53312 344 1301 1 ATAAATATGGAAAGGATATA TGG 32.49959 345 1302 1 TAAATATGGAAAGGATATAT GGG 33.709 346 1351 1 AAAGCTGATGAGAAAGAATG TGG 66.11521 347 1356 1 TGATGAGAAAGAATGTGGTT TGG 50.94728 348 1357 1 GATGAGAAAGAATGTGGTTT GGG 37.02061 349 1358 1 ATGAGAAAGAATGTGGTTTG GGG 59.03998 350 1379 1 GGATGAATTTTGAATGATGA AGG 48.10583 351 1380 1 GATGAATTTTGAATGATGAA GGG 53.0293 352 1386 1 TTTTGAATGATGAAGGGATG AGG 52.46871 353 1398 1 AAGGGATGAGGCTGTGTGTG TGG 55.94352 354 1421 -1 GGGACATGCTATAGCTAGCA GGG 60.76795 355 1422 -1 GGGGACATGCTATAGCTAGC AGG 49.84652 356 1441 -1 TTTTAATGGTGGGACAAAAG GGG 54.93961 357 1442 -1 ATTTTAATGGTGGGACAAAA GGG 35.09294 358 1443 -1 CATTTTAATGGTGGGACAAA AGG 27.24033 359 1451 -1 GAGGTGGCCATTTTAATGGT GGG 62.03513 360 1452 -1 TGAGGTGGCCATTTTAATGG TGG 64.3868 361 1455 1 CTTTTGTCCCACCATTAAAA TGG 29.86891 362 1455 -1 GTGTGAGGTGGCCATTTTAA TGG 29.73425 363 1467 -1 AAAACCTTCTTAGTGTGAGG TGG 69.54075 364 1470 -1 GTGAAAACCTTCTTAGTGTG AGG 60.43941 365 1474 1 ATGGCCACCTCACACTAAGA AGG 56.86845 366 1543 1 ATATATACATACACATGTAT AGG 45.18352 367 1544 1 TATATACATACACATGTATA GGG 55.90543 368 1612 -1 CTATGTTCGAATTCAAATTC GGG 34.62224 369 1613 -1 TCTATGTTCGAATTCAAATT CGG 36.42467 370 1635 1 TTCGAACATAGACTCAGATT TGG 35.59295 371 1648 -1 AGGGTTCAGGGTCGAATTTA GGG 28.94531 372 1649 -1 CAGGGTTCAGGGTCGAATTT AGG 26.64298 373 1660 -1 AGTTCGTGTTTCAGGGTTCA GGG 43.58548 374 1661 -1 AAGTTCGTGTTTCAGGGTTC AGG 26.7905 375 1667 -1 GAGTCTAAGTTCGTGTTTCA GGG 35.03576 376 1668 -1 TGAGTCTAAGTTCGTGTTTC AGG 15.64056 377 1686 1 CACGAACTTAGACTCAGACC TGG 55.22454 378 1692 1 CTTAGACTCAGACCTGGACC TGG 54.41072 379 1693 -1 TTGGGTCAAGGTCCAGGTCC AGG 45.45826 380 1699 -1 CGGGGTTTGGGTCAAGGTCC AGG 43.16961 381 1705 -1 TCGGGTCGGGGTTTGGGTCA AGG 38.7222 382 1711 -1 CGGGGTTCGGGTCGGGGTTT GGG 29.12979 383 1712 -1 TCGGGGTTCGGGTCGGGGTT TGG 16.04517 384 1717 -1 TCGAGTCGGGGTTCGGGTCG GGG 28.40008 385 1718 -1 TTCGAGTCGGGGTTCGGGTC GGG 28.3022 386 1719 -1 GTTCGAGTCGGGGTTCGGGT CGG 44.74237 387 1723 -1 CGGGGTTCGAGTCGGGGTTC GGG 33.72492 388 1724 -1 TCGGGGTTCGAGTCGGGGTT CGG 29.847 389 1729 -1 TAGTTTCGGGGTTCGAGTCG GGG 54.61581 390 1730 -1 CTAGTTTCGGGGTTCGAGTC GGG 35.21533 391 1731 -1 TCTAGTTTCGGGGTTCGAGT CGG 47.1652 392 1741 -1 CCAGGTCCAGTCTAGTTTCG GGG 59.72421 393 1742 -1 TCCAGGTCCAGTCTAGTTTC GGG 29.76351 394 1743 -1 GTCCAGGTCCAGTCTAGTTT CGG 32.43822 395 1746 1 CTCGAAC CC CGAAACTAGAC TGG 41.77216 396 1752 1 CCCCGAAACTAGACTGGACC TGG 44.07102 397 1759 1 ACTAGACTGGACCTGGACTC TGG 56.19546 398 1759 -1 TAGGTCCTAGGCCAGAGTCC AGG 53.14281 399 1765 1 CTGGACCTGGACTCTGGC CT AGG 55.36929 400 1771 -1 CTAGACCCGAGCTAGGTCCT AGG 55.5272 401 1776 1 CTCTGGCCTAGGACCTAGCT CGG 44.89925 402 1777 1 TCTGGCCTAGGACCTAGCTC GGG 55.94427 403 1778 -1 CTGAACTCTAGACCCGAGCT AGG 64.71982 404 1790 1 CTAGCTCGGGTCTAGAGTTC AGG 38.28516 405 1800 1 TCTAGAGTTCAGGTCCAGTC CGG 44.68981 406 1801 1 CTAGAGTTCAGGTCCAGTCC GGG 51.19192 407 1802 1 TAGAGTTCAGGTCCAGTCCG GGG 64.76165 408 1803 -1 CCTGACCTCGGACCCCGGAC TGG 42.34928 409 1808 -1 CAGATCCTGACCTCGGACCC CGG 54.44243 410 1809 1 CAGGTCCAGTCCGGGGTCCG AGG 55.03576 411 1814 1 CCAGTCCGGGGTCCGAGGTC AGG 39.80181 412 1815 -1 ACGAACCCAGATCCTGACCT CGG 70.13695 413 1820 1 CGGGGTCCGAGGTCAGGATC TGG 27.07 414 1821 1 GGGGTCCGAGGTCAGGATCT GGG 45.99404 415 1833 1 CAGGATCTGGGTTCGTGTTC TGG 13.13476 416 1834 1 AGGATCTGGGTTCGTGTTCT GGG 32.34104 417 1835 1 GGATCTGGGTTCGTGTTCTG GGG 61.73636 418 1841 1 GGGTTCGTGTTCTGGGGTTC AGG 32.84982 419 1845 1 TCGTGTTCTGGGGTTCAGGT TGG 32.94333 420 1846 1 CGTGTTCTGGGGTTCAGGTT GGG 37.64639 421 1850 1 TTCTGGGGTTCAGGTTGGGT TGG 36.37666 422 1851 1 TCTGGGGTTCAGGTTGGGTT GGG 39.38005 423 1856 1 GGTTCAGGTTGGGTTGGGTC TGG 33.59531 424 1863 1 GTTGGGTTGGGTCTGGAGTC TGG 19.45666 425 1864 1 TTGGGTTGGGTCTGGAGTCT GGG 39.24034 426 1870 1 TGGGTCTGGAGTCTGGGTCT AGG 26.29809 427 1871 1 GGGTCTGGAGTCTGGGTCTA GGG 46.06491 428 1883 1 TGGGTCTAGGGTCCAGATTC AGG 36.11531 429 1884 -1 CCTGAAC CC GATCCTGAATC TGG 41.73702 430 1888 1 CTAGGGTCCAGATTCAGGAT CGG 53.25207 431 1889 1 TAGGGTCCAGATTCAGGATC GGG 49.77329 432 1895 1 CCAGATTCAGGATCGGGTTC AGG 32.80081 433 1901 1 TCAGGATCGGGTTCAGGTTA AGG 33.91225 434 1919 1 TAAGGTTTGAGTCTGAGTCC AGG 59.59865 435 1925 1 TTGAGTCTGAGTCCAGGTAT AGG 52.86993 436 1926 -1 TCCCGACCAGAACCTATACC TGG 43.98773 437 1931 1 CTGAGTCCAGGTATAGGTTC TGG 45.95561 438 1935 1 GTCCAGGTATAGGTTCTGGT CGG 54.99259 439 1936 1 TCCAGGTATAGGTTCTGGTC GGG 55.16665 440 1964 1 AGTTCGAGAGTTTGAATTCA AGG 34.8731 441 1974 1 TTTGAATTCAAGGTCCAATT TGG 30.63103 442 1977 -1 GAACTCATCCAACTCCAAAT TGG 38.88806 443 1980 1 TTCAAGGTCCAATTTGGAGT TGG 43.86757 444 1997 1 AGTTGGATGAGTTCATGTCA TGG 67.34083 445 2063 -1 TTTAAAATTTTAATAGTGTT TGG 34.83327 446 2179 1 ATTCATAATTTTTAAATTAG AGG 42.99532 447 2180 1 TTCATAATTTTTAAATTAGA GGG 36.49527 448 2196 -1 TTATTTTTATCTTACTTATA GGG 25.50447 449 2197 -1 ATTATTTTTATCTTACTTAT AGG 20.4776 450 2308 -1 TTTACTGTACCGAATATTCA CGG 41.6042 451 2310 1 TTGTAGTTACCGTGAATATT CGG 32.91515 452 2325 1 ATATTCGGTACAGTAAATTA AGG 39.9063 453 2329 1 TCGGTACAGTAAATTAAGGA TGG 57.49355 454 2410 -1 ATATATAAAAATATAAATTG TGG 59.46361 455 2441 1 TATATTATTAATCTAGATAA TGG 49.32964 456 2493 -1 ATAATTATACTATATATTAT AGG 26.08693 457 2523 1 TTATAATAATTATACATGTT TGG 35.08265 458 2538 1 ATGTTTGGCAATTTCAATTT AGG 24.64794 459 2542 1 TTGGCAATTTCAATTTAGGT TGG 51.99459 460 2558 1 AGGTTGGTGACTGATATTCC TGG 39.09227 461 2565 -1 AAGCTTGGCCCGGTAGTTCC AGG 41.23058 462 2567 1 ACTGATATTCCTGGAACTAC CGG 48.93325 463 2568 1 CTGATATTCCTGGAACTACC GGG 62.10752 464 2575 -1 CGGCTCACCGAAGCTTGGCC CGG 48.9322 465 2579 1 GGAACTACCGGGCCAAGCTT CGG 46.22153 466 2580 -1 ATGAACGGCTCACCGAAGCT TGG 59.46928 467 2595 -1 GTATTATTATGAAGTATGAA CGG 57.67305 468 2666 1 ACGCTGTAAACAAAATAGTG CGG 67.72415 469 2768 1 ATTAATTGTTTATTATGTGT AGG 49.28958 470 2776 1 TTTATTATGTGTAGGACAAG AGG 55.5504 471 2779 1 ATTATGTGTAGGACAAGAGG TGG 65.45521 472 2799 1 TGGTGTGCTACGAGAACCCG CGG 69.35789 473 2804 -1 GAATCCCCACCGTCGGCCGC GGG 51.25283 474 2805 -1 TGAATCCCCACCGTCGGCCG CGG 46.05954 475 2806 1 CTACGAGAACCCGCGGCCGA CGG 55.45967 476 2809 1 CGAGAACCCGCGGCCGACGG TGG 52.81179 477 2810 1 GAGAACCCGCGGCCGACGGT GGG 56.14245 478 2811 1 AGAACCCGCGGCCGACGGTG GGG 60.30596 479 2811 -1 TACCGATGAATCCCCACCGT CGG 68.50256 480 2820 1 GGCCGACGGTGGGGATTCAT CGG 48.29492 481 2841 1 GGTATGTATTTGTGTTGTTC CGG 42.62051 482 2848 1 ATTTGTGTTGTTCCGGCAAT TGG 33.46831 483 2849 1 TTTGTGTTGTTCCGGCAATT GGG 46.97766 484 2849 -1 CCGTTTGCCTTCCCAATTGC CGG 39.46262 485 2853 1 TGTTGTTCCGGCAATTGGGA AGG 55.97024 486 2860 1 CCGGCAATTGGGAAGGCAAA CGG 54.12282 487 2872 1 AAGGCAAACGGTGTTCGCGC CGG 41.65064 488 2873 1 AGGCAAACGGTGTTCGCGCC GGG 37.60685 489 2874 1 GGCAAACGGTGTTCGCGCCG GGG 56.96599 490 2877 1 AAACGGTGTTCGCGCCGGGG TGG 55.4399 491 2880 -1 TTGAAGTTCTGACGCCACCC CGG 59.80253 492 2905 -1 ATAAAGCTCAGCAAAGTCTT TGG 43.946 493 2924 1 TTTGCTGAGCTTTATAACCT TGG 44.20648 494 2930 -1 GGGCAGCAACAGGCAAACCA AGG 68.3844 495 2940 -1 TTGTAATAAAGGGCAGCAAC AGG 46.09672 496 2950 -1 CCTTTGGCAGTTGTAATAAA GGG 32.56095 497 2951 -1 CCCTTTGGCAGTTGTAATAA AGG 29.14878 498 2961 1 CCCTTTATTACAACTGCCAA AGG 54.00419 499 2962 1 CCTTTATTACAACTGCCAAA GGG 58.42829 500 2966 -1 CCCCAGATCCTGTCTCCCTT TGG 39.01106 501 2969 1 TACAACTGCCAAAGGGAGAC AGG 46.03039 502 2975 1 TGCCAAAGGGAGACAGGATC TGG 35.48964 503 2976 1 GCCAAAGGGAGACAGGATCT GGG 44.90032 504 2977 1 CCAAAGGGAGACAGGATCTG GGG 58.20173 505 2978 1 CAAAGGGAGACAGGATCTGG GGG 63.61687 506 2982 1 GGGAGACAGGATCTGGGGGA AGG 54.73323 507 2985 1 AGACAGGATCTGGGGGAAGG AGG 49.86207 508 2998 -1 ATATATATATGTCTATGTGG AGG 65.35548 509 3001 -1 TATATATATATATGTCTATG TGG 58.96155 510 3084 1 GATTCTTAATGATGATATCA TGG 42.04328 511 3110 -1 AAAGCTTATTATATTAATAA TGG 32.75644 512 3167 1 AAGATGAAGAAGAAGAAAAC AGG 48.09653 513 3253 1 ATTATTGTACTTAATTCAGC TGG 42.31373 514 3275 -1 TACATAATATAATATTAGCA AGG 60.27522 515 3312 1 ATGAGATACACACATATATA TGG 43.91874 516 Table 5: gRNA sequences targeted for CsSP5G-2 Position Strand Sequence PAM Specificity Efficiency SEQ.
ID.
on SEQ. Score Score NO.
ID.
NO:13 263 1 TATAATTAATTAAGAATATA TGG 22.23226 32.43589 517 274 1 AAGAATATATGGCTAGAGAT AGG 79.37203 45.23675 518 275 1 AGAATATATGGCTAGAGATA GGG 68.00411 53.98613 519 287 1 TAGAGATAGGGACCCTCTTG TGG 94.26857 52.53234 520 288 -1 TTACTCTACCAACCACAAGA GGG 86.20032 67.79474 521 289 -1 ATTACTCTACCAACCACAAG AGG 78.08364 59.18615 522 291 1 GATAGGGACCCTCTTGTGGT TGG 83.63088 56.67036 523 303 1 CTTGTGGTTGGTAGAGTAAT AGG 79.58025 35.27607 524 314 1 TAGAGTAATAGGAGATGTTT TGG 65.94111 32.95839 525 328 1 ATGTTTTGGATCCTTTTACA AGG 70.02417 55.39173 526 328 -1 AGAGAGACTGACCTTGTAAA AGG 61.64142 42.08228 527 354 1 GTCTCTCTTAGAGTGAGTTA TGG 77.15915 47.06405 528 364 1 GAGTGAGTTATGGTAATGAG AGG 88.2009 66.48722 529 375 1 GGTAATGAGAGGTCAACAGA TGG 69.50771 64.33275 530 400 -1 GGTTGGTTAACAATTTGGGA AGG 71.89396 61.46045 531 404 -1 ACGAGGTTGGTTAACAATTT GGG 74.7603 32.45336 532 405 -1 CACGAGGTTGGTTAACAATT TGG 47.58272 23.81735 533 417 -1 CACCAATATCAACACGAGGT TGG 87.81265 61.80226 534 421 -1 TCACCACCAATATCAACACG AGG 83.79878 75.84355 535 426 1 AACCAACCTCGTGTTGATAT TGG 89.69152 44.46178 536 429 1 CAACCTCGTGTTGATATTGG TGG 86.47779 58.81318 537 442 1 ATATTGGTGGTGATGACCTA AGG 85.41543 60.13671 538 447 -1 CCAAAGTGTAGAAGGTCCTT AGG 92.80423 42.09425 539 455 -1 TTAATTTACCAAAGTGTAGA AGG 72.5548 48.09029 540 458 1 CCTAAGGACCTTCTACACTT TGG 96.23278 50.47553 541 493 -1 TGAAATCATTATGAATATTG AGG 48.22121 50.25272 542 570 1 TCATATATTGAAATTATTAC AGG 52.69845 36.5369 543 576 1 ATTGAAATTATTACAGGTCA TGG 77.63024 44.4074 544 579 1 GAAATTATTACAGGTCATGG TGG 68.71918 67.377 545 585 1 ATTACAGGTCATGGTGGATC CGG 91.58358 50.20677 546 593 -1 TTGCTAGGGCTAGGAGCATC CGG 91.68151 49.91113 547 602 -1 AGATTGGGGTTGCTAGGGCT AGG 88.79083 44.68519 548 607 -1 CCCTTAGATTGGGGTTGCTA GGG 95.67441 47.67473 549 608 -1 TCCCTTAGATTGGGGTTGCT AGG 93.93984 44.15699 550 616 -1 GCAAATACTCCCTTAGATTG GGG 90.51159 58.46921 551 617 1 GCCCTAGCAACCCCAATCTA AGG 96.11133 36.0562 552 617 -1 TGCAAATACTCCCTTAGATT GGG 80.38169 30.97985 553 618 1 CCCTAGCAACCCCAATCTAA GGG 91.08172 45.48361 554 618 -1 ATGCAAATACTCCCTTAGAT TGG 83.72207 38.79211 555 632 1 ATCTAAGGGAGTATTTGCAT TGG 72.37072 59.37908 556 691 1 TATTATTATTATAATAGATG AGG 33.94489 49.0571 557 692 1 ATTATTATTATAATAGATGA GGG 46.17269 54.14818 558 824 1 TTTAATTTTGTATAAAAATT TGG 30.69736 25.68348 559 941 -1 TTCATGCACACAACACATGT TGG 73.37183 55.60825 560 1002 -1 CAAAAGATAAAGACATATTT TGG 40.5511 14.24454 561 1014 1 CAAAATATGTCTTTATCTTT TGG 49.25714 18.66841 562 1049 1 CATTTTATAAAGATGTTAGT TGG 62.22119 33.17467 563 1050 1 ATTTTATAAAGATGTTAGTT GGG 55.65628 40.20867 564 1235 1 TTTTAGTGTCAGTTTTGAAT TGG 56.17701 29.61892 565 1253 -1 ACAAAATTCTGTAATTATTA GGG 40.47765 26.7988 566 1254 -1 TACAAAATTCTGTAATTATT AGG 42.0428 13.97206 567 1286 -1 ACAAATTAAAACAAGCTTTA GGG 61.75581 23.11439 568 1287 -1 AACAAATTAAAACAAGCTTT AGG 57.55848 32.2923 569 1310 1 TTTAATTTGTTAAAGTGACT AGG 43.2 54.58344 1358 -1 AAACATGTAAAAGAATTTAA GGG 28.72101 30.13118 571 1359 -1 TAAACATGTAAAAGAATTTA AGG 36.55236 17.03894 572 1386 -1 TAGCTAGCATATATGGATTG TGG 85.80949 58.5557 573 1393 -1 ATTTATATAGCTAGCATATA TGG 67.09149 25.17453 574 1414 1 TAGCTATATAAATATAAATA TGG 34.98771 35.14496 575 1419 1 ATATAAATATAAATATGGAA AGG 38.5034 60.53312 576 1427 1 ATAAATATGGAAAGGATATA TGG 50.85775 32.49959 577 1428 1 TAAATATGGAAAGGATATAT GGG 55.43276 33.709 578 1476 1 AAAGCTGATGAGAAAGAATG TGG 59.04283 66.42004 579 1481 1 TGATGAGAAAGAATGTGGTT TGG 56.44644 50.94728 580 1482 1 GATGAGAAAGAATGTGGTTT GGG 45.55022 37.02061 581 1483 1 ATGAGAAAGAATGTGGTTTG GGG 32.76114 59.03998 582 1504 1 GGATGAATTTTGAATGATGA AGG 57.96939 48.10583 583 1505 1 GATGAATTTTGAATGATGAA GGG 51.67398 53.0293 584 1511 1 TTTTGAATGATGAAGGGATG AGG 74.87655 52.46871 585 1523 1 AAGGGATGAGGCTGTGTGTG TGG 91.76706 55.94352 586 1545 -1 GGGACATGCTATAGCTAGCA GGG 93.3302 60.76795 587 1546 -1 GGGGACATGCTATAGCTAGC AGG 96.3887 49.84652 588 1565 -1 ATTTTAATGGGGGACAAAAG GGG 75.18965 50.95202 589 1566 -1 CATTTTAATGGGGGACAAAA GGG 70.07484 28.24863 590 1567 -1 CC ATTTTAATGGGGGACAAA AGG 72.90401 29.61281 1575 -1 TGAGGTGGCCATTTTAATGG GGG 88.0117 64.57289 592 1576 -1 GTGAGGTGGCCATTTTAATG GGG 88.89714 54.06851 593 1577 -1 TGTGAGGTGGCCATTTTAAT GGG 86.9284 20.4018 594 1578 1 CCTTTTGTCCCCCATTAAAA TGG 84.04315 22.66327 595 1578 -1 GTGTGAGGTGGCCATTTTAA TGG 90.68493 23.47984 596 1590 -1 AAAACCTTCTTAGTGTGAGG TGG 86.37634 69.54075 597 1593 -1 GTGAAAACCTTCTTAGTGTG AGG 80.43362 60.43941 598 1597 1 ATGGCCACCTCACACTAAGA AGG 96.3153 56.86845 599 1665 1 ATATATACATACACATGTAT AGG 43.76861 45.18352 600 1666 1 TATATACATACACATGTATA GGG 22.29849 55.90543 601 1733 -1 CTATGTTCGAATTCAAATTC GGG 59.04622 34.62224 602 1734 -1 TCTATGTTCGAATTCAAATT CGG 55.60998 36.42467 603 1756 1 TTCGAACATAGACTCAGATT TGG 67.83184 35.59295 604 1769 -1 CAGGGTTCAGGGTCGAATTA GGG 96.49558 35.95472 605 1770 -1 TCAGGGTTCAGGGTCGAATT AGG 93.28847 30.0118 606 1780 -1 AGTTCGTGTTTCAGGGTTC A GGG 91.81519 43.58548 1781 -1 AAGTTCGTGTTTCAGGGTTC AGG 94.75493 26.7905 608 1787 -1 GAGTCTAAGTTCGTGTTTC A GGG 82.9438 35.03576 609 1788 -1 TGAGTCTAAGTTCGTGTTTC AGG 86.51258 15.64056 610 1806 1 CACGAACTTAGACTCAGACC TGG 97.32676 55.22454 611 1811 1 ACTTAGACTCAGACCTGGAC TGG 94.91925 50.92057 612 1813 -1 TTTGGGTCAAGGTCCAGTCC AGG 94.63584 46.64457 613 1824 -1 TCGGGTCGGGGTTTGGGTCA AGG 85.77517 38.7222 614 1830 -1 CGGGGTTCGGGTCGGGGTTT GGG 93.19713 29.12979 615 1831 -1 TCGGGGTTCGGGTCGGGGTT TGG 90.00364 16.04517 616 1836 -1 TCGAGTCGGGGTTCGGGTCG GGG 94.47451 28.40008 617 1837 -1 TTCGAGTCGGGGTTCGGGTC GGG 89.12695 28.3022 618 1838 -1 GTTCGAGTCGGGGTTCGGGT CGG 93.33815 44.74237 619 1842 -1 CGGGGTTCGAGTCGGGGTTC GGG 97.78823 33.72492 620 1843 -1 TCGGGGTTCGAGTCGGGGTT CGG 95.88259 29.847 621 1848 -1 TAGTTTCGGGGTTCGAGTCG GGG 89.32043 54.61581 622 1849 -1 CTAGTTTCGGGGTTCGAGTC GGG 95.12488 35.21533 623 1850 -1 TCTAGTTTCGGGGTTCGAGT CGG 89.32442 47.1652 624 1860 -1 CC AGGTCCAGTCTAGTTTCG GGG 97.39694 59.72421 1861 -1 TCCAGGTCCAGTCTAGTTTC GGG 95.56199 29.76351 626 1862 -1 GTCCAGGTCCAGTCTAGTTT CGG 93.75616 32.43822 627 1865 1 CTCGAACCCCGAAACTAGAC TGG 95.01385 41.77216 628 1871 1 CC CC GAAACTAGACTGGACC TGG 99.6993 44.07102 629 1878 1 ACTAGACTGGACCTGGACTC TGG 98.4311 56.19546 630 1878 -1 TAGGTCCTAGGCCAGAGTCC AGG 98.14848 53.14281 631 1884 1 CTGGACCTGGACTCTGGCCT AGG 98.33955 55.36929 632 1890 -1 CTAGACCCGAGCTAGGTCCT AGG 99.08664 55.22238 633 1895 1 CTCTGGCCTAGGACCTAGCT CGG 97.78611 44.89925 634 1896 1 TCTGGCCTAGGACCTAGCTC GGG 98.32979 55.94427 635 1897 -1 GCCTGACCTAGACCCGAGCT AGG 99.73282 62.86618 636 1902 1 CTAGGACCTAGCTCGGGTCT AGG 99.47133 48.45849 637 1907 1 ACCTAGCTCGGGTCTAGGTC AGG 98.95846 46.63816 638 1914 1 TCGGGTCTAGGTCAGGCGTC CGG 98.55442 40.11313 639 1915 1 CGGGTCTAGGTCAGGCGTCC GGG 99.96756 43.65285 640 1922 1 AGGTCAGGCGTCCGGGTCCG AGG 99.22245 53.42575 641 1922 -1 CC CAGATCTGACCTCGGACC CGG 99.30512 55.96585 1928 -1 CACGAACCCAGATCTGACCT CGG 96.77532 68.87557 643 1932 1 TCCGGGTCCGAGGTCAGATC TGG 99.24263 31.05262 644 1933 1 CCGGGTCCGAGGTCAGATCT GGG 97.78879 47.515 645 1945 1 TCAGATCTGGGTTCGTGTTC TGG 98.00119 23.41796 646 1946 1 CAGATCTGGGTTCGTGTTCT GGG 90.6576 34.46177 647 1947 1 AGATCTGGGTTCGTGTTCTG GGG 72.74378 61.20728 648 1953 1 GGGTTCGTGTTCTGGGGTTC AGG 96.0542 32.84982 649 1957 1 TCGTGTTCTGGGGTTCAGGT TGG 96.79548 32.94333 650 1958 1 CGTGTTCTGGGGTTCAGGTT GGG 97.29827 37.64639 651 1962 1 TTCTGGGGTTCAGGTTGGGT TGG 95.13759 36.37666 652 1963 1 TCTGGGGTTCAGGTTGGGTT GGG 88.17509 39.38005 653 1968 1 GGTTCAGGTTGGGTTGGGTC TGG 80.71071 33.59531 654 1975 1 GTTGGGTTGGGTCTGGAGTC TGG 89.11478 19.45666 655 1976 1 TTGGGTTGGGTCTGGAGTCT GGG 91.69313 39.24034 656 1982 1 TGGGTCTGGAGTCTGGGTCT AGG 93.64853 26.29809 657 1983 1 GGGTCTGGAGTCTGGGTCTA GGG 95.12459 46.06491 658 1995 1 TGGGTCTAGGGTCCAGATTC AGG 81.74099 36.11531 659 1996 -1 CCTGAACCCGATCCTGAATC TGG 95.331 41.73702 660 2000 1 CTAGGGTCCAGATTCAGGAT CGG 92.37173 53.25207 661 2001 1 TAGGGTCCAGATTCAGGATC GGG 89.64241 49.77329 662 2007 1 CC AGATTCAGGATCGGGTTC AGG 95.93196 32.80081 2013 1 TCAGGATCGGGTTCAGGTTA AGG 87.67606 33.91225 664 2031 1 TAAGGTTTGAGTCTGAGTCC AGG 94.41411 59.59865 665 2037 1 TTGAGTCTGAGTCCAGGTAT AGG 90.1772 52.86993 666 2038 -1 TCCCGACCAGAACCTATACC TGG 87.92881 43.98773 667 2043 1 CTGAGTCCAGGTATAGGTTC TGG 92.36672 45.95561 668 2047 1 GTCCAGGTATAGGTTCTGGT CGG 94.39855 54.99259 669 2048 1 TCCAGGTATAGGTTCTGGTC GGG 94.37527 55.16665 670 2075 1 GAGTTCAGAGTTTGAATTCA AGG 63.71772 35.78178 671 2085 1 TTTGAATTCAAGGTCCAATT TGG 68.36156 30.63103 672 2088 -1 GAACTCATCCAACTCCAAAT TGG 63.82882 38.88806 673 2091 1 TTCAAGGTCCAATTTGGAGT TGG 83.895 43.86757 674 2108 1 AGTTGGATGAGTTCATGTC A TGG 82.12315 67.34083 2174 -1 TTTAAAATTTTAATAGTGTT TGG 46.71727 34.83327 676 2291 1 TTCATAATTTTTAAAATTAG AGG 22.81635 37.38885 677 2292 1 TCATAATTTTTAAAATTAGA GGG 36.07909 41.07827 678 2308 -1 TATTTTTATCTTTACTTATA GGG 47.52393 23.50322 679 2309 -1 TTATTTTTATCTTTACTTAT AGG 46.63947 16.34323 680 2421 -1 TTTACTGTACCGAATATTCA CGG 79.17329 41.6042 681 2423 1 TTGTAGTTACCGTGAATATT CGG 84.39107 32.91515 682 2438 1 ATATTCGGTACAGTAAATTA AGG 78.81327 39.9063 683 2442 1 TCGGTACAGTAAATTAAGGA TGG 89.21293 57.49355 684 2523 -1 ATATATAAAAATATAAATTG TGG 25.27378 59.76844 685 2552 1 TATATTATTAATCTAGATAA TGG 50.26283 44.83698 686 2604 -1 TTATAATTATACTAATATAT AGG 35.57578 34.45299 687 2632 1 TTATAATAATTATACATGTT TGG 44.45843 35.08265 688 2648 1 TGTTTGGCAATTTCAATTTT AGG 48.67666 19.55566 689 2652 1 TGGCAATTTCAATTTTAGGT TGG 62.81544 45.19466 690 2668 1 AGGTTGGTGACTGATATTCC TGG 90.67651 39.09227 691 2675 -1 AAGCTTGGCCCGGTAGTTCC AGG 100 41.23058 692 2677 1 ACTGATATTCCTGGAACTAC CGG 83.0177 48.93325 693 2678 1 CTGATATTCCTGGAACTACC GGG 96.95467 62.10752 694 2685 -1 CGGCTCACCGAAGCTTGGCC CGG 98.17534 48.9322 695 2689 1 GGAACTACCGGGCCAAGCTT CGG 98.10529 46.22153 696 2690 -1 ATGAACGGCTCACCGAAGCT TGG 97.02801 59.46928 697 2705 -1 GTATTATTATGAAGTATGAA CGG 54.7198 57.67305 698 2776 1 ACGCTGTAAACAAAATAGTG CGG 81.23666 66.20102 699 2870 1 ATTAATTGTTTATTATGTGT AGG 37.44309 49.28958 700 2878 1 TTTATTATGTGTAGGACAAG AGG 75.38758 55.5504 701 2881 1 ATTATGTGTAGGACAAGAGG TGG 76.37859 65.45521 702 2901 1 TGGTGTGCTACGAGAACCCG CGG 98.37073 69.35789 703 2905 1 GTGCTACGAGAACCCGCGGC CGG 100 49.67584 704 2906 1 TGCTACGAGAACCCGCGGCC GGG 99.48738 51.94565 705 2906 -1 ATGAATCCCCACCCGGCCGC GGG 99.3641 52.12455 706 2907 -1 GATGAATCCCCACCCGGCCG CGG 99.86331 48.71998 707 2909 1 TACGAGAACCCGCGGCCGGG TGG 99.98179 55.19917 708 2910 1 ACGAGAACCCGCGGCCGGGT GGG 100 48.89766 709 2911 1 CGAGAACCCGCGGCCGGGTG GGG 99.8583 44.45536 710 2913 -1 CATAC CGATGAATCC CC ACC CGG 91.97498 55.96558 2920 1 GCGGCCGGGTGGGGATTCAT CGG 96.23188 49.33169 712 2941 1 GGTATGTATTTGTGTTGTTC CGG 49.44229 42.62051 713 2948 1 ATTTGTGTTGTTCCGGCAAT TGG 93.12907 33.46831 714 2949 1 TTTGTGTTGTTCCGGCAATT GGG 95.02167 46.97766 715 2949 -1 CCGTTTGCCTTCCCAATTGC CGG 95.00883 39.46262 716 2953 1 TGTTGTTCCGGCAATTGGGA AGG 88.65091 55.97024 717 2960 1 CCGGCAATTGGGAAGGCAAA CGG 93.92485 54.12282 718 2972 1 AAGGCAAACGGTGTTCGCGC CGG 99.47463 41.65064 719 2973 1 AGGCAAACGGTGTTCGCGCC GGG 99.96331 37.60685 720 2974 1 GGCAAACGGTGTTCGCGCCG GGG 99.57812 56.96599 721 2977 1 AAACGGTGTTCGCGCCGGGG TGG 99.29854 55.4399 722 2980 -1 TTGAAGTTCTGACGCCACCC CGG 97.94034 59.80253 723 3005 -1 ATAAAGCTCAGCAAAGTCTT TGG 76.47984 43.946 724 3024 1 TTTGCTGAGCTTTATAACCT TGG 70.03213 44.20648 725 3030 -1 GGGCAGCAACAGGCAAACCA AGG 96.32764 68.3844 726 3040 -1 TTGTAATAAAGGGCAGCAAC AGG 93.04706 46.09672 727 3050 -1 CCTTTGGCAGTTGTAATAAA GGG 84.67114 32.56095 728 3051 -1 CC CTTTGGCAGTTGTAATAA AGG 86.81624 29.14878 3061 1 CC CTTTATTACAACTGCCAA AGG 95.16301 54.00419 3062 1 CCTTTATTACAACTGCCAAA GGG 87.58701 58.42829 731 3066 -1 CC CC AGATCCTGTCTCCC TT TGG 97.97572 39.01106 3069 1 TACAACTGCCAAAGGGAGAC AGG 93.01226 46.03039 733 3075 1 TGCCAAAGGGAGACAGGATC TGG 98.02303 35.48964 734 3076 1 GCCAAAGGGAGACAGGATCT GGG 98.25717 44.90032 735 3077 1 CC AAAGGGAGAC AGGATCTG GGG 94.1697 58.20173 736 3078 1 CAAAGGGAGACAGGATCTGG GGG 96.85202 63.61687 737 3082 1 GGGAGACAGGATCTGGGGGA AGG 98.34396 54.73323 738 3085 1 AGACAGGATCTGGGGGAAGG AGG 95.84931 49.86207 739 3098 -1 ATATATATATGTCTATGTGG AGG 57.02276 65.35548 740 3101 -1 TATATATATATATGTCTATG TGG 42.14695 58.96155 741 3185 1 GATTCTTAATGATGATATCA TGG 35.38161 43.13298 742 3213 -1 AAAGCTTATTATATTAATAA TGG 35.17605 32.75644 743 3250 1 TATATATATATAGAATAAGA TGG 45.50324 48.36613 744 3261 1 AGAATAAGATGGAAGAAAAC AGG 48.6601 44.82802 745 Table 6: gRNA sequences targeted for CsSP5G-3 Position Strand Sequence PAM Efficiency SEQ. ID.
on SEQ. Score NO.
ID.
NO:16 1007 -1 AACTTGGCTGTGGTGGAAGG AGG 62.58111 746 1010 -1 AGGAACTTGGCTGTGGTGGA AGG 53.35214 747 1014 -1 CAAAAGGAACTTGGCTGTGG TGG 59.7649 748 1017 -1 TGCCAAAAGGAACTTGGCTG TGG 49.9405 749 1023 -1 TTCAACTGCCAAAAGGAACT TGG 48.07818 750 1026 1 CACCACAGCCAAGTTCCTTT TGG 32.23959 751 1030 -1 CGTTTACTTCAACTGCCAAA AGG 54.51517 752 1052 1 TTGAAGTAAACGACAGCAAC AGG 41.20281 753 1062 1 CGACAGCAACAGGCAATCCA AGG 59.31913 754 1068 -1 TTTGCTGAGATCTACAACCT TGG 39.26965 755 1112 1 TTGAAGTTATGTCGCCACCC AGG 64.35662 756 1115 -1 AGACAGTGTATGCACCTGGG TGG 69.83127 757 1118 -1 GGCAGACAGTGTATGCACCT GGG 69.43409 758 1119 -1 AGGCAGACAGTGTATGCACC TGG 46.20011 759 1139 -1 TTCTGTTTCGACAGTTAGGA AGG 47.13328 760 1143 -1 TTTGTTCTGTTTCGACAGTT AGG 49.53212 761 1181 1 TAGCGATGTATTCCCACCGT CGG 68.69506 762 1182 -1 GAGAGCCCTAGACCGACGGT GGG 72.28535 763 1183 -1 CGAGAGCCCTAGACCGACGG TGG 62.48353 764 1186 -1 CTACGAGAGCCCTAGACCGA CGG 67.63038 765 1187 1 TGTATTCCCACCGTCGGTCT AGG 37.13047 766 1188 1 GTATTCCCACCGTCGGTCTA GGG 52.6053 767 1224 -1 GTGTATATACGCATATATGT AGG 53.19418 768 1367 1 TTAACTTATCGTTAACTTAT TGG 21.66841 769 1452 1 AGTGTATTAATATGTACCAA AGG 64.2117 770 1457 -1 GCAACTACGGGAACAGCCTT TGG 43.45249 771 1469 -1 ACTGATATTCCCGCAACTAC GGG 56.57771 772 1470 1 AAAGGCTGTTCCCGTAGTTG CGG 54.3468 773 1470 -1 GACTGATATTCCCGCAACTA CGG 47.03522 774 1471 1 AAGGCTGTTCCCGTAGTTGC GGG 43.75953 775 1494 -1 AGTGTATAACTTTTTCAGGT TGG 45.74911 776 1498 -1 TTTAAGTGTATAACTTTTTC AGG 12.25519 777 1553 1 ATAAAATTAATAATTATTAG TGG 40.90627 778 1569 1 TTAGTGGCACACACTATTGA TGG 50.84478 779 1583 -1 AATATTACTAACGAATGACA AGG 71.2185 780 1633 -1 TGTACTTAGTAATATCATTT CGG 39.17215 781 1658 -1 ATCTTAAAGAATATTTGCAT TGG 51.43028 782 1673 1 TGCAAATATTCTTTAAGATT AGG 33.25661 783 1682 1 TCTTTAAGATTAGGTTCGCT AGG 47.68808 784 1683 1 CTTTAAGATTAGGTTCGCTA GGG 53.86326 785 1688 1 AGATTAGGTTCGCTAGGGTT AGG 44.74391 786 1713 -1 ACTGTTAATGTTGCAGGTTA TGG 38.16734 787 1719 -1 ATTAATACTGTTAATGTTGC AGG 25.17433 788 1839 1 CGTATACTTAATTTTATGCT AGG 51.56297 789 1980 1 TATTTGTGCAATTATAAGTT AGG 41.35824 790 1992 -1 GTCAAAGACCCACAACAATA AGG 47.19197 791 1994 1 TAAGTTAGGCCTTATTGTTG TGG 52.99944 792 1995 1 AAGTTAGGCCTTATTGTTGT GGG 35.64647 793 2051 1 TTAACAGTGTTAATTTTAAA CGG 27.14498 794 2196 -1 TTGGTTTATGAAAAATTAGT GGG 43.4587 795 2197 -1 CTTGGTTTATGAAAAATTAG TGG 50.90688 796 2215 -1 TGCCTATATAACTAAATTCT TGG 33.54393 797 2224 1 AACCAAGAATTTAGTTATAT AGG 34.54892 798 2274 -1 TCTCAGGACTTTCTACACTT TGG 48.01676 799 2290 -1 AGATTGGTGGAGATGATCTC AGG 51.3529 800 2303 -1 TCACCTAGGGTTGAGATTGG TGG 50.45115 801 2306 -1 AACTCACCTAGGGTTGAGAT TGG 52.86728 802 2311 1 TCTCCACCAATCTCAACCCT AGG 61.53779 803 2316 -1 TCAAGTTGCTAACTCACCTA GGG 50.65517 804 2317 -1 CTCAAGTTGCTAACTCACCT AGG 56.33574 805 2332 1 GGTGAGTTAGCAACTTGAGA AGG 55.71603 806 2339 1 TAGCAACTTGAGAAGGTCTA AGG 50.2432 807 2357 -1 GGGATTAGGGCTATTAAC AA TGG 57.57407 808 2370 -1 AGTATCATATAGTGGGATTA GGG 45.56512 809 2371 -1 AAGTATCATATAGTGGGATT AGG 31.11638 810 2377 -1 CTCTTAAAGTATCATATAGT GGG 47.53587 811 2378 -1 TCTCTTAAAGTATCATATAG TGG 49.25586 812 2407 1 AGAGAGACAGTTTTTGTGAA AGG 50.90759 813 2408 1 GAGAGACAGTTTTTGTGAAA GGG 39.7937 814 2421 -1 GAGAGTGATAAGTGATGTGT TGG 59.68802 815 2443 -1 ATAGGGATCCTCTTGTGGTT GGG 49.32786 816 2444 -1 AATAGGGATCCTCTTGTGGT TGG 40.13389 817 2446 1 ATCACTCTCCCAACCACAAG AGG 54.01665 818 2448 -1 GGCTAATAGGGATCCTCTTG TGG 57.16178 819 2460 -1 ATATATATAAATGGCTAATA GGG 36.81696 820 2461 -1 TATATATATAAATGGCTAAT AGG 23.14055 821 2469 -1 ATAGTTGTTATATATATAAA TGG 34.2573 822 2520 1 AGAGAGATAGAGAGAAGAAG AGG 53.49955 823 2521 1 GAGAGATAGAGAGAAGAAGA GGG 63.65805 824 2537 1 AAGAGGGTTTGATGAGTTTT TGG 25.7462 825 2550 1 GAGTTTTTGGTTGTATAATT TGG 28.78721 826 2553 1 TTTTTGGTTGTATAATTTGG TGG 50.48919 827 2574 1 GGCTGACATTCAACAATTTA TGG 15.68184 828 Table 7: gRNA sequences targeted for CsSP5G-4 Position on SEQ. Strand Sequence PAM Efficiency SEQ.
ID. NO:19 Score ID. NO.
732 -1 TAAAGTTATTGGGAGTTGTG TGG 66.8211 829 742 -1 CCGACTACTGTAAAGTTATT GGG 28.42176 830 743 -1 GCCGACTACTGTAAAGTTAT TGG 36.02326 831 753 1 CCCAATAACTTTACAGTAGT CGG 40.53914 832 765 -1 TTATATAATTCTTATAGCAA TGG 48.27692 833 819 -1 AAAGGTTCTAATATATATTG TGG 47.44654 834 837 -1 TTAGCTTTTGTAACATCAAA AGG 33.39633 835 893 -1 GGCTGACATTCAACAATTTA TGG 15.68184 836 914 -1 GTTTTGGTTATATAATTTGG TGG 57.11172 837 917 -1 TGAGTTTTGGTTATATAATT TGG 28.40639 838 930 -1 GAAGAGGGTTTGATGAGTTT TGG 34.58611 839 945 -1 TGTAGAGAGAGATCAGAAGA GGG 60.53333 840 946 -1 ATGTAGAGAGAGATCAGAAG AGG 54.06301 841 990 1 ATAGTTGTTATATATATAAA TGG 33.58298 842 998 1 TATATATATAAATGGCTAAT AGG 23.14055 843 999 1 ATATATATAAATGGCTAATA GGG 36.81696 844 1011 1 GGCTAATAGGGATCCTCTTG TGG 57.16178 845 1013 -1 ATCACTCTCCCAACCACAAG AGG 54.01665 846 1015 1 AATAGGGATCCTCTTGTGGT TGG 40.13389 847 1016 1 ATAGGGATCCTCTTGTGGTT GGG 49.32786 848 1038 1 GAGAGTGATAAGTGATGTGT TGG 59.68802 849 1051 -1 GAGACACAGTTTTTGTGAAA GGG 37.91168 850 1052 -1 AGAGACACAGTTTTTGTGAA AGG 52.69581 851 1081 1 TCTCTTAAAGTATCATATAG TGG 51.27177 852 1082 1 CTCTTAAAGTATCATATAGT GGG 49.44647 853 1088 1 AAGTATCATATAGTGGGAAT AGG 36.1639 854 1089 1 AGTATCATATAGTGGGAATA GGG 49.45047 855 1102 1 GGGAATAGGGCTATTAACAA TGG 57.4132 856 1120 -1 TAGCAACTTGAGAAGGTCTA AGG 50.2432 857 1127 -1 GGTGAGTTAGCAACTTGAGA AGG 55.71603 858 1142 1 CTCAAGTTGCTAACTCACCT AGG 56.33574 859 1143 1 TCAAGTTGCTAACTCACCTA GGG 50.65517 860 1148 -1 TCTCCACCAATCTCAACCCT AGG 61.53779 861 1153 1 AACTCACCTAGGGTTGAGAT TGG 52.86728 862 1156 1 TCACCTAGGGTTGAGATTGG TGG 50.45115 863 1169 1 AGATTGGTGGAGATGATCTC AGG 51.3529 864 1185 1 TCTCAGGACTTTCTACACTT TGG 48.01676 865 1268 1 ATTAATACTGTTAATGTTGC AGG 25.17433 866 1274 1 ACTGTTAATGTTGCAGGTTA TGG 38.16734 867 1291 -1 TCGCTAGGGTTAGGAGCATC AGG 47.478 868 1300 -1 AGATTAGGTTCGCTAGGGTT AGG 44.74391 869 1305 -1 CTTTAAGATTAGGTTCGC TA GGG 53.86326 870 1306 -1 TCTTTAAGATTAGGTTCGCT AGG 47.68808 871 1315 -1 TGCAAATATTCTTTAAGATT AGG 33.25661 872 1330 1 ATCTTAAAGAATATTTGCAT TGG 51.43028 873 1402 1 AACTTTTAGATATATTACTT AGG 42.90897 874 1412 1 TATATTACTTAGGAATCACA AGG 71.76671 875 1426 -1 TAGTGGCACACACTTATTGA TGG 44.48106 876 1443 -1 TAAAAATTAATAATTATTAG TGG 38.53536 877 1503 1 TTAAAGTGTATAATTTTGTC AGG 40.17797 878 1507 1 AGTGTATAATTTTGTCAGGT TGG 38.69861 879 1530 -1 AAGCCTGTTCCCGTAGTTGC GGG 43.75953 880 1531 1 GACTGATATTCCCGCAAC TA CGG 47.03522 881 1531 -1 AAAGCCTGTTCCCGTAGTTG CGG 53.51908 882 1532 1 ACTGATATTCCCGCAACTAC GGG 56.57771 883 1538 1 ATTCCCGCAACTACGGGAAC AGG 40.62686 884 1544 1 GCAACTACGGGAACAGGCTT TGG 40.90731 885 1634 -1 TTAACTTACCATTAACTTAT TGG 23.66032 886 1637 1 AAATAACACCAATAAGTTAA TGG 36.79107 887 1740 1 TGTGTGTGATGATTTAATGA TGG 55.89947 888 1741 1 GTGTGTGATGATTTAATGAT GGG 53.71776 889 1759 1 ATGGGCGTACGCATATATGT AGG 58.24576 890 1795 -1 GAATTCCCACCGTCGGTCTT GGG 43.57541 891 1796 -1 TGAATTCCCACCGTCGGTCT TGG 31.13209 892 1797 1 CTACGAGAGCCCAAGACCGA CGG 67.05847 893 1800 1 CGAGAGC CC AAGACCGACGG TGG 58.98627 894 1801 1 GAGAGCCCAAGACCGACGGT GGG 70.56006 895 1802 -1 AAGCGATGAATTCCCACCGT CGG 68.48212 896 1840 1 TTTGTTCTGTTTCGACAGTT AGG 49.53212 897 1844 1 TTCTGTTTCGACAGTTAGGA AGG 47.13328 898 1864 1 AGGCAGACAGTGTATGCACC CGG 53.95754 899 1865 1 GGCAGACAGTGTATGCACCC GGG 67.05624 900 1868 1 AGACAGTGTATGCACCCGGG TGG 71.26007 901 1871 -1 TTGAAGTTATGTCGCCAC CC GGG 64.36734 902 1872 -1 GTTGAAGTTATGTCGCCACC CGG 57.40751 903 1915 1 TTTGCTGAAATCTACAAC CT TGG 36.83098 904 1921 -1 CGGCAGCAACAGGCAATCCA AGG 56.65812 905 1931 -1 TTGAAGTAAACGGCAGCAAC AGG 42.77031 906 1941 -1 CTTTTGGCAGTTGAAGTAAA CGG 46.14269 907 1953 1 CGTTTACTTCAACTGCCAAA AGG 54.51517 908 1957 -1 C AC CACAGCCAAGTTCCTTT TGG 32.23959 909 1960 1 TTCAACTGCCAAAAGGAACT TGG 48.07818 910 1966 1 TGCCAAAAGGAACTTGGCTG TGG 49.9405 911 1969 1 CAAAAGGAACTTGGCTGTGG TGG 59.7649 912 1973 1 AGGAACTTGGCTGTGGTGGA AGG 53.35214 913 1976 1 AACTTGGCTGTGGTGGAAGG AGG 62.58111 914 2046 -1 GACATATATAGATAGATAGA TGG 51.68794 915 2351 1 TGTATCATCAATAATATATA TGG 32.79628 916 Reference is made to Table 8 presenting a summary of the sequences within the scope of the current invention.
Table 8: Summary of sequences within the scope of the present invention Sequence type CsSP-1 CsSP-2 CsSP-3 CsSP5G-1 CsSP5G-2 CsSP5G-3 CsSP5G-4 Genomic SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
sequences NO:1 NO:4 NO:7 NO:10 NO:13 NO:16 NO:19 (Fig. 2A) (Fig. 3A) (Fig. 4A) (Fig. 5A) (Fig. 6A) (Fig.
7A) (Fig. 8A) Coding sequences SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
(CDS) NO:2 NO:5 NO:8 NO:11 NO:14 NO:17 NO:20 (Fig. 2B) (Fig. 3B) (Fig. 4B) (Fig. 5B) (Fig. 6B) (Fig. 7B) (Fig. 8B) Amino acid SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
sequences NO:3 NO:6 NO:9 NO:12 NO:15 NO:18 NO:21 (Fig. 2C) (Fig. 3C) (Fig. 4C) (Fig. 5C) (Fig. 6C) (Fig. 7C) (Fig. 8C) gRNA sequences SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
NO:22- NO:127- NO:212- NO:284- NO:517- NO:746- NO:829-SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
NO:126 NO:211 NO:283 NO:516 NO:745 NO:828 NO:916 (Table 1) (Table 2) (Table 3) (Table 4) (Table 5) (Table 6) (Table 7) The above gRNA molecules have been cloned into suitable vectors and their sequence has been verified. In addition different Cas9 versions have been analyzed for optimal compatibility between the Cas9 protein activity and the gRNA molecule in the Cannabis plant.
The efficiency of the designed gRNA molecules have been validated by transiently transforming Cannabis tissue culture. A plasmid carrying a gRNA sequence together with the Cas9 gene has been transformed into Cannabis protoplasts. The protoplast cells have been grown for a short period of time and then were analyzed for existence of genome editing events.
The positive constructs have been subjected to the herein established stable transformation protocol into Cannabis plant tissue for producing genome edited Cannabis plants in SP and/or SP5G genes.
Stage 3: Transforming Cannabis plants using Agrobacterium or biolistics (gene gun) methods. For Agrobacterium and bioloistics, a DNA plasmid carrying (Cas9 + gene specific gRNA) can be used.
A vector containing a selection marker, Cas9 gene and relevant gene specific gRNA's is constructed. For biolistics, Ribonucleoprotein (RNP) complexes carrying (Cas9 protein + gene specific gRNA) are used. RNP complexes are created by mixing the Cas9 protein with relevant gene specific gRNA' s.
According to some embodiments of the present invention, transformation of various Cannabis tissues was performed using particle bombardment of:
= 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. 9 photographically presenting GUS staining after transient transformation of the following Cannabis tissues (A) axillary buds (B) leaf (C) calli, and (D) cotyledons.
Fig. 9 demonstrates that various Cannabis tissues have been successfully transiently transformed using biolistics system. Transformation has been performed into calli, leaves, axillary buds and cotyledons of Cannabis.
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. 10A-C 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. 11 showing PCR detection of Cas9 DNA in shoots of transformed Cannabis plants. DNA extracted from shoots of plants transformed with Cas9 using biolistics. This figure shows that three weeks post transformation, Cas9 DNA was detected in shoots of transformed plants.
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. 12 presenting results of in vitro analysis of CRISPR/Cas9 cleavage activity. Fig. 12A schematically shows the genomic area targeted for editing (PAM is marked in red) and amplified by the reverse and forward designed primers Fig. 12B
photographically presents a gel showing successful digestion of the resulted PCR amplicon containing the gene specific gRNA sequence, by RNP complex containing Cas9. The analysis included the following steps:
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 sp and sp5g related phenotypes as described above. It is within the scope that different gRNA promoters were tested in order to maximize editing efficiency.
References:
Sebastian Soyk, Niels A Muller, Soon Ju Park, Inga Schmalenbach, Ke Jiang, Ryosuke Hayama, Lei Zhang, Joyce Van Eck, Jose M Jimenez-Gomez & Zachary B Lippman "Variation in the flowering gene SELF PRUNING 5G promotes day-neutrality and early yield in tomato" Nature Genetics, 2017 49, 162-168.
Tingdong Li, Xinping Yang, Yuan Yu, Xiaomin Si, Xiawan Zhai, Huawei Zhang, Wenxia Dong, Caixia Gao and Cao Xu "Domestication of wild tomato is accelerated by genome editing" Nature Biotechnology, 2018 36, 1160-1163.
Agustin Zsogon, TomaS Cermak, Emmanuel Rezende Naves, Marcela Morato Notini, Kai H Edel, Stefan Weinl, Luciano Freschi, Daniel F Voytas, Jorg Kudla and Lazar Eustaquio Pereira Peres "De novo domestication of wild tomato using genome editing" Nature Biotechnology, 2018 36, 1211-1216.
Zachary H. Lemmon, Nathan T. Reem, Justin Dalrymple, Sebastian Soyk, Kerry E.
Swartwood, Daniel Rodriguez-Leal, Joyce Van Eck and Zachary B. Lippman "Rapid improvement of domestication traits in an orphan crop by genome editing" Nature Plants, 2018 4, 766-770.
Xie, K. and Yang Y. "RNA-guided genome editing in plants using a CRISPR¨Cas system."
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Claims (105)
1. A modified Cannabis plant exhibiting at least one improved domestication trait compared with wild type Cannabis, wherein said modified plant comprises at least one mutated Cannabis SELF PRUNING (SP) (CsSP) gene selected from the group consisting of CsSP-1 having a genomic nucleotide sequence as set forth in SEQ ID NO:1 or a functional variant thereof, CsSP-2 having a genomic nucleotide sequence as set forth in SEQ ID
NO:4 or a functional variant thereof, CsSP-3 having a genomic nucleotide sequence as set forth in SEQ ID NO:7 or a functional variant thereof and any combination thereof, and/or at least one mutated Cannabis SELF PRUNING 5G (SP5G) (CsSP5G) gene selected from the group consisting of CsSP5G-1 having a genomic nucleotide sequence as set forth in SEQ ID
NO:10 or a functional variant thereof, CsSP5G-2 having a genomic nucleotide sequence as set forth in SEQ ID NO:13 or a functional variant thereof, CsSP5G-3 having a genomic nucleotide sequence as set forth in SEQ ID NO:16 or a functional variant thereof, CsSP5G-4 having a genomic nucleotide sequence as set forth in SEQ ID NO:19 or a functional variant thereof and any combination thereof.
NO:4 or a functional variant thereof, CsSP-3 having a genomic nucleotide sequence as set forth in SEQ ID NO:7 or a functional variant thereof and any combination thereof, and/or at least one mutated Cannabis SELF PRUNING 5G (SP5G) (CsSP5G) gene selected from the group consisting of CsSP5G-1 having a genomic nucleotide sequence as set forth in SEQ ID
NO:10 or a functional variant thereof, CsSP5G-2 having a genomic nucleotide sequence as set forth in SEQ ID NO:13 or a functional variant thereof, CsSP5G-3 having a genomic nucleotide sequence as set forth in SEQ ID NO:16 or a functional variant thereof, CsSP5G-4 having a genomic nucleotide sequence as set forth in SEQ ID NO:19 or a functional variant thereof and any combination thereof.
2. The modified Cannabis plant according to claim 1, wherein said functional variant has at least 75% sequence identity to said CsSP or said CsSP5G nucleotide sequence.
3. The modified Cannabis plant according to claim 1, wherein said mutation is introduced using mutagenesis, small interfering RNA (siRNA), microRNA (miRNA), artificial miRNA
(amiRNA), DNA introgression, endonucleases or any combination thereof.
(amiRNA), DNA introgression, endonucleases or any combination thereof.
4. The modified Cannabis plant according to claim 1, wherein said mutation is introduced using targeted genome modification.
5. The modified Cannabis plant according to claim 4, wherein said mutation 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.
6. The modified Cannabis plant according to claim 5, wherein said Cas gene is selected from the group consisting of Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, AMENDED SHEET IPEA/IL
Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cast 10d, Cas12, Cas13, Cas14, CasX, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn 1, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl, Csb 1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Cszl, Csx15, Csfl, Csf2, Csf3, Csf4, and Cu1966and any combination thereof.
Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cast 10d, Cas12, Cas13, Cas14, CasX, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn 1, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl, Csb 1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Cszl, Csx15, Csfl, Csf2, Csf3, Csf4, and Cu1966and any combination thereof.
7. The modified Cannabis plant according to claim 1, wherein the mutated CsSP
or CsSP5G
gene is a CRISPR/Cas9- induced heritable mutated allele.
or CsSP5G
gene is a CRISPR/Cas9- induced heritable mutated allele.
8. The modified Cannabis plant according to claim 1, wherein said mutation is a missense mutation, nonsense mutation, insertion, deletion, indel, substitution or duplication.
9. The modified Cannabis plant of claim 8, wherein the insertion or the deletion produces a gene comprising a frameshift.
10. The modified Cannabis plant of claim 1, wherein said plant is homozygous for said at list one CsSP mutated gene.
11. The modified Cannabis plant of claim 1, wherein said plant is homozygous for said at list one CsSP5G mutated gene.
12. The modified Cannabis plant of claim 1, wherein said plant is a Cssp Cssp5g double mutant.
13. The modified Cannabis plant according to claim 7, wherein said mutation is in the coding region of said allele, a mutation in the regulatory region of said allele, or an epigenetic factor.
14. The modified Cannabis plant according to claim 1, wherein said mutation is a silencing mutation, a knockdown mutation, a knockout mutation, a loss of function mutation or any combination thereof.
15. The modified Cannabis plant according to claim 1 wherein said mutation is generated in planta.
AMENDED SHEET IPEA/IL
AMENDED SHEET IPEA/IL
16. The modified Cannabis plant according to claim 1 wherein said mutation 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:22-SEQ ID NO:916 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA
sequence selected from the group consisting of SEQ ID NO:22-916 and any combination thereof.
sequence selected from the group consisting of SEQ ID NO:22-SEQ ID NO:916 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA
sequence selected from the group consisting of SEQ ID NO:22-916 and any combination thereof.
17. The modified Cannabis plant according to claim 1 wherein said mutation in said CsSP-1 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:22-SEQ ID NO:126 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:22-126 and any combination thereof.
sequence selected from the group consisting of SEQ ID NO:22-SEQ ID NO:126 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:22-126 and any combination thereof.
18. The modified Cannabis plant according to claim 1 wherein said mutation in said CsSP-2 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:127-SEQ ID NO:211 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:127-211 and any combination thereof.
sequence selected from the group consisting of SEQ ID NO:127-SEQ ID NO:211 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:127-211 and any combination thereof.
19. The modified Cannabis plant according to claim 1 wherein said mutation in said CsSP-3 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:212-SEQ ID NO:283 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:212-283 and any combination thereof.
sequence selected from the group consisting of SEQ ID NO:212-SEQ ID NO:283 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:212-283 and any combination thereof.
20. The modified Cannabis plant according to claim 1 wherein said mutation in said CsSP5G-1 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:284-SEQ ID NO:516 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 284-516 and any combination thereof.
AMENDED SHEET IPEA/IL
sequence selected from the group consisting of SEQ ID NO:284-SEQ ID NO:516 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 284-516 and any combination thereof.
AMENDED SHEET IPEA/IL
21. The modified Cannabis plant according to claim 1 wherein said mutation in said CsSP5G-2 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:517-SEQ ID NO:745 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:517-745 and any combination thereof.
sequence selected from the group consisting of SEQ ID NO:517-SEQ ID NO:745 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:517-745 and any combination thereof.
22. The modified Cannabis plant according to claim 1 wherein said mutation in said CsSP5G-3 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:746-SEQ ID NO:828 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 746-828 and any combination thereof.
sequence selected from the group consisting of SEQ ID NO:746-SEQ ID NO:828 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 746-828 and any combination thereof.
23. The modified Cannabis plant according to claim 1 wherein said mutation in said CsSP5G-4 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:829-SEQ ID NO:916 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 829-916 and any combination thereof.
sequence selected from the group consisting of SEQ ID NO:829-SEQ ID NO:916 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 829-916 and any combination thereof.
24. The modified Cannabis plant according to any one of claims 16-23 wherein said gRNA
sequence comprises a 3' NGG Protospacer Adjacent Motif (PAM).
sequence comprises a 3' NGG Protospacer Adjacent Motif (PAM).
25. The modified Cannabis plant according to any one of claims 16-23 wherein said construct is introduced into the plant cells via Agrobacterium infiltration, virus based plasmids for delivery of the genome editing molecules or mechanical insertion such as polyethylene glycol (PEG) mediated DNA transformation, electroporation or gene gun biolistics.
26. The modified Cannabis plant according to claim 1, wherein said plant has decreased expression levels of at least one of said CsSP genes.
AMENDED SHEET IPEA/IL
AMENDED SHEET IPEA/IL
27. The modified Cannabis plant according to claim 26, wherein the sequence of said expressed CsSP gene is selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:9 or a functional variant thereof.
NO:5, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:9 or a functional variant thereof.
28. The modified Cannabis plant according to claim 1, wherein said plant has decreased expression levels of at least one of said CsSP5G genes.
29. The modified Cannabis plant according to claim 28, wherein the sequence of said expressed CsSP5G gene is selected from the group consisting of: SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20 and SEQ
ID NO:21 or a functional variant thereof.
NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20 and SEQ
ID NO:21 or a functional variant thereof.
30. The modified Cannabis plant according to claim 1, wherein said plant is semi-determinant.
31. The modified Cannabis plant according to claim 1, wherein said plant has determinant growth habit.
32. The modified Cannabis plant according to claim 1, wherein said plant flowers earlier than a corresponding wild type cannabis plant.
33. The modified Cannabis plant according to claim 1, wherein said plant exhibits improved earliness as compared to a corresponding wild type cannabis plant.
34. The modified Cannabis plant according to claim 1, wherein said plant exhibits suppressed sympodial shoot termination as compared to a corresponding wild type cannabis plant.
35. The modified Cannabis plant according to claim 1, wherein said plant exhibits similar sympodial shoot termination as compared to a corresponding wild type cannabis plant.
36. The modified Cannabis plant according to claim 1, wherein said plant exhibits suppressed or reduced day-length sensitivity as compared to a corresponding wild type cannabis plant.
37. The modified Cannabis plant according to claim 1 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.
AMENDED SHEET IPEA/IL
indica, C. ruderalis and any hybrid or cultivated variety of the genus Cannabis.
AMENDED SHEET IPEA/IL
38. The modified Cannabis plant according to claim 1, wherein said domestication trait is selected from the group consisting of reduced flowering time, earliness, synchronous flowering, reduced day-length sensitivity, determinant or semi-determinant architecture, early termination of sympodial cycling, earlier axillary shoot flowering, compact growth habit, reduced height, reduced number of sympodial units, adaptation to mechanical harvest, higher harvest index and any combination thereof.
39. The modified Cannabis plant according to claim 1 wherein said Cssp Cssp5g double mutant is characterized by having a more than additive effect on a trait selected from the group consisting of compactness, earlier axillary shoot flowering, earlier termination of sympodial cycling, harvest index and any combination thereof as compared to wild type and/or sp mutant Cannabis plants.
40. A Cannabis plant, plant part or plant cell according to claim 1 wherein said plant does not comprise a transgene.
41. A plant part, plant cell or plant seed of a plant according to claim 1.
42. A tissue culture of regenerable cells, protoplasts or callus obtained from the modified Cannabis plant according to claim 1.
43. The modified Cannabis plant according to claim 1 wherein said plant genotype is obtainable by deposit under accession number with NCIMB Aberdeen AB21 9YA, Scotland, UK
or with ATCC.
or with ATCC.
44. A method for producing a modified Cannabis plant exhibiting at least one improved domestication trait compared with wild type Cannabis, said method comprises steps of genetically modifying at least one Cannabis SELF PRUNING (SP) (CsSP) gene selected from the group consisting of CsSP-1 having a genomic nucleotide sequence as set forth in SEQ ID NO:1 or a functional variant thereof, CsSP-2 having a genomic nucleotide sequence as set forth in SEQ ID NO:4 or a functional variant thereof, CsSP-3 having a genomic nucleotide sequence as set forth in SEQ ID NO:7 or a functional variant thereof and any combination thereof, and/or at least one Cannabis SELF PRUNING 5G (SP5G) (CsSP5G) gene selected from the group consisting of CsSP5G-1 having a genomic nucleotide AMENDED SHEET IPEA/IL
sequence as set forth in SEQ ID NO:10 or a functional variant thereof, CsSP5G-2 having a genomic nucleotide sequence as set forth in SEQ ID NO:13 or a functional variant thereof, CsSP5G-3 having a genomic nucleotide sequence as set forth in SEQ ID NO:16 or a functional variant thereof, CsSP5G-4 having a genomic nucleotide sequence as set forth in SEQ ID NO:19 or a functional variant thereof and any combination thereof.
sequence as set forth in SEQ ID NO:10 or a functional variant thereof, CsSP5G-2 having a genomic nucleotide sequence as set forth in SEQ ID NO:13 or a functional variant thereof, CsSP5G-3 having a genomic nucleotide sequence as set forth in SEQ ID NO:16 or a functional variant thereof, CsSP5G-4 having a genomic nucleotide sequence as set forth in SEQ ID NO:19 or a functional variant thereof and any combination thereof.
45. A method for producing a modified Cannabis plant exhibiting at least one improved domestication trait compared with wild type Cannabis by targeted genome modification, said method comprises steps of genetically introducing a loss of function mutation in at least one Cannabis SELF PRUNING (SP) (CsSP) gene selected from the group consisting of CsSP-1 having a genomic nucleotide sequence as set forth in SEQ ID NO:1 or a functional variant thereof, CsSP-2 having a genomic nucleotide sequence as set forth in SEQ ID NO:4 or a functional variant thereof, CsSP-3 having a genomic nucleotide sequence as set forth in SEQ ID NO:7 or a functional variant thereof and any combination thereof, and/or at least one Cannabis SELF PRUNING 5G (SP5G) (CsSP5G) gene selected from the group consisting of CsSP5G-1 having a genomic nucleotide sequence as set forth in SEQ ID
NO:10 or a functional variant thereof, CsSP5G-2 having a genomic nucleotide sequence as set forth in SEQ ID NO:13 or a functional variant thereof, CsSP5G-3 having a genomic nucleotide sequence as set forth in SEQ ID NO:16 or a functional variant thereof, CsSP5G-4 having a genomic nucleotide sequence as set forth in SEQ ID NO:19 or a functional variant thereof and any combination thereof.
NO:10 or a functional variant thereof, CsSP5G-2 having a genomic nucleotide sequence as set forth in SEQ ID NO:13 or a functional variant thereof, CsSP5G-3 having a genomic nucleotide sequence as set forth in SEQ ID NO:16 or a functional variant thereof, CsSP5G-4 having a genomic nucleotide sequence as set forth in SEQ ID NO:19 or a functional variant thereof and any combination thereof.
46. A method of improving at least one domestication trait compared with wild type Cannabis, comprising steps of producing a modified Cannabis plant, seed or plant part thereof, that is homozygous for at least one mutated CsSP5G gene selected from the group consisting of CsSP5G-1 having a genomic nucleotide sequence as set forth in SEQ ID NO:10 or a functional variant thereof, CsSP5G-2 having a genomic nucleotide sequence as set forth in SEQ ID NO:13 or a functional variant thereof, CsSP5G-3 having a genomic nucleotide sequence as set forth in SEQ ID NO:16 or a functional variant thereof, CsSP5G-4 having a genomic nucleotide sequence as set forth in SEQ ID NO:19 or a functional variant thereof and any combination thereof in a sp background and enabling growth of said Cannabis plant, seed or plant part thereof.
AMENDED SHEET IPEA/IL
AMENDED SHEET IPEA/IL
47. The method according to any one of claims 44 -46, wherein said method comprises steps of:
a. identifying at least one Cannabis SP (CsSP) and/or at least one Cannabis (CsSP5G) allele;
b. synthetizing at least one guide RNA (gRNA) comprising a nucleotide sequence complementary to said at least one identified CsSP and/or CsSP5G allele;
c. transforming Cannabis plant cells with a construct comprising (a) Cas nucleotide sequence operably linked to said at least one gRNA, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and said at least one gRNA;
d. screening the genome of said transformed plant cells for induced targeted loss of function mutation in at least one of said CsSP and/or CsSP5G allele;
e. regenerating Cannabis plants carrying said loss of function mutation in at least one of said CsSP and/or CsSP5G allele; and f. screening said regenerated plants for a Cannabis plant with improved domestication trait.
a. identifying at least one Cannabis SP (CsSP) and/or at least one Cannabis (CsSP5G) allele;
b. synthetizing at least one guide RNA (gRNA) comprising a nucleotide sequence complementary to said at least one identified CsSP and/or CsSP5G allele;
c. transforming Cannabis plant cells with a construct comprising (a) Cas nucleotide sequence operably linked to said at least one gRNA, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and said at least one gRNA;
d. screening the genome of said transformed plant cells for induced targeted loss of function mutation in at least one of said CsSP and/or CsSP5G allele;
e. regenerating Cannabis plants carrying said loss of function mutation in at least one of said CsSP and/or CsSP5G allele; and f. screening said regenerated plants for a Cannabis plant with improved domestication trait.
48. The method according to claim 47, wherein said step of screening the genome of said transformed plant cells for induced targeted loss of function mutation further comprises steps of obtaining a nucleic acid sample of said transformed plant and performing a nucleic acid amplification and optionally restriction enzyme digestion to detect a mutation in said at least one of said CsSP and/or CsSP5G allele.
49. The method according to any one of claims 44 -46, wherein said functional variant has at least 75% sequence identity to said CsSP or said CsSP5G nucleotide sequence.
50. The method according to any one of claims 44 -46, wherein said mutation is introduced using mutagenesis, small interfering RNA (siRNA), microRNA (miRNA), artificial miRNA
(amiRNA), DNA introgression, endonucleases or any combination thereof.
AMENDED SHEET IPEA/IL
(amiRNA), DNA introgression, endonucleases or any combination thereof.
AMENDED SHEET IPEA/IL
51. The method according to any one of claims 44 -46, wherein said mutation is introduced using targeted genome modification.
52. The method according to claim 51, wherein said mutation 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.
(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.
53. The method according to claim 52, 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, Cast l0d, Cas12, Cas13, Cas14, CasX, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc 1 , Csc2, Csa5, Csnl, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl, Csb 1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz 1, Csx15, Csfl, Csf2, Csf3, Csf4, and Cu1966and any combination thereof.
54. The method according to any one of claims 44 -46, wherein the mutated CsSP
or CsSP5G
gene is a CRISPR/Cas9- induced heritable mutated allele.
or CsSP5G
gene is a CRISPR/Cas9- induced heritable mutated allele.
55. The method according to any one of claims 44 -46, wherein said mutation is a missense mutation, nonsense mutation, insertion, deletion, indel, substitution or duplication.
56. The method according to claim 55, wherein the insertion or the deletion produces a gene comprising a frameshift.
57. The method according to any one of claims 44 -46, wherein said plant is homozygous for said at list one CsSP mutated gene.
58. The method according to any one of claims 44 -46, wherein said plant is homozygous for said at list one CsSP5G mutated gene.
59. The method according to any one of claims 44 -46, wherein said plant is a Cssp Cssp5g double mutant.
AMENDED SHEET IPEA/IL
AMENDED SHEET IPEA/IL
60. The method according to any one of claims 44 -46, wherein said mutation is in the coding region of said allele, a mutation in the regulatory region of said allele, or an epigenetic factor
61. The method according to any one of claims 44 -46, wherein said mutation is a silencing mutation, a knockdown mutation, a knockout mutation, a loss of function mutation or any combination thereof.
62. The method according to any one of claims 44 -46, wherein said mutation is generated in planta.
63. The method according to any one of claims 44 -46, wherein said mutation 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:22-SEQ ID NO:916 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA
sequence selected from the group consisting of SEQ ID NO:22-916 and any combination thereof.
sequence selected from the group consisting of SEQ ID NO:22-SEQ ID NO:916 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA
sequence selected from the group consisting of SEQ ID NO:22-916 and any combination thereof.
64. The method according to claim 44- 46 wherein said mutation in said CsSP-1 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:22-SEQ ID NO:126 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA
sequence selected from the group consisting of SEQ ID NO:22-126 and any combination thereof.
sequence selected from the group consisting of SEQ ID NO:22-SEQ ID NO:126 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA
sequence selected from the group consisting of SEQ ID NO:22-126 and any combination thereof.
65. The method according to claim 44- 46 wherein said mutation in said CsSP-2 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:127-SEQ ID NO:211 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA
sequence selected from the group consisting of SEQ ID NO:127-211 and any combination thereof.
sequence selected from the group consisting of SEQ ID NO:127-SEQ ID NO:211 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA
sequence selected from the group consisting of SEQ ID NO:127-211 and any combination thereof.
66. The method according to claim 44- 46 wherein said mutation in said CsSP-3 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:212-SEQ ID NO:283 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA
AMENDED SHEET IPEA/IL
sequence selected from the group consisting of SEQ ID NO:212-283 and any combination thereof.
sequence selected from the group consisting of SEQ ID NO:212-SEQ ID NO:283 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA
AMENDED SHEET IPEA/IL
sequence selected from the group consisting of SEQ ID NO:212-283 and any combination thereof.
67. The method according to claim 44- 46 wherein said mutation in said CsSP5G-1 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:284-SEQ ID NO:516 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 284-516 and any combination thereof.
sequence selected from the group consisting of SEQ ID NO:284-SEQ ID NO:516 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 284-516 and any combination thereof.
68. The method according to claim 44- 46 wherein said mutation in said CsSP5G-2 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:517-SEQ ID NO:745 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:517-745 and any combination thereof.
sequence selected from the group consisting of SEQ ID NO:517-SEQ ID NO:745 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO:517-745 and any combination thereof.
69. The method according to claim 44- 46 wherein said mutation in said CsSP5G-3 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:746-SEQ ID NO:828 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 746-828 and any combination thereof.
sequence selected from the group consisting of SEQ ID NO:746-SEQ ID NO:828 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 746-828 and any combination thereof.
70. The method according to claim 44- 46 wherein said mutation in said CsSP5G-4 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:829-SEQ ID NO:916 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 829-916 and any combination thereof.
sequence selected from the group consisting of SEQ ID NO:829-SEQ ID NO:916 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA sequence selected from the group consisting of SEQ ID NO: 829-916 and any combination thereof.
71. The method according to any one of claims 64-70 wherein said gRNA sequence comprises a 3' NGG Protospacer Adjacent Nlotif (PAM).
AMENDED SHEET IPEA/IL
AMENDED SHEET IPEA/IL
72. The method according to any one of claims 64-70 wherein said construct is introduced into the plant cells via Agrobacterium infiltration, virus based plasmids for delivery of the genome editing molecules or mechanical insertion such as polyethylene glycol (PEG) mediated DNA transformation, electroporation or gene gun biolistics.
73. The method according to any one of claims 44-46, wherein said plant has decreased expression levels of at least one of said CsSP genes.
74. The method according to claim 73, wherein the sequence of said expressed CsSP gene is selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ
ID NO:6, SEQ ID NO:8 and SEQ ID NO:9 or a functional variant thereof.
ID NO:6, SEQ ID NO:8 and SEQ ID NO:9 or a functional variant thereof.
75. The method according to any one of claims 44 -46, wherein said plant has decreased expression levels of at least one of said CsSP5G genes.
76. The method according to claim 75, wherein the sequence of said expressed CsSP5G gene is selected from the group consisting of: SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:21 or a functional variant thereof.
NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:21 or a functional variant thereof.
77. The method according to any one of claims 44-46, wherein said plant is semi-determinant.
78. The method according to any one of claims 44 -46, wherein said plant has determinant growth habit.
79. The method according to any one of claims 44 -46, wherein said plant flowers earlier than a corresponding wild type cannabis plant.
80. The method according to any one of claims 44 -46, wherein said plant exhibits improved earliness as compared to a corresponding wild type cannabis plant.
81. The method according to any one of claims 44 -46, wherein said plant exhibits suppressed sympodial shoot termination as compared to a corresponding wild type cannabis plant.
AMENDED SHEET IPEA/IL
AMENDED SHEET IPEA/IL
82. The method according to any one of claims 44 -46, wherein said plant exhibits similar sympodial shoot termination as compared to a corresponding wild type cannabis plant.
83. The method according to any one of claims 44 -46, wherein said plant exhibits suppressed or reduced day-length sensitivity as compared to a corresponding wild type cannabis plant.
84. The method according to any one of claims 44 -46, 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.
indica, C. ruderalis and any hybrid or cultivated variety of the genus Cannabis.
85. A Cannabis plant, plant part or plant cell produced by the method according to any one of claims 44 -46, wherein said plant does not comprise a transgene.
86. A plant part, plant cell or plant seed of a plant produced by the method according to any one of claims 44 -46.
87. A tissue culture of regenerable cells, protoplasts or callus obtained from the modified Cannabis plant produced by the method according to any one of claims 44 -46.
88. The method according to any one of claims 44 -46, wherein said plant genotype is obtainable by deposit under accession number with NCIMB Aberdeen AB21 9YA, Scotland, UK
or with ATCC.
or with ATCC.
89. The method according to any one of claims 44 -46, wherein said at least one domestication trait is selected from the group consisting of reduced flowering time, earliness, synchronous flowering, reduced day-length sensitivity, determinant or semi-determinant architecture, early termination of sympodial cycling, earlier axillary shoot flowering, compact growth habit, reduced height, reduced number of sympodial units, adaptation to mechanical harvest, higher harvest index and any combination thereof.
90. The method according to any one of claims 44 -46, wherein said Cssp Cssp5g double mutant is characterized by having a more than additive effect on a trait selected from the group consisting of compactness, earlier axillary shoot flowering, earlier termination of sympodial cycling, harvest index and any combination thereof as compared to wild type and/or sp mutant Cannabis plants.
AMENDED SHEET IPEA/IL
AMENDED SHEET IPEA/IL
91. An isolated nucleotide sequence having at least 75% sequence identity to a CsSP genomic nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:4 and SEQ ID NO:7.
NO:4 and SEQ ID NO:7.
92. An isolated nucleotide sequence having at least 75% sequence identity to a CsSP5G
genomic nucleotide sequence selected from the group consisting of SEQ ID
NO:10, SEQ
ID NO:13, SEQ ID NO:16 and SEQ ID NO:19.
genomic nucleotide sequence selected from the group consisting of SEQ ID
NO:10, SEQ
ID NO:13, SEQ ID NO:16 and SEQ ID NO:19.
93. An isolated nucleotide sequence having at least 75% sequence identity to a CsSP nucleotide coding sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8.
94. An isolated nucleotide sequence having at least 75% sequence identity to a CsSP5G
nucleotide coding sequence selected from the group consisting of SEQ ID NO:11, SEQ ID
NO:14, SEQ ID NO:17 and SEQ ID NO:20.
nucleotide coding sequence selected from the group consisting of SEQ ID NO:11, SEQ ID
NO:14, SEQ ID NO:17 and SEQ ID NO:20.
95. An isolated amino acid sequence having at least 75% sequence similarity to a CsSP amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6 and SEQ
ID NO:9.
ID NO:9.
96. An isolated amino acid sequence having at least 75% sequence similarity to a CsSP5G
amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID
NO:15, SEQ ID NO:18 and SEQ ID NO:21.
amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID
NO:15, SEQ ID NO:18 and SEQ ID NO:21.
97. An isolated nucleotide sequence having at least 75% sequence identity to a CsSP-targeted gRNA nucleotide sequence as set forth in SEQ ID NO:22-283.
98. An isolated nucleotide sequence having at least 75% sequence identity to a CsSP5G-targeted gRNA nucleotide sequence as set forth in SEQ ID NO:284-916.
99. Use of a nucleotide sequence as set forth in at least one of SEQ ID NO:22-126 and any combination thereof for targeted genome modification of Cannabis SP-1 (CsSP-1) allele.
100. Use of a nucleotide sequence as set forth in at least one of SEQ ID
NO:127-211 and any combination thereof for targeted genome modification of Cannabis SP-2 (CsSP-2) allele.
AMENDED SHEET IPEA/IL
NO:127-211 and any combination thereof for targeted genome modification of Cannabis SP-2 (CsSP-2) allele.
AMENDED SHEET IPEA/IL
101. Use of a nucleotide sequence as set forth in at least one of SEQ ID
NO:212-283 and any combination thereof for targeted genome modification of Cannabis SP-3 (CsSP-3) allele.
NO:212-283 and any combination thereof for targeted genome modification of Cannabis SP-3 (CsSP-3) allele.
102. Use of a nucleotide sequence as set forth in at least one of SEQ ID
NO:284-516 and any combination thereof for targeted genome modification of Cannabis SP5G-1 (CsSP5G-1) allele.
NO:284-516 and any combination thereof for targeted genome modification of Cannabis SP5G-1 (CsSP5G-1) allele.
103. Use of a nucleotide sequence as set forth in at least one of SEQ ID
NO:517-745 and any combination thereof for targeted genome modification of Cannabis SP5G-2 (CsSP5G-2) allele.
NO:517-745 and any combination thereof for targeted genome modification of Cannabis SP5G-2 (CsSP5G-2) allele.
104. Use of a nucleotide sequence as set forth in at least one of SEQ ID
NO:746-828 and any combination thereof for targeted genome modification of Cannabis SP5G-3 (CsSP5G-3) allele.
NO:746-828 and any combination thereof for targeted genome modification of Cannabis SP5G-3 (CsSP5G-3) allele.
105. Use of a nucleotide sequence as set forth in at least one of SEQ ID
NO:829-916 and any combination thereof for targeted genome modification of Cannabis SP5G-4 (CsSP5G-4) allele.
AMENDED SHEET IPEA/IL
NO:829-916 and any combination thereof for targeted genome modification of Cannabis SP5G-4 (CsSP5G-4) allele.
AMENDED SHEET IPEA/IL
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201962854036P | 2019-05-29 | 2019-05-29 | |
| US62/854,036 | 2019-05-29 | ||
| PCT/IL2020/050592 WO2020240560A1 (en) | 2019-05-29 | 2020-05-27 | Cannabis plants with improved yield |
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| Publication Number | Publication Date |
|---|---|
| CA3142241A1 true CA3142241A1 (en) | 2020-12-03 |
Family
ID=73551993
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA3142241A Pending CA3142241A1 (en) | 2019-05-29 | 2020-05-27 | Cannabis plants with improved yield |
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| Country | Link |
|---|---|
| EP (1) | EP3975698A4 (en) |
| CN (1) | CN115942868A (en) |
| AU (1) | AU2020284184A1 (en) |
| CA (1) | CA3142241A1 (en) |
| CO (1) | CO2021016207A2 (en) |
| IL (1) | IL288219A (en) |
| MX (1) | MX2021014688A (en) |
| WO (1) | WO2020240560A1 (en) |
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| EP4156912A4 (en) * | 2020-05-27 | 2024-01-03 | Canbreed Ltd | Cannabis plants with improved agronomic traits |
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| EP2573178A3 (en) * | 2007-07-10 | 2013-07-24 | Monsanto Technology LLC | Transgenic plants with enhanced agronomic traits |
| US20160177404A1 (en) * | 2011-08-18 | 2016-06-23 | Courtagen Life Sciences Inc. | Cannabis genomes and uses thereof |
| IL262264B1 (en) | 2016-04-11 | 2024-04-01 | Cold Spring Harbor Laboratory | Florigen pathway toolkit |
| MX2019001968A (en) * | 2016-08-18 | 2019-10-02 | Canopy Growth Corp | Plants and methods for increasing and decreasing synthesis of cannabinoids. |
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2020
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2021
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| MX2021014688A (en) | 2022-01-18 |
| CN115942868A (en) | 2023-04-07 |
| EP3975698A1 (en) | 2022-04-06 |
| AU2020284184A2 (en) | 2022-01-06 |
| EP3975698A4 (en) | 2022-08-17 |
| WO2020240560A1 (en) | 2020-12-03 |
| ZA202110886B (en) | 2023-05-31 |
| CO2021016207A2 (en) | 2021-12-10 |
| AU2020284184A1 (en) | 2021-12-23 |
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