Note: Descriptions are shown in the official language in which they were submitted.
METHODS FOR INCREASING POWDERY MILDEW RESISTANCE IN CANNABIS
FIELD OF THE INVENTION
The present disclosure relates to conferring pathogen resistance in Cannabis
plants. More
particularly, the current invention pertains to producing fungal resistant
Cannabis plants by
controlling genes conferring susceptibility to such pathogens.
BACKGROUND OF THE INVENTION
Cannabis is one of the oldest domesticated plants with evidence of being used
by a vast array of
ancient cultures. It is thought to have originated from central Asia from
which it was spread by
humans to China, Europe, the Middle East and the Americas. Thus, Cannabis has
been bred by
many different cultures for various uses such as food, fiber and medicine
since the dawn of
agricultural societies. In the last few decades, Cannabis breeding has stopped
as it became illegal
and non-economic to do so. With the recent legislation converting Cannabis
back to legality, there
is a growing need for the implementation of new and advanced breeding
techniques in future
Cannabis breeding programs. This will allow speeding up the long process of
classical breeding
and accelerate reaching new and genetically improved Cannabis varieties for
fiber, food and
medicine products. Developing and implementing molecular biology tools to
support the breeders,
will allow creating new fungal resistant traits and tracking the movement of
such desired traits
across breeders germplasm.
Currently, breeding of Cannabis plants is mostly done by small Cannabis
growers. There is very
limited if any molecular tools supporting or leading the breeding process.
Traditional Cannabis
breeding is done by mixing breeding material with hope to find the desired
traits and phenotypes
by random crosses. These methods have allowed the construction of the leading
Cannabis varieties
on the market today. As the cultivation of Cannabis intensifies in protected
structures such as
greenhouses and closed growth chambers, such an environment encourages the
prevalence of
certain diseases, with the lead cause being fungi.
Powdery mildew is a fungal disease that affects a wide range of plants.
Powdery mildew diseases
are caused by many different species of fungi in the order Erysiphales, with
Podosphaera xanthii
being the most commonly reported cause. Powdery mildew is one of the easier
plant diseases to
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Date Recue/Date Received 2022-06-30
identify, as its symptoms are quite distinctive. Infected plants display white
powdery spots on the
leaves and stems. The lower leaves are the most affected, but the mildew can
appear on any above-
ground part of the plant. As the disease progresses, the spots get larger and
denser as large numbers
of asexual spores are formed, and the mildew may spread up and reduce the
length of the plant.
Powdery mildew grows well in environments with high humidity and moderate
temperatures.
Greenhouses provide an ideal moist, temperate environment for the spread of
the disease. This
causes harm to agricultural and horticultural practices where powdery mildew
may thrive in a
greenhouse setting. In an agricultural or horticultural setting, the pathogen
can be controlled using
chemical methods, bio organic methods, and genetic resistance. It is important
to be aware of
powdery mildew and its management as the resulting disease can significantly
reduce important
crop yields.
MLO proteins function as negative regulators of plant defense to powdery
mildew disease. Loss-
of-function mlo alleles in barley, Arabidopsis and tomato have been reported
to lead to broad-
spectrum and durable resistance to the fungal pathogen causing powdery mildew.
US6211433 and US6576814 describe modulating the expression of Mb o genes in
Maize by
producing transgenic plants comprising mutation-induced recessive alleles of
maize Mb.
However, such methods require genetically modifying the plant genome,
particularly transforming
plants with external foreign genes that enhance disease resistance.
US2018208939 discloses the generation of mutant wheat lines with mutations
inactivating MLO
alleles which confer heritable resistance to powdery mildew fungus.
Cannabis cultivation has always suffered from fungal diseases due to high
humidity growing
conditions in growth rooms or greenhouses.
In view of the above there is a heightened immediate need for the development
of Cannabis plants
that carry genetic resistance to fungal diseases, thereby reducing or
eliminating the need for
fungicide use in the cultivation of Cannabis. In addition, there is a need for
non-GMO, advanced
breeding programs of Cannabis for food, medicine and fiber (Hemp) production.
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Date Recue/Date Received 2022-06-30
SUMMARY OF THE INVENTION
It is therefore one object of the present invention to disclose a modified
Cannabis plant exhibiting
enhanced resistance to powdery mildew (PM), wherein the modified plant
comprises a mutated
Cannabis mlol (Csmlol) allele, the mutated allele comprising a genomic
modification selected
from an indel of 14 bp at position corresponding to position 12 of SEQ ID NO:
882, or a fraction
thereof, or a nucleic acid insertion at position corresponding to position 104-
105 of SEQ ID NO:
882, or a combination thereof.
It is another object of the present invention to disclose the modified
Cannabis plant as defined
above, wherein the indel comprises a sequence as set forth in SEQ ID NO:883 or
a fraction thereof.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the Csmlol mutant allele comprises a nucleic acid
sequence
corresponding to the sequence as set forth in SEQ ID NO:884, or a nucleic acid
sequence
corresponding to the sequence as set forth in SEQ ID NO:885, or a nucleic acid
sequence
corresponding to the sequence as set forth in SEQ ID NO:886, or a homologue
having at least 80%
sequence identity to the nucleic acid sequence of the mutated Csmlol allele,
or a complementary
sequence thereof, or any combination thereof.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the Csmlol mutant allele comprises a nucleic acid
sequence
corresponding to the sequence as set forth in SEQ ID NO:886, or a homologue
having at least 80%
sequence identity to the nucleic acid sequence of the mutated Csmlol allele.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the mutated Csmlol allele confers an enhanced
resistance to powdery
mildew as compared to a Cannabis plant comprising a wild type CsML01 allele
having a nucleic
acid sequence as set forth in SEQ ID NO:882 and/or having a nucleic acid
sequence as set forth in
SEQ ID NO:1 or a functional variant thereof.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the functional variant has at least 80% sequence
identity to the
corresponding CsML01 nucleotide sequence.
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Date Recue/Date Received 2022-06-30
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the mutated allele comprising a deletion of 14 bp at
position 389 of SEQ
ID NO: 1, or a nucleic acid insertion at position 482-483 of SEQ ID NO: 1, or
a combination
thereof.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the mutated Csmlol allele is generated using genome
editing.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the plant has decreased expression levels of Mlol
protein, relative to a
Cannabis plant lacking the mutated Csmlol allele.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the mutated Csmlol allele is generated using
mutagenesis, small
interfering RNA (siRNA), microRNA (miRNA), artificial miRNA (amiRNA), DNA
introgression,
endonucleases or any combination thereof.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the endonuclease is selected from the group
consisting of CRISPR
(Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-
associated (Cas) gene
(CRISPR/Cas), Transcription activator-like effector nuclease (TALEN), Zinc
Finger Nuclease
(ZFN), meganuclease or any combination thereof.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the Cas gene is selected from the group consisting
of Cas3, Cas4, Cas5,
Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9,
Cas10, Castl Od,
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, Csnl, 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 Cu1966,
bacteriophages Cas such
as Cas1T3$ (Cas-phi) and any combination thereof.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the plant comprises a DNA construct, the DNA
construct comprising a
promoter operably linked to a nucleotide sequence encoding a plant optimized
Cas9 endonuclease,
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Date Recue/Date Received 2022-06-30
wherein the plant optimized Cas9 endonuclease is capable of binding to and
creating a double
strand break in a genomic target sequence of the plant genome.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the DNA construct further comprises gRNA targeted to
at least one
CsML01 allele.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the gRNA has a nucleic acid sequence corresponding
to a sequence
selected from the group consisting of SEQ ID NO:17, SEQ ID NO:43 and SEQ ID
NO:50 or a
complementary sequence thereof, or any combination thereof.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the genome modification is a silencing mutation, a
knockdown mutation,
a knockout mutation, a loss of function mutation or any combination thereof.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the genome modification is an insertion, deletion,
indel or substitution.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the genome modification is an induced mutation in
the coding region of
the allele, a mutation in the regulatory region of the allele, a mutation in a
gene downstream in
the MLO pathogen response pathway and/or an epigenetic factor.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the genome modification is generated via
introduction (a) Cos DNA and
gRNA sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:43
and SEQ
ID NO:50 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex
comprising Cas
protein and gRNA sequence selected from the group consisting of SEQ ID NO:17,
SEQ ID NO:43
and SEQ ID NO:50 and any combination thereof.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the gRNA sequence comprises a 3' Protospacer
Adjacent Motif (PAM)
selected from the group consisting of NGG (SpCas), NNNNGATT (NmeCas9),
NNAGAAW (StCas9), NAAAAC (TdCas9), NNGRRT (SaCas9) and TBN (Cas-phi).
Date Recue/Date Received 2022-06-30
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the PM is selected from the group consisting of
Golovinomyces
cichoracearum, Golovinomyces ambrosiae and a mixture thereof.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the Cannabis plant is selected from the group of
species that includes,
but is not limited to, Cannabis saliva (C. saliva), C. indica, C. ruderalis
and any hybrid or
cultivated variety of the genus Cannabis.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the Cannabis plant does not comprise a transgene.
It is another object of the present invention to disclose a modified Cannabis
plant, progeny plant,
plant part or plant cell as defined in any of the above.
It is another object of the present invention to disclose a plant part, plant
cell or plant seed of a
modified plant as defined in any of the above.
It is another object of the present invention to disclose a tissue culture of
regenerable cells,
protoplasts or callus obtained from the modified Cannabis plant as defined in
any of the above.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the plant genotype is obtainable by deposit under
accession number with
NCIMB Aberdeen AB21 9YA, Scotland, UK.
It is another object of the present invention to disclose a modified Cannabis
plant exhibiting
enhanced resistance to powdery mildew (PM), wherein the modified plant
comprises a targeted
genome modification conferring reduced expression of a Cannabis ML01 (CsML01)
gene as
compared to a Cannabis plant lacking the targeted genome modification, the
targeted genome
modification generates a mutated Cannabis mlol (Csmlol) allele comprising a
deletion of a
nucleic acid sequence as set forth in SEQ ID NO:883 or a fraction thereof as
compared to the wild
type CsML01 allele comprising a sequence as set forth in SEQ ID NO:1, or a
nucleic acid insertion
at position 482-483 of SEQ ID NO:1, or a combination thereof.
It is another object of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein the mutated allele comprising a deletion of 14 bp at
position 389 of SEQ
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Date Recue/Date Received 2022-06-30
ID NO: 1, or a nucleic acid insertion at position 482-483 of SEQ ID NO: 1, or
a combination
thereof.
It is another object of the present invention to disclose a method for
producing a modified Cannabis
plant as defined in any of the above, the method comprises introducing using
targeted genome
modification, at least one genomic modification conferring reduced expression
of at least one
Cannabis ML01 (CsML01) allele as compared to a Cannabis plant lacking the
targeted genome
modification, the genomic modification generates a mutated Cannabis mlol
(Csmlol) allele
comprising an indel of 14 bp at a position corresponding to position 12 of SEQ
ID NO: 882 or a
fraction thereof, or a nucleic acid insertion at position corresponding to
position 104-105 of SEQ
ID NO: 882, or a combination thereof.
It is another object of the present invention to disclose the method as
defined above, comprises
steps of introducing a loss of function mutation into the CsML01 allele using
targeted genome
modification.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the indel comprises a sequence as set forth in SEQ ID NO:883 or a
fraction thereof.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the Csmlol mutant allele comprises a nucleic acid sequence
corresponding to the
sequence as set forth in SEQ ID NO:884, or a nucleic acid sequence
corresponding to the sequence
as set forth in SEQ ID NO:885, or a nucleic acid sequence corresponding to the
sequence as set
forth in SEQ ID NO:886, or a homologue having at least 80% sequence identity
to the nucleic acid
sequence of the mutated Csmlol allele, or a complementary sequence thereof, or
any combination
thereof.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the Csmlol mutant allele comprises a nucleic acid sequence
corresponding to the
sequence as set forth in SEQ ID NO:886, or a homologue having at least 80%
sequence identity
to the nucleic acid sequence of the mutated Csmlol allele.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the mutated Csmlol allele confers an enhanced resistance to powdery
mildew as
compared to a Cannabis plant comprising a wild type CsML01 allele having a
nucleic acid
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Date Recue/Date Received 2022-06-30
sequence as set forth in SEQ ID NO:882 and/or having a nucleic acid sequence
as set forth in SEQ
ID NO:1 or a functional variant thereof.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the functional variant has at least 80% sequence identity to the
corresponding CsML01
nucleotide sequence.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the mutated allele comprising a deletion of 14 bp at position 389 of
SEQ ID NO: 1, or a
nucleic acid insertion at position 482-483 of SEQ ID NO: 1, or a combination
thereof.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the modified plant has decreased levels of at least one Mb o protein
as compared to wild
type Cannabis plant.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the genome modification is introduced using CRISPR (Clustered
Regularly Interspaced
Short Palindromic Repeats) and CRISPR-associated (Cas) gene (CRISPR/Cas),
Transcription
activator-like effector nuclease (TALEN), Zinc Finger Nuclease (ZFN),
meganuclease or any
combination thereof.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the Cas gene is selected from the group consisting of Cas3, Cas4,
Cas5, Cas5e (or CasD),
Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Castl0d,
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, Csnl, 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 Cu1966, bacteriophages Cas such as
Cas(13$ (Cas-phi) and
any combination thereof.
It is another object of the present invention to disclose the method as
defined in any of the above,
comprising steps of introducing an expression vector comprising a promoter
operably linked to a
nucleotide sequence encoding a plant optimized Cas9 endonuclease and gRNA
targeted to at least
one CsML01 allele.
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Date Recue/Date Received 2022-06-30
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the gRNA nucleotide sequence targeting the CsML01 allele is selected
from the group
consisting of SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary
sequence
thereof.
It is another object of the present invention to disclose the method as
defined in any of the above,
comprising steps of introducing and co-expressing in a Cannabis plant Cas9 and
gRNA targeted
to CsML01 gene and screening for induced targeted mutations conferring reduced
expression of
the CsML01 gene.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the genomic modification is in the coding region of the allele, a
mutation in the regulatory
region of the allele, a mutation in a gene downstream in the MLO pathogen
response pathway or
an epigenetic factor.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the genomic modification is selected from the group consisting of a
silencing mutation, a
knockdown mutation, a knockout mutation, a loss of function mutation and any
combination
thereof.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the genomic modification is an insertion, deletion, indel or
substitution mutation.
It is another object of the present invention to disclose the method as
defined in any of the above,
further comprising steps of selecting a plant resistant to powdery mildew from
plants comprising
mutated Csmlol allele.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the selected plant is characterized by enhanced resistance to powdery
mildew as compared
to a Cannabis plant comprising a CsML01 nucleic acid comprising a nucleic acid
sequence as set
forth in SEQ ID NO:882.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the genetic modification in the CsML01 is generated in planta via
introduction of a
construct comprising (a) Cas DNA and gRNA sequence selected from the group
consisting of SEQ
ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence thereof,
and any
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Date Recue/Date Received 2022-06-30
combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas
protein and gRNA
sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:43 and
SEQ ID NO:50
or a complementary sequence thereof, and any combination thereof.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the gRNA nucleotide sequence comprises a 3' Protospacer Adjacent Motif
(PAM)
selected from the group consisting of NGG (SpCas), NNNNGATT (NmeCas9),
NNAGAAW (StCas9), NAAAAC (TdCas9), NNGRRT (SaCas9) and TBN (Cas-phi).
It is another object of the present invention to disclose the method as
defined in any of the above,
further comprising steps of regenerating a plant carrying the genomic
modification.
It is another object of the present invention to disclose the method as
defined in any of the above,
further comprising steps of screening the regenerated plants for a plant
resistant to powdery
mildew.
It is another object of the present invention to disclose the method as
defined a method for
conferring powdery mildew resistance to a Cannabis plant comprising producing
a plant according
to the method as defined in any of the above.
It is another object of the present invention to disclose a plant, plant part,
plant cell, tissue culture
or a seed obtained or obtainable by the method as defined in any of the above.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the powdery mildew is selected from the group of species consisting of
Golovinomyces
cichoracearum, Golovinomyces ambrosiae and a mixture thereof.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the Cannabis plant is selected from the group of species that
includes, but is not limited
to, Cannabis saliva (C. saliva), C. indica, C. ruderalis and any hybrid or
cultivated variety of the
genus Cannabis.
It is another object of the present invention to disclose a method of
determining the presence of a
mutant Csmlol allele in a Cannabis plant comprising assaying the Cannabis
plant for at least one
of the presence of an indel comprising a nucleic acid sequence as set forth in
SEQ ID NO:883, an
insertion at position 104-105 of SEQ ID NO: 882, a nucleic acid sequence
corresponding to the
sequence as set forth in SEQ ID NO:884, a nucleic acid sequence corresponding
to the sequence
Date Recue/Date Received 2022-06-30
as set forth in SEQ ID NO:885, a nucleic acid sequence corresponding to the
sequence as set forth
in SEQ ID NO:886, or a homologue having at least 80% sequence identity to the
nucleic acid
sequence of the mutated Csmlol allele, a complementary sequence thereof, or
any combination
thereof.
It is another object of the present invention to disclose a method for
identifying a Cannabis plant
with resistance to powdery mildew, the method comprises steps of: (a)
screening the genome of
the Cannabis plant for a mutated Csmlol allele, the mutated allele comprises a
genomic
modification selected from an indel of 14 bp at a position corresponding to
position 12 of SEQ ID
NO: 882 or a fraction thereof, or a nucleic acid insertion at position
corresponding to position 104-
105 of SEQ ID NO: 882, or a combination thereof; (b) optionally, regenerating
plants carrying the
genetic modification; and (c) optionally, screening the regenerated plants for
a plant resistant to
powdery mildew.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the genomic modification is a loss of function mutation.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the indel comprises a sequence as set forth in SEQ ID NO:883 or a
fraction and/or a
complementary sequence thereof.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the Csmlol mutant allele comprises a nucleic acid sequence
corresponding to the
sequence as set forth in SEQ ID NO:884, or a nucleic acid sequence
corresponding to the sequence
as set forth in SEQ ID NO:885, or a nucleic acid sequence corresponding to the
sequence as set
forth in SEQ ID NO:886, or a homologue having at least 80% sequence identity
to the nucleic acid
sequence of the mutated Csmlol allele, or a complementary sequence thereof, or
any combination
thereof.
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the method comprises steps of screening the Cannabis plant for the
presence of a deletion
in CsML01 gene comprising a nucleic acid sequence as set forth in SEQ ID NO:1,
the deletion
comprising a nucleotide sequence as set forth in SEQ ID NO: 883.
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Date Recue/Date Received 2022-06-30
It is another object of the present invention to disclose the method as
defined in any of the above,
wherein the modified Cannabis plant comprising a mutant Csmlol nucleic acid
conferring
enhanced resistance to powdery mildew as compared to a Cannabis plant
comprising a wild type
CsML01 nucleic acid.
It is another object of the present invention to disclose a method for down
regulation of Cannabis
ML01 (CsML01) gene, which comprises utilizing the nucleotide sequence as set
forth in at least
one of SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence thereof, and
a
combination thereof, for introducing a loss of function mutation into the
CsML01 gene using
targeted genome modification.
It is another object of the present invention to disclose an isolated amino
acid sequence having at
least 80% sequence identity to a nucleic acid sequence selected from the group
consisting of SEQ
ID NO:882-886, SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary
sequence or any combination thereof.
It is another object of the present invention to disclose use of a nucleotide
sequence as set forth in
SEQ ID NO: 883-886, SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 for
generating,
identifying and/or screening for a Cannabis plant comprising within its genome
mutant Csmlo
allele conferring resistance to PM.
It is another object of the present invention to disclose the use as defined
in any of the above,
wherein the presence of at least one nucleic acid sequence selected from the
group consisting of
SEQ ID NO:1, SEQ ID NO:883, SEQ ID NO:882 indicates that the Cannabis plant
comprises a
wild type CsML01 allele, and the presence of at least one nucleic acid
sequence selected from the
group consisting of SEQ ID NO:884, SEQ ID NO:885 and SEQ ID NO:886 indicates
that the
Cannabis plant comprises a mutant Csmoll allele.
It is another object of the present invention to disclose use of a nucleotide
sequence as set forth in
at least one of SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary
sequence
or any combination thereof for targeted genome modification of Cannabis ML01
(CsML01) gene.
It is another object of the present invention to disclose a detection kit for
determining the presence
or absence of a mutant Csmlo 1 allele in a Cannabis plant, comprising a
nucleic acid fragment
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Date Recue/Date Received 2022-06-30
comprising a sequence selected from SEQ ID NO:882-886, SEQ ID NO:17, SEQ ID
NO:43 and
SEQ ID NO:50 or a complementary sequence or any combination thereof.
It is another object of the present invention to disclose the detection kit as
defined above, wherein
the kit is useful for identifying a Cannabis plant with enhanced resistance to
powdery mildew.
BRIEF DESCRIPTION OF THE FIGURES
Exemplary non-limited embodiments of the disclosed subject matter will be
described, with
reference to the following description of the embodiments, in conjunction with
the figures. The
figures are generally not shown to scale and any sizes are only meant to be
exemplary and not
necessarily limiting. Corresponding or like elements are optionally designated
by the same
numerals or letters.
Figs 1A-C is presenting a photographic illustration of an infected Cannabis
plant leaf exhibiting
PM symptoms of white powdery spots on the leaves (Fig. 1A), an enlarged view
(X4) of fungal
asexual spore-carrying bodies (conidia) of Golovinomyces cichoracearum on
Cannabis leaf tissue
(Fig. 1B), and a microscopic imaging of Golovinomyces cichoracearum spores
(Fig. 1C);
Fig. 2A-B is schematically presenting WT plant cell penetrated by the fungal
appressorium leading
to haustorium establishment and infection by secondary hyphae (Fig. 2A), and
mlo knockout plant
cell into which the fungal spores are incapable of penetrating (Fig. 2B);
Fig. 3 is schematically presenting CRISPR/Cas9 mode of action as depicted by
Xie, Kabin, and
Yinong Yang. "RNA-guided genome editing in plants using a CRISPR¨Cas system."
Molecular
plant 6.6 (2013): 1975-1983;
Fig. 4A-D is photographically presenting GUS staining after transient
transformation of Cannabis
axillary buds (Fig. 4A), leaves (Fig. 4B), calli (Fig. 4C), and cotyledons
(Fig. 4D);
Fig. 5 is presenting regenerated Cannabis tissue;
Fig. 6 is photographically presenting PCR detection of Cas9 DNA in shoots of
Cannabis plants
transformed using biolistics;
Fig. 7A-B is illustrating in vitro cleavage activity of CRISPR/Cas9; a scheme
of genomic area
targeted for editing is shown in Fig. 7A, and a gel showing digestion of PCR
amplicon containing
13
Date Recue/Date Received 2022-06-30
the gRNA sequence by RNP complex containing Cas9 and gene specific gRNA is
shown in Fig.
7B;
Fig. 8 is presenting a schematic illustration of a DNA plasmid containing a
plant codon optimized
Cas9 nuclease from Streptococcus pyogenes (pcoSpCas9) and sgRNA sequences used
for
transformation, as embodiments of the present invention;
Fig. 9 schematically presents genomic localization of sgRNAs used for
targeting CsML01 first
exon, as embodiments of the present invention;
Fig. 10 presents genomic nucleotide sequence of the first exon (exon 1) of
wild type CsML01
targeted by three gRNA sequences;
Fig. 11 presents amino acid sequence of the first exon (exon 1) of wild type
CsML01;
Fig. 12 photographically presents detection of CsML01 PCR products showing
length variation
as a result of Cas9- mediated genome editing;
Fig. 13 schematically presents genome edited CsML01 DNA fragments produced by
the present
invention; and
Fig. 14 schematically presents nucleic acid sequence comparison of WT CsML01
and genome
edited Csmlol d1411 fragments produced by the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
In the following detailed description of the preferred embodiments, reference
is made to the
accompanying drawings that form a part hereof, and in which are shown by way
of illustration
specific embodiments in which the invention may be practiced. It is understood
that other
embodiments may be utilized and structural changes may be made without
departing from the
scope of the present invention. The present invention may be practiced
according to the claims
without some or all of these specific details. For the purpose of clarity,
technical material that is
known in the technical fields related to the invention has not been described
in detail so that the
present invention is not unnecessarily obscured.
The present invention provides a modified Cannabis plant exhibiting enhanced
resistance to
powdery mildew (PM), wherein the plant comprises a targeted genome
modification conferring
14
Date Recue/Date Received 2022-06-30
reduced expression of at least one Cannabis MLO (CsMLO) allele as compared to
a Cannabis
plant lacking said targeted genome modification.
The present invention is aimed at showing that lack of mildew resistance loci
0 (MLO) genes in
Cannabis is correlated with resistance to PM. It is herein disclosed that MLO
deletions are likely
to increase PM resistance in Cannabis. According to further aspects of the
invention, lack of certain
MLO genes is used as markers for pathogen resistance and may accelerate
breeding for more
resistant Cannabis lines.
According to one embodiment, the present invention provides a modified
Cannabis plant
exhibiting enhanced resistance to powdery mildew (PM), wherein said modified
plant comprises
a mutated Cannabis mlol (Csmlol) allele, said mutated allele comprising a
genomic modification
selected from an indel of 14 bp at position corresponding to position 12 of
SEQ ID NO: 882, or a
fraction thereof, or a nucleic acid insertion at position corresponding to
position 104-105 of SEQ
ID NO: 882, or a combination thereof.
According to a further embodiment of the present invention, the indel
comprises a sequence as set
forth in SEQ ID NO:883 or a fraction thereof.
According to a further embodiment of the present invention, the Csmlol mutant
allele comprises
a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID
NO:884, or a nucleic
acid sequence corresponding to the sequence as set forth in SEQ ID NO: 885, or
a nucleic acid
sequence corresponding to the sequence as set forth in SEQ ID NO:886, or a
homologue having
at least 80% sequence identity to the nucleic acid sequence of said mutated
Csmlol allele, or a
complementary sequence thereof, or any combination thereof.
According to a further embodiment of the present invention, the Csmlol mutant
allele comprises
a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID
NO:886, or a
homologue having at least 80% sequence identity to the nucleic acid sequence
of said mutated
Csmlol allele.
According to a further embodiment of the present invention, the mutated Csmlol
allele confers an
enhanced resistance to powdery mildew as compared to a Cannabis plant
comprising a wild type
CsML01 allele having a nucleic acid sequence as set forth in SEQ ID NO:882
and/or having a
nucleic acid sequence as set forth in SEQ ID NO:1 or a functional variant
thereof.
Date Recue/Date Received 2022-06-30
According to a further embodiment of the present invention, the functional
variant has at least 80%
sequence identity to the corresponding CsML01 nucleotide sequence.
According to a further embodiment of the present invention, the mutated allele
comprising a
deletion of 14 bp at position 389 of SEQ ID NO: 1, or a nucleic acid insertion
at position 482-483
of SEQ ID NO: 1, or a combination thereof.
According to a further embodiment of the present invention, the mutated Csmlol
allele is
generated using genome editing.
It is further within the scope of the present invention to provide, a modified
Cannabis plant
exhibiting enhanced resistance to powdery mildew (PM), wherein said modified
plant comprises
a targeted genome modification conferring reduced expression of a Cannabis
ML01 (CsML01)
gene as compared to a Cannabis plant lacking said targeted genome
modification, said targeted
genome modification generates a mutated Cannabis mlol (C sml ol) allele
comprising a deletion
of a nucleic acid sequence as set forth in SEQ ID NO:883 or a fraction thereof
as compared to the
wild type CsML01 allele comprising a sequence as set forth in SEQ ID NO:1, or
a nucleic acid
insertion at position 482-483 of SEQ ID NO:1, or a combination thereof.
According to a further aspect of the present invention, a method for producing
a modified Cannabis
plant as defined in any of the above is provided. The method comprises
introducing using targeted
genome modification, at least one genomic modification conferring reduced
expression of at least
one Cannabis ML01 (CsML01) allele as compared to a Cannabis plant lacking said
targeted
genome modification, said genomic modification generates a mutated Cannabis
mlol (C sml ol)
allele comprising an indel of 14 bp at a position corresponding to position 12
of SEQ ID NO: 882
or a fraction thereof, or a nucleic acid insertion at position corresponding
to position 104-105 of
SEQ ID NO: 882, or a combination thereof.
According to further aspects of the present invention, a method of determining
the presence of a
mutant Csmlol allele in a Cannabis plant is provided. The method comprising
assaying the
Cannabis plant for at least one of the presence of an indel comprising a
nucleic acid sequence as
set forth in SEQ ID NO:883, an insertion at position 104-105 of SEQ ID NO:
882, a nucleic acid
sequence corresponding to the sequence as set forth in SEQ ID NO:884, a
nucleic acid sequence
corresponding to the sequence as set forth in SEQ ID NO:885, a nucleic acid
sequence
corresponding to the sequence as set forth in SEQ ID NO:886, or a homologue
having at least 80%
16
Date Recue/Date Received 2022-06-30
sequence identity to the nucleic acid sequence of said mutated Csmlol allele,
a complementary
sequence thereof, or any combination thereof.
It is further within the scope to provide a method for identifying a Cannabis
plant with resistance
to powdery mildew, said method comprises steps of: (a) screening the genome of
said Cannabis
plant for a mutated Csmlol allele, said mutated allele comprises a genomic
modification selected
from an indel of 14 bp at a position corresponding to position 12 of SEQ ID
NO: 882 or a fraction
thereof, or a nucleic acid insertion at position corresponding to position 104-
105 of SEQ ID NO:
882, or a combination thereof; (b) optionally, regenerating plants carrying
said genetic
modification; and (c) optionally, screening said regenerated plants for a
plant resistant to powdery
mildew.
It is further within the scope of the present invention to provide a method
for down regulation of
Cannabis ML01 (CsML01) gene, which comprises utilizing the nucleotide sequence
as set forth
in at least one of SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence
thereof, and a
combination thereof, for introducing a loss of function mutation into said
CsML01 gene using
targeted genome modification.
The present invention further provides an isolated amino acid sequence having
at least 80%
sequence identity to a nucleic acid sequence selected from the group
consisting of SEQ ID
NO:882-886, SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary
sequence
or any combination thereof.
It is also within the scope to disclose a use of a nucleotide sequence as set
forth in SEQ ID NO:
883-886, SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 for generating,
identifying and/or
screening for a Cannabis plant comprising within its genome mutant Csmlo
allele conferring
resistance to PM.
It is also within the scope to disclose a use of a nucleotide sequence as set
forth in at least one of
SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence or any
combination thereof for targeted genome modification of Cannabis ML01 (CsML01)
gene.
According to further aspects, the present invention provides a detection kit
for determining the
presence or absence of a mutant Csmlol allele in a Cannabis plant, comprising
a nucleic acid
17
Date Recue/Date Received 2022-06-30
fragment comprising a sequence selected from SEQ ID NO:882-886, SEQ ID NO:17,
SEQ ID
NO:43 and SEQ ID NO:50 or a complementary sequence or any combination thereof.
According to an embodiment of the present invention, the targeted genome
modification is in a
CsMLO allele having a wild type genomic nucleotide sequence selected from the
group consisting
of CsML01 having a sequence as set forth in SEQ ID NO:1 or a functional
variant thereof,
CsML02 having a sequence as set forth in SEQ ID NO:4 or a functional variant
thereof and
CsML03 having a sequence as set forth in SEQ ID NO:7 or a functional variant
thereof.
According to a further embodiment of the present invention, the functional
variant has at least
75%, preferably 80% sequence identity to the corresponding CsMLO nucleotide
sequence.
According to a further embodiment of the present invention, the modified
Cannabis plant has
decreased expression levels of at least one Mb o protein, relative to a
Cannabis plant lacking the at
least one genome modification.
According to a further embodiment of the present invention, the genome
modification is
introduced using mutagenesis, small interfering RNA (siRNA), microRNA (miRNA),
artificial
miRNA (amiRNA), DNA introgression, endonucleases or any combination thereof.
According to a further embodiment of the present invention, the genetic
modification is introduced
using targeted genome modification, preferably said genetic modification is
introduced using an
endonuclease.
According to a further embodiment of the present invention, the genome
modification is
introduced using guide RNA, e.g. single guide RNA (sgRNA) designed and
targeted to mutate
CsML01 gene, said sgRNA nucleotide sequence is selected from the group
consisting of SEQ ID
NO:17, SEQ ID NO:43 and SEQ ID NO:50.
According to a further embodiment of the present invention, the modified
Cannabis plant
comprises at least one mutated CsML01 allele comprising a nucleotide sequence
selected from
the group consisting of a nucleotide sequence as set forth in SEQ ID NO:875, a
nucleotide
sequence as set forth in SEQ ID NO:877, a nucleotide sequence as set forth in
SEQ ID NO:880 or
a homologue having at least 80% sequence identity to the nucleotide sequence
of said at least one
mutated CsML01 allele or a combination thereof.
18
Date Recue/Date Received 2022-06-30
According to a further embodiment of the present invention, the modified
Cannabis plant
comprises at least one silencing mutation, a knockdown mutation, a knockout
mutation, a loss of
function mutation or any combination thereof in at least one gene or allele
selected from the group
consisting of CsML01, CsML02 and CsML03.
According to a further embodiment of the present invention the mutated CsML01
allele comprises
a deletion having a nucleotide sequence as set forth in SEQ ID NO. :876, SEQ
ID NO. :879 or SEQ
ID NO.:881.
According to a further embodiment of the present invention, the mutated CsML01
allele confers
an enhanced resistance to powdery mildew as compared to a Cannabis plant
comprising a wild
type CsML01 allele sequence.
According to a further embodiment of the present invention, the wild type
CsML01 allele
comprises a nucleic acid sequence as set forth in at least one of SEQ ID
NO:873, SEQ ID NO:876,
SEQ ID NO: 879 or SEQ ID NO:881. According to a further embodiment of the
present invention
the present invention provides modified Cannabis plant exhibiting enhanced
resistance to powdery
mildew (PM) compared to wild type Cannabis plant, wherein the modified plant
comprises a
genetic modification conferring reduced expression of at least one Cannabis
MLO (CsMLO) allele.
The present invention further provides methods for producing the
aforementioned modified
Cannabis plant using genome editing or modification techniques.
Powdery mildew (PM) is a major fungal disease that threatens thousands of
plant species. Powdery
mildew is commonly controlled by frequent applications of fungicides, having
negative effects on
the environment, and leading to additional costs for growers. To reduce the
amount of chemicals
required to control this pathogen, the development of resistant crop varieties
is a priority.
It is herein acknowledged that PM pathogenesis is associated with up-
regulation of specific MLO
genes during early stages of infection, causing down-regulation of plant
defense pathways. These
up-regulated genes are responsible for PM susceptibility (S-genes) and their
knock-out cause
durable and broad-spectrum resistance.
As the Cannabis legal market is expanding worldwide, this agricultural crop
will gradually move
from indoor growing facilities to simple low cost greenhouses to enable mass
production at
reduced operational costs. One of the major challenges facing this transition
is the lack of
19
Date Recue/Date Received 2022-06-30
compatible genetics (strains) adapted for green house growth and more
specifically genetic fungal
resistances. Cannabis susceptibility to fungal diseases results in damages and
losses to the grower
and forces the widespread use of fungicides. Excessive fungicide use poses
health threats to
Cannabis consumers.
To date, there are no fungal disease resistant Cannabis varieties on the
market. Classical breeding
programs dedicated to the end of creating fungal disease resistant Cannabis
varieties are virtually
impossible due to limited genetic variation, legal constraints on import and
export of genetic
material and limited academic knowledge and gene banks involved is such
projects. In addition,
traditional breeding is a long process with low rates of success and
certainty, as it is based on trial
and error.
The solution proposed by the current invention is using genome editing such as
the CRISPR/Cas
system in order to create fungal disease resistant Cannabis varieties.
Breeding using genome
editing allows a precise and significantly shorter breeding process in order
to achieve these goals
with a much higher success rate. Thus genome editing, has the potential to
generate improved
varieties faster and at a lower cost. By using genome editing to generate
Powdery Mildew (PM)
resistant Cannabis varieties, the current disclosure will allow growers
worldwide to supply a safer
product to Cannabis consumers.
It is further noted that using genome editing is considered as non GMO by the
Israeli regulator and
in the US, the USDA has already classified a dozen of genome edited plant as
non regulated and
non GMO (https ://www.usda.gov/m edi a/pres s-rel eases/20 1 8/03/2 8/s
ecretary-perdue-i ssues-usda-
statement-plant-breeding-innovation).
The Cannabis industry's value chain is based on a steady supply of high
quality consistent product.
Due to lack of suitable genetics adapted for intensive agriculture production,
most growing
methods are based on cloning as a mean of vegetative propagation in order to
ensure genetic
consistency of the plant material. These methods are outdated, expensive and
not fit for purpose.
The lack of Cannabis strains that are disease resistant, stable and uniform,
pose a threat to the
ability of supplying the industry with the raw material needed to support
itself.
Legal limitations and outdated breeding techniques significantly hamper the
efforts of generating
new and improved Cannabis varieties fit for intensive agriculture.
Date Recue/Date Received 2022-06-30
Cannabis legalization in certain countries has increased significantly the
number of Cannabis
growers and area used for growing. One possible solution is moving growing
Cannabis into
greenhouses (protected growing facilities) like the vegetable industry has
been doing for the last
few decades. Unlike the vegetable industry, Cannabis is based on vegetative
propagation while
vegetables are grown through seeds. In addition, Cannabis growers are using
Cannabis strains that
were bred for indoor cultivation and are now using those for their greenhouse
operations. This
situation is obviously not ideal and causes many logistic issues for the
growers. For example, since
Cannabis plants require short days for the induction of flowering, growers
install darkening
curtains in the greenhouse to control day length for the plants. This
artificial darkening results in
increased humidity in the greenhouse thus creating optimal conditions for
fungal pathogens to
spread and thrive. These conditions force growers to intensively use
fungicides to control pathogen
populations. With strict regulatory constraints in place across the legalized
states, these conditions
pose a great challenge for sustainable Cannabis production and consumer
health.
The next step for the Cannabis industry is the adoption and use of hybrid
seeds for propagation,
which is common practice in the conventional seed industry (from field crops
to vegetables). In
addition, breeding for basic agronomic traits that are completely lacking in
currently available
Cannabis varieties (with an emphasis on disease resistances) will
significantly increase grower's
productivity. This will allow growing and supplying high quality raw material
for the Cannabis
industry.
In order to generate a reproducible product, Cannabis growers are currently
using vegetative
propagation (cloning or tissue culture). However, in conventional
agricultural, genetic stability of
field crops and vegetables is maintained by using Fl hybrid seeds. These
hybrids are generated by
crossing homozygous parental lines.
Currently, breeding of Cannabis plants is mostly done by small Cannabis
growers. There is very
limited if any molecular tools supporting or leading the breeding process.
Traditional Cannabis
breeding is done by mixing breeding material with hope to find the desired
traits and phenotypes
by random crosses.
The present invention provides for the first time enhanced resistant Cannabis
plants to fungal
diseases. The current invention disclose the generation of non-transgenic
Cannabis plant resistant
to the powdery mildew fungal disease, using the genome editing technology,
e.g., the
21
Date Recue/Date Received 2022-06-30
CRISPR/Cas9 tool. The generated mutations can be readily introduced into elite
or locally adapted
Cannabis lines rapidly, with relatively minimal effort and investment.
As used herein the term "about" denotes 25% of the defined amount or measure
or value.
As used herein the term "similar" denotes a correspondence or resemblance
range of about
20%, particularly 15%, more particularly about 10% and even more
particularly about 5%.
A "plant" as used herein refers to any plant at any stage of development,
particularly a seed plant.
The term "plant" includes the whole plant or any parts or derivatives thereof,
such as plant cells,
seeds, plant protoplasts, plant cell tissue culture from which tomato plants
can be regenerated,
plant callus or calli, meristematic cells, microspores, embryos, immature
embryos, pollen, ovules,
anthers, fruit, flowers, leaves, cotyledons, pistil, seeds, seed coat, roots,
root tips and the like.
The term "plant cell" used herein refers to a structural and physiological
unit of a plant,
comprising a protoplast and a cell wall. The plant cell may be in a form of an
isolated single cell
or a cultured cell, or as a part of higher organized unit such as, for
example, plant tissue, a plant
organ, or a whole plant.
The term "plant cell culture" as used herein means cultures of plant units
such as, for example,
protoplasts, regenerable cells, cell culture, cells, cells in plant tissues,
pollen, pollen tubes, ovules,
embryo sacs, zygotes and embryos at various stages of development, leaves,
roots, root tips,
anthers, meristematic cells, microspores, flowers, cotyledons, pistil, fruit,
seeds, seed coat or any
combination thereof.
The term "plant material" or "plant part" used herein refers to leaves, stems,
roots, root tips,
flowers or flower parts, f uits, pollen, egg cells, zygotes, seeds, seed coat,
cuttings, cell or tissue
cultures, or any other part or product of a plant or a combination thereof.
A "plant organ" as used herein means a distinct and visibly structured and
differentiated part of
a plant such as a root, stem, leaf, flower, flower bud, or embryo.
The term "Plant tissue" as used herein means a group of plant cells organized
into a structural
and functional unit. Any tissue of a plant in planta or in culture is
included. This term includes,
but is not limited to, whole plants, plant organs, plant seeds, tissue
culture, protoplasts,
meristematic cells, calli and any group of plant cells organized into
structural and/or functional
units. The use of this term in conjunction with, or in the absence of, any
specific type of plant
22
Date Recue/Date Received 2022-06-30
tissue as listed above or otherwise embraced by this definition is not
intended to be exclusive of
any other type of plant tissue.
As used herein, the term "progeny" or "progenies" refers in a non limiting
manner to offspring
or descendant plants. According to certain embodiments, the term "progeny" or
"progenies" refers
to plants developed or grown or produced from the disclosed or deposited seeds
as detailed inter
alia. The grown plants preferably have the desired traits of the disclosed or
deposited seeds, i.e.
reduced expression of at least one CsMLO gene.
The term "Cannabis" refers hereinafter to a genus of flowering plants in the
family Cannabaceae.
Cannabis is an annual, dioecious, flowering herb that includes, but is not
limited to three different
species, Cannabis saliva, Cannabis indica and Cannabis ruderalis . The term
also refers to hemp.
Cannabis plants produce a group of chemicals called cannabinoids.
Cannabinoids, terpenoids, and
other compounds are secreted by glandular trichomes that occur most abundantly
on the floral
calyxes and bracts of female Cannabis plants.
As used herein the term "genetic modification" or "genomic modification"
refers hereinafter
to genetic manipulation or modulation, which is the manipulation of an
organism's genes using biotechnology. It also refers to a set of technologies
used to change the
genetic makeup of cells, including the transfer of genes within and across
species, targeted
mutagenesis and genome editing technologies to produce improved organisms.
According to main
embodiments of the present invention, modified Cannabis plants with increased
resistance to PM
are generated using genome editing mechanism. This technique enables to
achieve in planta
modification of specific genes that relate to and/or control the infection of
powdery mildew in the
Cannabis plant.
The term "genome editing", or "genome/genetic modification" or "genome
engineering"
generally refers hereinafter to a type of genetic engineering in which DNA is
inserted, deleted,
modified or replaced in the genome of a living organism. Unlike previous
genetic engineering
techniques that randomly insert genetic material into a host genome, genome
editing targets the
insertions to site specific locations.
It is within the scope of the present invention that the common methods for
such editing
use engineered nucleases, or "molecular scissors". These nucleases create site-
specific double-
strand breaks (DSBs) at desired locations in the genome. The induced double-
strand breaks
23
Date Recue/Date Received 2022-06-30
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. coil protein involved in DNA
repair
dCAS9 = nuclease-deficient Cas9 sgRNA = single guide RNA
DSB = Double-Stranded Break tracrRNA, rRNA = 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
It is noted that it is within the scope of the current invention that the term
gRNA also refers to or
means single guide RNA (sgRNA).
According to specific aspects of the present invention, the CRISPR (Clustered
Regularly
Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are
used for the first
time for generating genome modification in targeted genes in the Cannabis
plant. It is herein
acknowledged that the functions of CRISPR (Clustered Regularly Interspaced
Short Palindromic
Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity
in select bacteria
and archaea, enabling the organisms to respond to and eliminate invading
genetic material. These
repeats were initially discovered in the 1980s in E. coil. Without wishing to
be bound by theory,
reference is now made to a type of CRISPR mechanism, in which invading DNA
from viruses or
24
Date Recue/Date Received 2022-06-30
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 Csnl) is
required for gene silencing. Cas9 participates in the processing of crRNAs,
and is responsible for
the destruction of the target DNA. Cas9's function in both of these steps
relies on the presence of
two nuclease domains, a RuvC-like nuclease domain located at the amino
terminus and a HNH-
like nuclease domain that resides in the mid-region of the protein. To achieve
site-specific DNA
recognition and cleavage, Cas9 is complexed with both a crRNA and a separate
trans-activating
crRNA (tracrRNA or trRNA), that is partially complementary to the crRNA. The
tracrRNA is
required for crRNA maturation from a primary transcript encoding multiple pre-
crRNAs. This
occurs in the presence of RNase III and Cas9.
Without wishing to be bound by theory, it is herein acknowledged that during
the destruction of
target DNA, the HNH and RuvC-like nuclease domains cut both DNA strands,
generating double-
stranded breaks (DSBs) at sites defined by a 20-nucleotide target sequence
within an associated
crRNA transcript. The HNH domain cleaves the complementary strand, while the
RuvC domain
cleaves the noncomplementary strand.
It is further noted that the double-stranded endonuclease activity of Cas9
also requires that a short
conserved sequence, (2-6 nucleotides) known as protospacer-associated motif
(PAM), follows
immediately 3"- of the crRNA complementary sequence.
According to further aspects of the invention, a two-component system may be
used by the current
invention, combining trRNA and crRNA into a single synthetic single guide RNA
(sgRNA) for
guiding targeted gene alterations.
It is further within the scope that Cas9 nuclease variants include wild-type
Cas9, Cas9D10A and
nuclease-deficient Cas9 (dCas9).
Reference is now made to Fig. 3 schematically presenting an example of
CRISPR/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
Date Recue/Date Received 2022-06-30
figure, the Cas9 endonuclease forms a complex with a chimeric RNA (called
guide RNA or
gRNA), replacing the crRNA¨transcrRNA heteroduplex, and the gRNA could be
programmed to
target specific sites. The gRNA¨Cas9 should comprise at least 15-base-pairing
(gRNA seed
region) without mismatch between the 5'-end of engineered gRNA and targeted
genomic site, and
a motif called protospacer-adjacent motif or PAM that follows the base-pairing
region in the
complementary strand of the targeted DNA. The commonly-used Cas9 from
Streptococcus
pyogenes (SpCas9) recognizes the PAM sequence 5'-NGG-3' (where "N" can be any
nucleotide
base).
Other Cas variants and their PAM sequences (5' to 3') within the scope of the
current invention
include NmeCas9 (isolated from Neisseria meningitides) recognizing NNNNGATT,
StCas9
(isolated from Streptococcus thermophiles) recognizing NNAGAAW, TdCas9
(isolated from
Treponema denticola) recognizing NAAAAC, SaCas9 (isolated from Staphylococcus
aureus)
recognizing NNGRRT or NGRRT or NGRRN and TBN (Cas-phi).
The term "meganucleases" as used herein refers
hereinafter
to endodeoxyribonucleases characterized by a large recognition site (double-
stranded DNA
sequences of 12 to 40 base pairs); as a result this site generally occurs only
once in any
given genome. Meganucleases are therefore considered to be the most specific
naturally
occurring restriction enzymes.
The term "protospacer adjacent motif" or "PAM" as used herein refers
hereinafter to a 2-6 base
pair DNA sequence immediately following the DNA sequence targeted by the Cas9
nuclease in
the CRISPR bacterial adaptive immune system. PAM is a component of the
invading virus or
plasmid, but is not a component of the bacterial CRISPR locus. PAM is an
essential targeting
component which distinguishes bacterial self from non-self DNA, thereby
preventing the CRISPR
locus from being targeted and destroyed by nuclease.
The term "Next-generation sequencing" or "NGS" as used herein refers
hereinafter to massively,
parallel, high- throughput or deep sequencing technology platforms that
perform sequencing of
millions of small fragments of DNA in parallel. Bioinformatics analyses are
used to piece together
these fragments by mapping the individual reads to the reference genome.
The term "gene knockdown" as used herein refers hereinafter to an experimental
technique by
which the expression of one or more of an organism's genes is reduced. The
reduction can occur
26
Date Recue/Date Received 2022-06-30
through genetic modification, i.e. targeted genome editing or by treatment
with a reagent such as
a short DNA or RNA oligonucleotide that has a sequence complementary to either
gene or an
mRNA transcript. The reduced expression can be at the level of RNA or at the
level of protein. It
is within the scope of the present invention that the term gene knockdown also
refers to a loss of
function mutation and /or gene knockout mutation in which an organism's genes
is made
inoperative or nonfunctional.
The term "gene silencing" or "silence" or silencing" as used herein refers
hereinafter to
the regulation of gene expression in a cell to prevent the expression of a
certain gene. Gene
silencing can occur during either transcription or translation. In certain
aspects of the invention,
gene silencing is considered to have a similar meaning as gene knockdown. When
genes are
silenced, their expression is reduced. In contrast, when genes are knocked
out, they are completely
not expressed. Gene silencing may be considered a gene knockdown mechanism
since the methods
used to silence genes, such as RNAi, CRISPR, or siRNA, generally reduce the
expression of a
gene by at least 70% but do not completely eliminate it. In some embodiments
of the present
invention, gene silencing by targeted genome modification results in non-
functional gene products,
such as transcripts or proteins, for example non-functional CsML01 exon 1
fragments.
The term "microRNAs" or "miRNAs" refers hereinafter to small non-coding RNAs
that have
been found in most of the eukaryotic organisms. They are involved in the
regulation of gene
expression at the post-transcriptional level in a sequence specific manner.
MiRNAs are produced
from their precursors by Dicer-dependent small RNA biogenesis pathway. MiRNAs
are candidates
for studying gene function using different RNA-based gene silencing
techniques. For example,
artificial miRNAs (amiRNAs) targeting one or several genes of interest is a
potential tool in
functional genomics.
The term "in planta" means in the context of the present invention within the
plant or plant cells.
More specifically, it means introducing CRISPR/Cas complex into plant material
comprising a
tissue culture of several cells, a whole plant, or into a single plant cell,
without introducing a
foreign gene or a mutated gene. It also used to describe conditions present in
a non-laboratory
environment (e.g. in vivo).
As used herein, the term "powdery mildew" or "PM" refers hereinafter to fungi
that are obligate,
biotrophic parasites of the phylum Ascomycota of Kingdom Fungi. The diseases
they cause are
27
Date Recue/Date Received 2022-06-30
common, widespread, and easily recognizable. Infected plants display white
powdery spots on the
leaves and stems Infection by the fungus is favored by high humidity but not
by free water.
Powdery mildew fungi tend to grow superficially, or epiphytically, on plant
surfaces. During the
growing season, hyphae are produced preferably on both upper and lower leaf
surfaces. Infections
can also occur on stems, flowers, or fruit. Specialized absorption cells,
termed haustoria, extend
into the plant epidermal cells to obtain nutrition.
Powdery mildew fungi can reproduce both sexually and asexually. Sexual
reproduction is via
chasmothecia (cleistothecium), a type of ascocarp where the genetic material
recombines. Within
each ascocarp are several asci. Under optimal conditions, ascospores mature
and are released to
initiate new infections Conidia (asexual spores) are also produced on plant
surfaces during the
growing season. They develop either singly or in chains on specialized hyphae
called
conidiophores. Conidiophores arise from the epiphytic hyphae, or in the case
of endophytic
hyphae, the conidiophores emerge through leaf stomata. It should be noted that
powdery mildew
fungi must be adapted to their hosts to be able to infect them. The present
invention provides for
the first time Cannabis plants with enhanced resistance or tolerance to PM
disease. The enhanced
resistance to PM is generated by genome editing techniques targeted at
silencing at least one
Cannabis Mildew Locus 0 (MLO) gene. The modified resulted Cannabis plant
exhibits enhanced
resistance to PM as compared to a Cannabis plant lacking the targeted
modification.
The term "MILO" or "Mb" or "ml" refers hereinafter to the Mildew Locus 0 (MLO)
gene
family encoding for plant-specific proteins harboring several transmembrane
domains,
topologically reminiscent of metazoan G-protein coupled receptors. It is
within the scope of the
present invention that specific homologs of the MLO family act as
susceptibility genes towards
PM fungi. It is emphasized that the present invention provides for the first
time the identification
of MLO orthologous alleles in the Cannabis plant. Three Cannabis MLO alleles
or genes (i.e.
ML01, ML02, ML03) have been herein identified, namely CsML01, CsML02 and
CsML03.
The term "orthologue" as used herein refers hereinafter to one of two or more
homologous gene
sequences found in different species.
The term "functional variant" or "functional variant of a nucleic acid or
protein sequence" as used
herein, for example with reference to SEQ ID NOs: 1, 2 or 3 refers to a
variant gene sequence or
part of the gene sequence which retains the biological function of the full
non-variant allele (e.g.
28
Date Recue/Date Received 2022-06-30
CsMLO allele) and hence has the activity of modulating response to PM. A
functional variant also
comprises a variant of the gene of interest encoding a polypeptide which has
sequence alterations
that do not affect function of the resulting protein, for example in non-
conserved residues. Also
encompassed is a variant that is substantially identical, i.e. has only some
sequence variations, for
example in non-conserved residues, to the wild type nucleic acid sequences of
the alleles as shown
herein and is biologically active.
The term "variety" or "cultivar" used herein means a group of similar plants
that by structural
features and performance can be identified from other varieties within the
same species.
The term "allele" used herein means any of one or more alternative or variant
forms of a gene or
a genetic unit at a particular locus, all of which alleles relate to one trait
or characteristic at a
specific locus. In a diploid cell of an organism, alleles of a given gene are
located at a specific
location, or locus (loci plural) on a chromosome. Alternative or variant forms
of alleles may be the
result of single nucleotide polymorphisms, insertions, inversions,
translocations or deletions, or
the consequence of gene regulation caused by, for example, by chemical or
structural modification,
transcription regulation Or post-translational
modification/regulation.
An allele associated with a qualitative trait may comprise alternative or
variant forms of various
genetic units including those mat are identical or associated with a single
gene or multiple genes
or their products or even a gene disrupting or controlled by a genetic factor
contributing to the
phenotype represented by the locus. According to further embodiments, the term
"allele"
designates any of one or more alternative forms of a gene at a particular
locus. Heterozygous alleles
are two different alleles at the same locus. Homozygous alleles are two
identical alleles at a
particular locus. A wild type allele is a naturally occurring allele. In the
context of the current
invention, the term allele refers to the three identified Cannabis MLO genes,
namely CsML01,
CsML02 and CsML03 having the genomic nucleotide sequence as set forth in SEQ
ID NOs: 1, 2
or 3, respectively.
As used herein, the term "locus" (loci plural) means a specific place or
places or region or a site
on a chromosome where for example a gene or genetic marker element or factor
is found. In
specific embodiments, such a genetic element is contributing to a trait.
29
Date Recue/Date Received 2022-06-30
As used herein, the term "homozygous" refers to a genetic condition or
configuration existing
when two identical or like alleles reside at a specific locus, but are
positioned individually on
corresponding pairs of homologous chromosomes in the cell of a diploid
organism.
Conversely, as used herein, the term "heterozygous" means a genetic condition
or configuration
existing when two different or unlike alleles reside at a specific locus, but
are positioned
individually on corresponding pairs of homologous chromosomes in the cell of a
diploid organism.
In specific embodiments, the tomato plants of the present invention comprise
heterozygous
configuration of the genetic markers associated with the high yield
characteristics.
The term "corresponding" or "corresponding to" or "corresponding to nucleotide
sequence" or
"corresponding to position" as used herein, refers in the context of the
present invention to
sequence homology or sequence identity. These terms relate to two or more
nucleic acid or protein
sequences, that are the same or have a specified percentage of amino acid
residues or nucleotides
that are the same, when compared and aligned for maximum correspondence, as
measured using
one of the available sequence comparison algorithms or by visual inspection.
If two sequences,
which are to be compared with each other, differ in length, sequence identity
preferably relates to
the percentage of the nucleotide residues of the shorter sequence, which are
identical with the
nucleotide residues of the longer sequence. As used herein, the percent of
identity or homology
between two sequences is a function of the number of identical positions
shared by the sequences,
taking into account the number of gaps, and the length of each gap, which
needs to be introduced
for optimal alignment of the two sequences. The comparison of sequences and
determination of
identity percent between two sequences can be accomplished using a
mathematical algorithm as
known in the relevant art. According to further aspects of the invention, the
term "corresponding
to the nucleotide sequence" or "corresponding to position", refers to
variants, homologues and
fragments of the indicated nucleotide sequence, which possess or perform the
same biological
function or correlates with the same phenotypic characteristic of the
indicated nucleotide sequence.
Another indication that two nucleic acid sequences are substantially identical
or that a sequence is
"corresponding to the nucleotide sequence" is that the two molecules hybridize
to each other under
stringent conditions. High stringency conditions, such as high hybridization
temperature and low
salt in hybridization buffers, permits only hybridization between nucleic acid
sequences that are
Date Recue/Date Received 2022-06-30
highly similar, whereas low stringency conditions, such as lower temperature
and high salt, allows
hybridization when the sequences are less similar.
In other embodiments of the invention, such substantially identical sequences
refer to
polynucleotide or amino acid sequences that share at least about 80%
similarity, preferably at least
about 90% similarity, alternatively, about 95%, 96%, 97%, 98% or 99%
similarity to the indicated
polynucleotide or amino acid sequences.
According to other aspects of the invention, the term "corresponding" refers
also to complementary
sequences or base pairing such that when they are aligned antiparallel to each
other, the nucleotide
bases at each position in the sequences will be complementary. The degree of
complementarity
between two nucleic acid strands may vary.
As used herein, the phrase "genetic marker" or "molecular marker" or
"biomarker" refers to a
feature in an individual's genome e.g., a nucleotide or a polynucleotide
sequence that is associated
with one or more loci or trait of interest In some embodiments, a genetic
marker is polymorphic
in a population of interest, or the locus occupied by the polymorphism,
depending on context.
Genetic markers or molecular markers include, for example, single nucleotide
polymorphisms
(SNPs), indels (i.e. insertions deletions), simple sequence repeats (SSRs),
restriction fragment
length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAFDs),
cleaved
amplified polymorphic sequence (CAPS) markers, Diversity Arrays Technology
(DArT) markers,
and amplified fragment length polymorphisms (AFLPs) or combinations thereof,
among many
other examples such as the DNA sequence per se. Genetic markers can, for
example, be used to
locate genetic loci containing alleles on a chromosome that contribute to
variability of phenotypic
traits. The phrase "genetic marker" or "molecular marker" or "biomarker" can
also refer to a
polynucleotide sequence complementary or corresponding to a genomic sequence,
such as a
sequence of a nucleic acid used as a probe or primer.
As used herein, the term "germplasm" refers to the totality of the genotypes
of a population or
other group of individuals (e.g., a species). The term "germplasm" can also
refer to plant material;
e.g., a group of plants that act as a repository for various alleles. Such
germplasm genotypes or
populations include plant materials of proven genetic superiority; e.g., for a
given environment or
geographical area, and plant materials of unknown or unproven genetic value;
that are not part of
31
Date Recue/Date Received 2022-06-30
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-
32
Date Recue/Date Received 2022-06-30
stranded or double-stranded. Such nucleic acids or polynucleotides include,
but are not limited to,
coding sequences of structural genes, anti-sense sequences, and non-coding
regulatory sequences
that do not encode mRNAs or protein products. These terms also encompass a
gene. The term
"gene", "allele" or "gene sequence" is used broadly to refer to a DNA nucleic
acid associated with
a biological function. Thus, genes may include introns and exons as in the
genomic sequence, or
may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in
combination
with regulatory sequences. Thus, according to the various aspects of the
invention, genomic DNA,
cDNA or coding DNA may be used. In one embodiment, the nucleic acid is cDNA or
coding
DNA.
The terms "peptide", "polypeptide" and "protein" are used interchangeably
herein and refer to
amino acids in a polymeric form of any length, linked together by peptide
bonds.
According to other aspects of the invention, a 'modified" or a "mutant" plant
is a plant that has
been altered compared to the naturally occurring wild type (WT) plant.
Specifically, the
endogenous nucleic acid sequences of each of the MLO homologs in Cannabis
(nucleic acid
sequences CsML01, CsML02 and CsML03) have been altered compared to wild type
sequences
using mutagenesis and/or genome editing methods as described herein. This
causes inactivation of
the endogenous Mb o genes and thus disables Mb o function. Such plants have an
altered phenotype
and show resistance or increased resistance to PM compared to wild type
plants. Therefore, the
resistance is conferred by the presence of at least one mutated endogenous
CsML01, CsML02
and CsML03 genes in the Cannabis plant genome which has been specifically
targeted using
targeted genome modification.
According to further aspects of the present invention, the increased
resistance to PM is not
conferred by the presence of transgenes expressed in Cannabis.
It should be noted that nucleic acid sequences of wild type alleles are
designated using capital
letters namely CsML01, CsML02 and CsML03. Mutant mlo nucleic acid sequences
use non-
capitalization. Cannabis plants of the invention are modified plants compared
to wild type plants
which comprise and express mutant mlo alleles.
It is further within the scope of the current invention that mlo mutations
that down-regulate or
disrupt functional expression of the wild-type Mb o sequence may be recessive,
such that they are
complemented by expression of a wild-type sequence.
33
Date Recue/Date Received 2022-06-30
A mlo mutant phenotype according to the invention is characterized by the
exhibition of an
increased resistance against PM. In other words, a mlo mutant according to the
invention confers
resistance to the pathogen causing PM, which is identified as described inter
alia.
It is further noted that a wild type Cannabis plant is a plant that does not
have any mutant Mbo
alleles.
Main aspects of the invention involve targeted mutagenesis methods,
specifically genome editing,
and exclude embodiments that are solely based on generating plants by
traditional breeding
methods. In a further embodiment of the current invention, as explained
herein, the disease
resistant trait is not due to the presence of a transgene.
The inventors have generated mutant Cannabis lines with mutations inactivating
at least one
CsMLO homoeoallele which confer heritable resistance to powdery mildew. In
this way no
functional CsMLO protein is made. Thus, the invention relates to these mutant
Cannabis lines and
related methods.
According to one embodiment, the present invention provides a modified
Cannabis plant
exhibiting enhanced resistance to powdery mildew (PM) compared to wild type
Cannabis plant.
The Cannabis plant of the present invention comprises a genetic modification
conferring reduced
expression of at least one Cannabis MLO (CsMLO) allele.
It is within the scope of the present invention that the CsMLO allele is
selected from the group
consisting of CsML01 having a nucleotide sequence as set forth in SEQ ID NO:1
or a fragment
or a functional variant thereof, CsML02 having a nucleotide sequence as set
forth in SEQ ID NO:4
or a fragment or a functional variant thereof and CsML03 having a nucleotide
sequence as set
forth in SEQ ID NO:7 or a fragment or a functional variant thereof.
According to a further embodiment of the present invention, the functional
variant has at least 75%
sequence identity to the CsMLO nucleotide sequence.
It is within the scope of the current invention that genome editing can be
achieved using sequence-
specific nucleases (SSNs) and results in chromosomal changes, such as
nucleotide deletions,
insertions or substitutions at specified genetic loci. Non limiting examples
of SSNs include zinc
finger nucleases (ZFNs), TAL effector nucleases (TALENs) and, clustered
regularly interspaced
short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system.
34
Date Recue/Date Received 2022-06-30
Non limiting examples Cas proteins used by the present invention include Csnl,
Cpfl Cas9, Cas12,
Cas13, Cas14, CasX and any combination thereof.
According to further aspects of the invention, Cannabis plant resistant to the
powdery mildew
fungal pathogen using the CRISPR/Cas9 technology is generated, which is based
on the Cas9
DNA nuclease guided to a specific DNA target by a single guide RNA (sgRNA).
It is herein acknowledged that wild-type alleles of MILDEW RESISTANT LOCUS 0
(Mb),
which encodes a membrane-associated protein with seven transmembrane domains,
confer
susceptibility to fungi causing the powdery mildew disease. Therefore,
homozygous loss-of-
function mutations (ml) result in resistance to powdery mildew.
According to certain embodiments of the present invention, in planta
modification of specific
genes that relate to and/or control the infection of powdery mildew in the
Cannabis plant is
achieved for the first time by the present invention, i.e. the Cannabis MLO
genes (CsMLO). More
specifically, but not limited to, the use of gene editing technologies, for
example the CRISPR/Cas
technology (e.g. Cas9 or Cpfl), in order to generate knockout alleles of genes
(i.e. MLO genes)
controlling the resistance to powdery mildew (PM) is disclosed for the
Cannabis plant. The above
in planta modification can be based on alternative gene editing technologies
such as Zinc Finger
Nucleases (ZFN's), Transcription activator-like effector nucleases (TALEN's),
RNA silencing
(amiRNA etc.) and/or meganucleases.
The loss of function mutation may be a deletion or insertion ("indels") with
reference the wild type
CsMLO allele sequence. The deletion may comprise 1-20 or more, for example 1,
2, 3, 4, 5, 6, 7,
8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18 or 20 nucleotides or more in one or
more strand. The insertion
may comprise 1-20 or more, for example 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 1, 12,
13, 14, 15, 16, 17, 18 or
20 or more nucleotides in one or more strand.
The plant of the invention includes plants wherein the plant is heterozygous
for the each of the
mutations. In a preferred embodiment however, the plant is homozygous for the
mutations.
Progeny that is also homozygous can be generated from these plants according
to methods known
in the art.
It is further within the scope that variants of a particular CsMLO nucleotide
or amino acid sequence
according to the various aspects of the invention will have at least about 50%-
99%, for example
Date Recue/Date Received 2022-06-30
at least 75%, for example at least 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%,
95%, 96%, 97%,
98% or 99% or more sequence identity to that particular non-variant CsMLO
nucleotide sequence
of the CsMLO allele as shown in SEQ ID NO 1, 2 or 3. Sequence alignment
programs to determine
sequence identity are well known in the art.
Also, the various aspects of the invention encompass not only a CsMLO nucleic
acid sequence or
amino acid sequence, but also fragments thereof. By "fragment" is intended a
portion of the
nucleotide sequence or a portion of the amino acid sequence and hence of the
protein encoded
thereby. Fragments of a nucleotide sequence may encode protein fragments that
retain the
biological activity of the native protein and hence act to modulate responses
to PM.
According to a further embodiment of the invention, the herein newly
identified Cannabis MLO
locus (CsMLO) have been targeted using the triple sgRNA strategy.
According to further embodiments of the present invention, DNA introduction
into the plant cells
can be done by Agrobacterium infiltration, virus based plasmids for delivery
of the genome editing
molecules and mechanical insertion of DNA (PEG mediated DNA transformation,
biolistics, etc.).
In addition, it is within the scope of the present invention that the Cas9
protein is directly inserted
together with a gRNA (ribonucleoprotein- RNP's) in order to bypass the need
for in vivo
transcription and translation of the Cas9+gRNA plasmid in planta to achieve
gene editing.
It is also possible to create a genome edited plant and use it as a rootstock.
Then, the Cas protein
and gRNA can be transported via the vasculature system to the top of the plant
and create the
genome editing event in the scion.
It is within the scope of the present invention that the usage of CRISPR/Cas
system for the
generation of PM resistant Cannabis plants, allows the modification of
predetermined specific
DNA sequences without introducing foreign DNA into the genome by GMO
techniques.
According to one embodiment of the present invention, this is achieved by
combining the Cas
nuclease (e.g. Cas9, 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. 3). The predefined
gene specific gRNA's are cloned into the same plasmid as the Cas gene and this
plasmid is inserted
into plant cells. Insertion of the aforementioned plasmid DNA can be done, but
not limited to,
36
Date Recue/Date Received 2022-06-30
using different delivery systems, biological and/or mechanical, e.g.
Agrobacterium infiltration,
virus based plasmids for delivery of the genome editing molecules and
mechanical insertion of
DNA (PEG mediated DNA transformation, biolistics, etc.).
It is further within the scope of the present invention that upon reaching the
specific predetermined
DNA sequence, the Cas9 nuclease cleaves both DNA strands to create double
stranded breaks
leaving blunt ends. This cleavage site is then repaired by the cellular non
homologous end joining
DNA repair mechanism resulting in insertions or deletions which eventually
create a mutation at
the cleavage site. For example, it is acknowledged that a deletion form of the
mutation consists of
at least 1 base pair deletion. As a result of this base pair deletion the gene
coding sequence is
disrupted and the translation of the encoded protein is compromised either by
a premature stop
codon or disruption of a functional or structural property of the protein.
Thus DNA is cut by the
Cas9 protein and re-assembled by the cell's DNA repair mechanism.
It is further within the scope that resistance to PM in Cannabis plants is
produced by generating
gRNA with homology to a specific site of predetermined genes in the Cannabis
genome i.e. MLO
genes, sub cloning this gRNA into a plasmid containing the Cas9 gene, and
insertion of the plasmid
into the Cannabis plant cells. In this way site specific mutations in the MLO
genes are generated
thus effectively creating non-active molecules, resulting in inability of
powdery mildew and
similar organisms of infecting the genome edited plant.
Reference is now made to Figs 1A-C schematically present Cannabis plant
infected by the fungal
pathogen Golovinomyces cichoracearum, causal agent of the Powdery Mildew
disease. More
specifically this figure shows (A) Cannabis plant leaf exhibiting PM symptoms
(B) Fungal asexual
spore-carrying bodies (conidia) of Golovinomyces cichoracearum on Cannabis
leaf tissue, and (C)
microscopic view of Golovinomyces cichoracearum spores.
Reference is now made to Fig. 2A-B schematically presenting PM resistance
suggested mode of
action. This figure shows (A) a WT plant cell penetrated by the PM fungus
(100). More
particularly, a WT plant cell 10 is infected by PM spore 20 producing germ
tubes 30 and penetrated
by the PM fungal appressorium 40 which then leads to haustorium 50
establishment and infection
by secondary hyphae; and (B) an mlo knockout cell 15 rendering fungal spores
incapable of
penetrating the plant cell (200).
37
Date Recue/Date Received 2022-06-30
According to one embodiment, the present invention provides a modified
Cannabis plant
exhibiting enhanced resistance to powdery mildew (PM), wherein said modified
plant comprises
a mutated Cannabis mlol (Csmlol) allele, said mutated allele comprising a
genomic modification
selected from an indel of 14 bp at position corresponding to position 12 of
SEQ ID NO: 882, or a
fraction thereof, or a nucleic acid insertion at position corresponding to
position 104-105 of SEQ
ID NO: 882, or a combination thereof.
According to a further embodiment of the present invention, the indel
comprises a sequence as set
forth in SEQ ID NO:883 or a fraction thereof.
According to a further embodiment of the present invention, the Csmlol mutant
allele comprises
a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID
NO:884, or a nucleic
acid sequence corresponding to the sequence as set forth in SEQ ID NO: 885, or
a nucleic acid
sequence corresponding to the sequence as set forth in SEQ ID NO:886, or a
homologue having
at least 80% sequence identity to the nucleic acid sequence of said mutated
Csmlol allele, or a
complementary sequence thereof, or any combination thereof.
According to a further embodiment of the present invention, the Csmlol mutant
allele comprises
a nucleic acid sequence corresponding to the sequence as set forth in SEQ ID
NO:886, or a
homologue having at least 80% sequence identity to the nucleic acid sequence
of said mutated
Csmlol allele.
According to a further embodiment of the present invention, the mutated Csmlol
allele confers an
enhanced resistance to powdery mildew as compared to a Cannabis plant
comprising a wild type
CsML01 allele having a nucleic acid sequence as set forth in SEQ ID NO:882
and/or having a
nucleic acid sequence as set forth in SEQ ID NO:1 or a functional variant
thereof.
According to a further embodiment of the present invention, the functional
variant has at least 80%
sequence identity to the corresponding CsML01 nucleotide sequence.
According to a further embodiment of the present invention, the mutated allele
comprising a
deletion of 14 bp at position 389 of SEQ ID NO: 1, or a nucleic acid insertion
at position 482-483
of SEQ ID NO: 1, or a combination thereof.
According to a further embodiment of the present invention, the mutated Csmlol
allele is
generated using genome editing.
38
Date Recue/Date Received 2022-06-30
It is further within the scope of the present invention to provide, a modified
Cannabis plant
exhibiting enhanced resistance to powdery mildew (PM), wherein said modified
plant comprises
a targeted genome modification conferring reduced expression of a Cannabis
ML01 (CsML01)
gene as compared to a Cannabis plant lacking said targeted genome
modification, said targeted
genome modification generates a mutated Cannabis mlol (Csmlol) allele
comprising a deletion
of a nucleic acid sequence as set forth in SEQ ID NO:883 or a fraction thereof
as compared to the
wild type CsML01 allele comprising a sequence as set forth in SEQ ID NO:1, or
a nucleic acid
insertion at position 482-483 of SEQ ID NO:1, or a combination thereof.
According to a further aspect of the present invention, a method for producing
a modified Cannabis
plant as defined in any of the above is provided. The method comprises
introducing using targeted
genome modification, at least one genomic modification conferring reduced
expression of at least
one Cannabis ML01 (CsML01) allele as compared to a Cannabis plant lacking said
targeted
genome modification, said genomic modification generates a mutated Cannabis
mlol (Csmlol)
allele comprising an indel of 14 bp at a position corresponding to position 12
of SEQ ID NO: 882
or a fraction thereof, or a nucleic acid insertion at position corresponding
to position 104-105 of
SEQ ID NO: 882, or a combination thereof.
According to further aspects of the present invention, a method of determining
the presence of a
mutant Csmlol allele in a Cannabis plant is provided. The method comprising
assaying the
Cannabis plant for at least one of the presence of an indel comprising a
nucleic acid sequence as
set forth in SEQ ID NO:883, an insertion at position 104-105 of SEQ ID NO:
882, a nucleic acid
sequence corresponding to the sequence as set forth in SEQ ID NO:884, a
nucleic acid sequence
corresponding to the sequence as set forth in SEQ ID NO:885, a nucleic acid
sequence
corresponding to the sequence as set forth in SEQ ID NO:886, or a homologue
having at least 80%
sequence identity to the nucleic acid sequence of said mutated Csmlol allele,
a complementary
sequence thereof, or any combination thereof.
It is further within the scope to provide a method for identifying a Cannabis
plant with resistance
to powdery mildew, said method comprises steps of: (a) screening the genome of
said Cannabis
plant for a mutated Csmlol allele, said mutated allele comprises a genomic
modification selected
from an indel of 14 bp at a position corresponding to position 12 of SEQ ID
NO: 882 or a fraction
thereof, or a nucleic acid insertion at position corresponding to position 104-
105 of SEQ ID NO:
39
Date Recue/Date Received 2022-06-30
882, or a combination thereof; (b) optionally, regenerating plants carrying
said genetic
modification; and (c) optionally, screening said regenerated plants for a
plant resistant to powdery
mildew.
It is further within the scope of the present invention to provide a method
for down regulation of
Cannabis ML01 (CsML01) gene, which comprises utilizing the nucleotide sequence
as set forth
in at least one of SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence
thereof, and a
combination thereof, for introducing a loss of function mutation into said
CsML01 gene using
targeted genome modification.
The present invention further provides an isolated amino acid sequence having
at least 80%
sequence identity to a nucleic acid sequence selected from the group
consisting of SEQ ID
NO:882-886, SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary
sequence
or any combination thereof.
It is also within the scope to disclose a use of a nucleotide sequence as set
forth in SEQ ID NO:
883-886, SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 for generating,
identifying and/or
screening for a Cannabis plant comprising within its genome mutant Csmlo
allele conferring
resistance to PM.
It is also within the scope to disclose a use of a nucleotide sequence as set
forth in at least one of
SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 or a complementary sequence or any
combination thereof for targeted genome modification of Cannabis ML01 (CsML01)
gene.
According to further aspects, the present invention provides a detection kit
for determining the
presence or absence of a mutant Csmlol allele in a Cannabis plant, comprising
a nucleic acid
fragment comprising a sequence selected from SEQ ID NO:882-886, SEQ ID NO:17,
SEQ ID
NO:43 and SEQ ID NO:50 or a complementary sequence or any combination thereof.
It is a further aspect of the present invention to disclose a modified
Cannabis plant exhibiting
enhanced resistance to powdery mildew (PM), wherein said plant comprises a
targeted genome
modification conferring reduced expression of at least one Cannabis MLO
(CsMLO) allele as
compared to a Cannabis plant lacking said targeted genome modification.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined
above, wherein said targeted genome modification is in a CsMLO allele having a
wild type
Date Recue/Date Received 2022-06-30
genomic nucleotide sequence selected from the group consisting of CsML01
having a sequence
as set forth in SEQ ID NO:1 or a functional variant thereof, CsML02 having a
sequence as set
forth in SEQ ID NO:4 or a functional variant thereof and CsML03 having a
sequence as set forth
in SEQ ID NO:7 or a functional variant thereof.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein said functional variant has at least 80% sequence
identity to the
corresponding CsMLO nucleotide sequence.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein said plant has decreased expression levels of at
least one Mb o protein,
relative to a Cannabis plant lacking said at least one genome modification.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein said genomic modification is introduced using
mutagenesis, small
interfering RNA (siRNA), microRNA (miRNA), artificial miRNA (amiRNA), DNA
introgression,
endonucleases or any combination thereof.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein said genetic modification is introduced using
targeted genome
modification, preferably said genetic modification is introduced using an
endonuclease.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above wherein said targeted genome modification is introduced using
CRISPR
(Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-
associated (Cas) gene
(CRISPR/Cas), Transcription activator-like effector nuclease (TALEN), Zinc
Finger Nuclease
(ZFN), meganuclease or any combination thereof.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above wherein said Cas gene is selected from the group consisting
of Cas3, Cas4, Cas5,
Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9,
Cas10, Castl Od,
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, Csnl, Csn2, Csm2,
Csm3, Csm4,
Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl, Csbl, Csb2, Csb3, Csx17,
Csx14, Csx10,
41
Date Recue/Date Received 2022-06-30
Csx16, CsaX, Csx3, Cszl, Csx15, Csfl, Csf2, Csf3, Csf4, and Cu1966,
bacteriophages Cas such
as Cas1T3$ (Cas-phi) and any combination thereof.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein said plant comprises a recombinant DNA construct,
said recombinant
DNA construct comprising a promoter operably linked to a nucleotide sequence
encoding a plant
optimized Cas9 endonuclease, wherein said plant optimized Cas9 endonuclease is
capable of
binding to and creating a double strand break in a genomic target sequence of
said plant genome.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein said DNA construct further comprises sgRNA targeted
to at least one
CsMLO allele selected from the group consisting of CsML01, CsML02 and CsML03.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein said sgRNA is targeted to mutate CsML01 gene, said
sgRNA nucleotide
sequence is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:43
and SEQ ID
NO:50.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein said plant comprises at least one mutated CsML01
allele comprising a
nucleotide sequence selected from the group consisting of a nucleotide
sequence as set forth in
SEQ ID NO:875, a nucleotide sequence as set forth in SEQ ID NO:877, a
nucleotide sequence as
set forth in SEQ ID NO:880, a homologue having at least 80% sequence identity
to the nucleotide
sequence of said at least one mutated CsML01 allele and a combination thereof.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein said mutation is a silencing mutation, a knockdown
mutation, a knockout
mutation, a loss of function mutation or any combination thereof.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein said genome modification is an insertion, deletion,
indel or substitution.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein said mutated CsML01 allele comprises a deletion
having a nucleotide
sequence as set forth in SEQ ID NO.:876, SEQ ID NO.:879 or SEQ ID NO.:881.
42
Date Recue/Date Received 2022-06-30
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein said mutated allele confers an enhanced resistance
to powdery mildew
as compared to a Cannabis plant comprising a wild type CsML01 allele sequence.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein said wild type CsML01 allele comprises a nucleic
acid sequence as set
forth in at least one of SEQ ID NO:873, SEQ ID NO:876, SEQ ID NO:879 or SEQ ID
NO:881.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above wherein said genome modification is an induced mutation in
the coding region
of said allele, a mutation in the regulatory region of said allele, a mutation
in a gene downstream
in the MLO pathogen response pathway and/or an epigenetic factor.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above wherein said genome modification is generated in planta.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above wherein said targeted genome modification is generated in
planta via introduction
of a construct comprising (a) Cas DNA and sgRNA sequence selected from the
group consisting
of SEQ ID NO:10-SEQ ID NO: 870 and any combination thereof, or (b) a
ribonucleoprotein (RNP)
complex comprising Cos protein and sgRNA sequence selected from the group
consisting of SEQ
ID NO:10-870 and any combination thereof.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above wherein said targeted genome modification in said CsML01 is
generated in
planta via introduction of a construct comprising (a) Cos DNA and sgRNA
sequence selected from
the group consisting of SEQ ID NO:10-SEQ ID NO:286 and any combination
thereof, or (b) a
ribonucleoprotein (RNP) complex comprising Cas protein and sgRNA sequence
selected from the
group consisting of SEQ ID NO:10-286 and any combination thereof.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above wherein said targeted genome modification in said CsML02 is
generated in
planta via introduction of a construct comprising (a) Cos DNA and sgRNA
sequence selected from
the group consisting of SEQ ID NO:287-SEQ ID NO:625 and any combination
thereof, or (b) a
43
Date Recue/Date Received 2022-06-30
ribonucleoprotein (RNP) complex comprising Cas protein and sgRNA sequence
selected from the
group consisting of SEQ ID NO:287-625 and any combination thereof.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein said targeted genome modification in said CsML03 is
generated in
planta via introduction of a construct comprising (a) Cos DNA and sgRNA
sequence selected from
the group consisting of SEQ ID NO:626-SEQ ID NO:870 and any combination
thereof, or (b) a
ribonucleoprotein (RNP) complex comprising Cos protein and gRNA sequence
selected from the
group consisting of SEQ ID NO:626-870 and any combination thereof.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein said sgRNA sequence comprises a 3' Protospacer
Adjacent
Motif (PAM) selected from the group consisting of NGG (SpCas), NNNNGATT
(NmeCas9),
NNAGAAW (StCas9), NAAAAC (TdCas9), NNGRRT (SaCas9) and TBN (Cas-phi).
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein said construct is introduced into the plant cells
via Agrobacterium
infiltration, virus based plasmids for delivery and/or expression of the
genome editing molecules
or mechanical insertion such as polyethylene glycol (PEG) mediated DNA
transformation,
electroporation or gene gun biolistics.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein said PM is selected from the group consisting of
Golovinomyces
cichoracearum, Golovinomyces ambrosiae and a mixture thereof.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein said Cannabis plant is selected from the group of
species that includes,
but is not limited to, Cannabis saliva (C. saliva), C. indica, C. ruderalis
and any hybrid or
cultivated variety of the genus Cannabis.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein said Cannabis plant does not comprise a transgene.
It is a further aspect of the present invention to disclose a modified
Cannabis plant, progeny plant,
plant part or plant cell as defined in any of the above.
44
Date Recue/Date Received 2022-06-30
It is a further aspect of the present invention to disclose a plant part,
plant cell or plant seed of a
modified plant as defined in any of the above.
It is a further aspect of the present invention to disclose a tissue culture
of regenerable cells,
protoplasts or callus obtained from the modified Cannabis plant as defined in
any of the above.
It is a further aspect of the present invention to disclose the modified
Cannabis plant as defined in
any of the above, wherein said plant genotype is obtainable by deposit under
accession number
with NCIMB Aberdeen AB21 9YA, Scotland, UK.
It is a further aspect of the present invention to disclose a method for
producing a modified
Cannabis plant with increased resistance to powdery mildew (PM) comprising
introducing using
targeted genome modification, at least one genomic modification conferring
reduced expression
of at least one Cannabis MLO (CsMLO) allele as compared to a Cannabis plant
lacking said
targeted genome modification.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said method comprises steps of introducing a targeted genome
modification to at least one
CsMLO allele having a wild type genomic nucleotide sequence selected from the
group consisting
of CsML01 comprising a sequence as set forth in SEQ ID NO:1 or a functional
variant thereof,
CsML02 comprising a sequence as set forth in SEQ ID NO:4 or a functional
variant thereof and
CsML03 comprising a sequence as set forth in SEQ ID NO:7 or a functional
variant thereof.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said functional variant has at least 80% sequence identity to the said
CsMLO nucleotide
sequence.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said method comprises steps of introducing a loss of function mutation
into at least one
of CsML01, CsML02 and CsML02 nucleic acid sequence.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said method comprises steps of introducing a deletion mutation into
the first exon of
CsML01 genomic sequence to produce a mutated CsML01 allele comprising a
nucleotide
sequence selected from the group consisting of a nucleotide sequence as set
forth in SEQ ID
NO:875, a nucleotide sequence as set forth in SEQ ID NO:877, a nucleotide
sequence as set forth
Date Recue/Date Received 2022-06-30
in SEQ ID NO:880, a homologue having at least 80% sequence identity to the
nucleotide sequence
of said at least one mutated CsML01 allele and a combination thereof.
It is a further aspect of the present invention to disclose the method as
defined in any of the
above, wherein said modified plant has decreased levels of at least one Mb o
protein as compared
to wild type Cannabis plant.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said modified plant has decreased levels of at least one Mb o protein
as compared to a
Cannabis plant comprising a wild type CsML01 allele sequence comprising a
nucleic acid
sequence as set forth in at least one of SEQ ID NO:873, SEQ ID NO:876, SEQ ID
NO:879 or SEQ
ID NO:881.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said genome modification is introduced using CRISPR (Clustered
Regularly Interspaced
Short Palindromic Repeats) and CRISPR-associated (Cas) gene (CRISPR/Cas),
Transcription
activator-like effector nuclease (TALEN), Zinc Finger Nuclease (ZFN),
meganuclease or any
combination thereof.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said Cas gene is selected from the group consisting of Cas3, Cas4,
Cas5, Cas5e (or CasD),
Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Castl0d,
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, Csnl, 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 Cu1966, bacteriophages Cas such as
Cas(13$ (Cas-phi) and
any combination thereof.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
comprising steps of introducing an expression vector comprising a promoter
operably linked to a
nucleotide sequence encoding a plant optimized Cas9 endonuclease and sgRNA
targeted to at least
one CsMLO allele selected from the group consisting of CsML01, CsML02 and
CsML03.
46
Date Recue/Date Received 2022-06-30
It is a further object of the present invention to disclose the method as
defined in any of the above,
wherein said sgRNA nucleotide sequence targeting CsML01 is selected from the
group consisting
of SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
comprising steps of introducing and co-expressing in a Cannabis plant Cas9 and
sgRNA targeted
to at least one of CsML01, CsML02 and CsML03 genes and screening for induced
targeted
mutations in at least one of CsML01, CsML02 and CsML03 genes.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
comprising steps of screening for induced targeted mutations in at least one
of CsML01, CsML02
and CsML03 genes comprising obtaining a nucleic acid sample from a transformed
plant and
carrying out nucleic acid amplification and optionally restriction enzyme
digestion to detect a
mutation in at least one of CsML01, CsML02 and CsML03.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said nucleic acid amplification for screening induced targeted
mutations in CsML01
genomic sequence uses primers having nucleic acid sequence as set forth in SEQ
ID NO: 871 and
SEQ ID NO: 872.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
further comprising steps of assessing PCR fragments or amplicons amplified
from the transformed
plants using a gel electrophoresis based assay.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
further comprising steps of confirming the presence of a mutation by
sequencing the at least one
of CsML01, CsML02 and CsML03 nucleic acid fragment or amlicon.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said mutation is in the coding region of said allele, a mutation in
the regulatory region of
said allele, a mutation in a gene downstream in the MLO pathogen response
pathway or an
epigenetic factor.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said mutation is selected from the group consisting of a silencing
mutation, a knockdown
mutation, a knockout mutation, a loss of function mutation and any combination
thereof.
47
Date Recue/Date Received 2022-06-30
It is a further aspect of the present invention to disclose the method as
defined in any of the
above, wherein said mutation is an insertion, deletion, indel or substitution
mutation.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said mutation is a deletion in the first exon of CsML01, said deletion
comprises nucleic
acid sequence selected from the group consisting of SEQ ID NO. :876, SEQ ID
NO. :879 or SEQ
ID NO.:881.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
further comprising steps of selecting a plant resistant to powdery mildew from
transformed plants
comprising mutated at least one of CsML01, CsML02 and CsML03 nucleic acid
fragment.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said selected plant is characterized by enhanced resistance to powdery
mildew as
compared to a Cannabis plant comprising a CsML01 nucleic acid comprising a
nucleic acid
sequence as set forth in SEQ ID NO:873.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said genetic modification in said CsML01 is generated in planta via
introduction of a
construct comprising (a) Cas DNA and gRNA sequence selected from the group
consisting of SEQ
ID NO:10-SEQ ID NO:286 and any combination thereof, or (b) a ribonucleoprotein
(RNP)
complex comprising Cos protein and gRNA sequence selected from the group
consisting of SEQ
ID NO:10-286 and any combination thereof.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said genetic modification in said CsML02 is generated in planta via
introduction of a
construct comprising (a) Cas DNA and gRNA sequence selected from the group
consisting of SEQ
ID NO:287-SEQ ID NO:625 and any combination thereof, or (b) a
ribonucleoprotein (RNP)
complex comprising Cos protein and gRNA sequence selected from the group
consisting of SEQ
ID NO:287-625 and any combination thereof.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said genetic modification in said CsML03 is generated in planta via
introduction of a
construct comprising (a) Cas DNA and gRNA sequence selected from the group
consisting of SEQ
ID NO:626-SEQ ID NO:870 and any combination thereof, or (b) a
ribonucleoprotein (RNP)
48
Date Recue/Date Received 2022-06-30
complex comprising Cos protein and gRNA sequence selected from the group
consisting of SEQ
ID NO:626-870 and any combination thereof.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said gRNA nucleotide sequence comprises a 3' Protospacer Adjacent
Motif (PAM), said
PAM is selected from the group consisting of: NGG (SpCas), NNNNGATT (NmeCas9),
NNAGAAW (StCas9), NAAAAC (TdCas9), NNGRRT (SaCas9) and TBN (Cas-phi).
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said construct is introduced into the plant cells using Agrobacterium
infiltration, virus
based plasmids for delivery of the genome editing molecules by or mechanical
insertion such as
polyethylene glycol (PEG) mediated DNA transformation, electroporation or gene
gun biolistics.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
further comprising steps of regenerating a plant carrying said genomic
modification.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
further comprising steps of screening said regenerated plants for a plant
resistant to powdery
mildew.
It is a further aspect of the present invention to disclose a method for
conferring resistance to
powdery mildew to a Cannabis plant comprising producing a plant as defined in
any of the above.
It is a further aspect of the present invention to disclose a plant, plant
part, plant cell, tissue culture
or a seed obtained or obtainable by the method as defined in any of the above.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said PM is selected from the group consisting of Golovinomyces
cichoracearum,
Golovinomyces ambrosiae and a mixture thereof.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said Cannabis plant is selected from the group of species that
includes, but is not limited
to, Cannabis saliva (C. saliva), C. indica, C. ruderalis and any hybrid or
cultivated variety of the
genus Cannabis.
It is a further aspect of the present invention to disclose a method for
producing a modified
Cannabis plant with increased resistance to powdery mildew compared to a
Cannabis wild type
plant using targeted genome modification comprising introducing at least one
genetic modification
49
Date Recue/Date Received 2022-06-30
conferring reduced expression of at least one Cannabis MLO (CsMLO) allele,
said method
comprises steps of: (a) identifying at least one Cannabis MLO (CsMLO)
orthologous allele; (b)
sequencing genomic DNA of said at least one identified CsMLO; (c) synthetizing
at least one
guide RNA (gRNA) comprising a nucleotide sequence complementary to said at
least one
identified CsMLO; (d) transforming Cannabis plant cells with a construct
comprising (a) Cas
nucleotide sequence and said gRNA, or (b) a ribonucleoprotein (RNP) complex
comprising Cas
protein and said gRNA; (e) screening the genome of said transformed plant
cells for induced
targeted mutations in at least one of said CsMLO alleles comprising obtaining
a nucleic acid
sample from said transformed plant and carrying out nucleic acid amplification
and optionally
restriction enzyme digestion to detect a mutation in said at least one of said
CsMLO allele; (f)
confirming the presence of said genetic mutation in the genome of said plant
cells by sequencing
said at least one CsMLO allele; (g) regenerating plants carrying said genetic
modification; and (h)
screening said regenerated plants for a plant resistant to powdery mildew.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said method comprises steps of introducing a targeted genome
modification to at least one
CsMLO allele having a wild type genomic nucleotide sequence selected from the
group consisting
of CsML01 comprising a sequence as set forth in SEQ ID NO:1 or a functional
variant thereof,
CsML02 comprising a sequence as set forth in SEQ ID NO:4 or a functional
variant thereof and
CsML03 comprising a sequence as set forth in SEQ ID NO:7 or a functional
variant thereof.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said functional variant has at least 80% sequence identity to the said
CsMLO nucleotide
sequence.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said plant has decreased levels of at least one Mb o protein.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
further comprising steps of introducing into said plant sgRNA targeted to
mutate CsML01 gene,
said sgRNA nucleotide sequence is selected from the group consisting of SEQ ID
NO:17, SEQ ID
NO:43 and SEQ ID NO:50.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said nucleic acid amplification for screening induced targeted
mutations in CsML01
Date Recue/Date Received 2022-06-30
genomic sequence uses primers having nucleic acid sequence as set forth in SEQ
ID NO: 871 and
SEQ ID NO: 872.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said plant comprises at least one mutated CsML01 allele comprising a
nucleotide
sequence selected from the group consisting of a nucleotide sequence as set
forth in SEQ ID
NO:875, a nucleotide sequence as set forth in SEQ ID NO:877, a nucleotide
sequence as set forth
in SEQ ID NO:880, a homologue having at least 80% sequence identity to the
nucleotide sequence
of said at least one mutatedCsML01 allele and a combination thereof.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said mutation is a silencing mutation, a knockdown mutation, a
knockout mutation, a loss
of function mutation or any combination thereof.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said mutated CsML01 allele comprises a deletion having a nucleotide
sequence as set
forth in SEQ ID NO.:876, SEQ ID NO.:879 or SEQ ID NO.:881.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said mutated allele confers an enhanced resistance to powdery mildew
as compared to a
Cannabis plant comprising a wild type CsML01 allele sequence.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said wild type CsML01 allele comprises a nucleic acid sequence as set
forth in at least
one of SEQ ID NO:873, SEQ ID NO:876, SEQ ID NO:879 or SEQ ID NO:881.
It is a further aspect of the present invention to disclose a method of
determining the presence of
a mutant CsML01 nucleic acid in a Cannabis plant comprising assaying said
Cannabis plant with
primers having nucleic acid sequence as set forth in SEQ ID NO: 871 and SEQ ID
NO: 872.
It is a further aspect of the present invention to disclose a method for
determining the presence or
absence of a mutant CsML01 nucleic acid or polypeptide in a Cannabis plant
comprising detecting
the presence or absence of a deletion of a nucleotide sequence as set forth in
SEQ ID NO. :876,
SEQ ID NO.:879 or SEQ ID NO.:881.
It is a further aspect of the present invention to disclose a method for
identifying a Cannabis plant
with resistance to powdery mildew, said method comprises steps of: (a)
screening the genome of
51
Date Recue/Date Received 2022-06-30
said Cannabis plant for induced targeted mutations in at least one of CsML01,
CsML02 and/or
CsML03 alleles having a wild type genomic nucleotide sequence selected from
the group
consisting of CsML01 comprising a sequence as set forth in SEQ ID NO:1 or a
functional variant
thereof, CsML02 comprising a sequence as set forth in SEQ ID NO:4 or a
functional variant
thereof and CsML03 comprising a sequence as set forth in SEQ ID NO:7 or a
functional variant
thereof; (b) confirming the presence of said genetic mutation in the genome of
said plant cells by
sequencing said at least one CsMLO allele; (c) regenerating plants carrying
said genetic
modification; and (d) screening said regenerated plants for a plant resistant
to powdery mildew.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said screening for the presence of mutated CsML01 allele is carried
out using a primer
pair having nucleic acid sequence as set forth in SEQ ID NO: 871 and SEQ ID
NO: 872.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said method comprises steps of screening for the presence of mutated
CsML01 allele
comprising a nucleic acid sequence selected from the group consisting of a
nucleotide sequence as
set forth in SEQ ID NO:875, a nucleotide sequence as set forth in SEQ ID
NO:877, a nucleotide
sequence as set forth in SEQ ID NO:880, a homologue having at least 80%
sequence identity to
the nucleotide sequence of said at least one mutated CsML01 allele and a
combination thereof.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said method comprises steps of screening said Cannabis plant for the
presence of a
deletion in CsML01 comprising a nucleotide sequence selected from the group
consisting of SEQ
ID NO.:876, SEQ ID NO.:879 and SEQ ID NO.:881.
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein the presence of at least one nucleic acid sequence selected from the
group consisting of
SEQ ID NO:873, SEQ ID NO:876, SEQ ID NO:879 and SEQ ID NO:881 indicates that
the
Cannabis plant comprises wild type CsML01 nucleic acid, and the presence of at
least one nucleic
acid sequence selected from the group consisting of SEQ ID NO:875, SEQ ID
NO:877 and SEQ
ID NO:880, optionally in combination with the absence of at least one nucleic
acid sequence
selected from the group consisting of SEQ ID NO:876, SEQ ID NO:879 and SEQ ID
NO:881
indicates that the Cannabis plant comprises a mutant CsML01 nucleic acid.
52
Date Recue/Date Received 2022-06-30
It is a further aspect of the present invention to disclose the method as
defined in any of the above,
wherein said Cannabis plant comprising a mutant CsML01 nucleic acid is
characterized by
enhanced resistance to powdery mildew as compared to a Cannabis plant
comprising said wild
type CsML01 nucleic acid.
It is a further aspect of the present invention to disclose an isolated
nucleotide sequence of a primer
or primer pair having at least 75% sequence identity to a nucleic acid
sequence selected from the
group consisting of SEQ ID NO:1, 2, 4, 5, 7, 8 and SEQ ID NO:10-873, 875, 876,
877, 879, 880
and 881.
It is a further aspect of the present invention to disclose an isolated amino
acid sequence having at
least 75% sequence similarity to an amino acid sequence selected from the
group consisting of
SEQ ID NO:3, SEQ ID NO:6,SEQ ID NO:9, SEQ ID NO:874, SEQ ID NO:878 and SEQ ID
NO: 887.
It is a further aspect of the present invention to disclose use of a
nucleotide sequence as set forth
in at least one of SEQ ID NO:871 and SEQ ID NO:872 as a primer or primer pair
for identifying
or screening for a Cannabis plant comprising within its genome mutant CsML01
nucleic acid
and/or polypeptide.
It is a further aspect of the present invention to disclose use of a
nucleotide sequence as set forth
in at least one of SEQ ID NO:871 and SEQ ID NO:872 as a primer or primer pair
for identifying
or screening for a Cannabis plant resistance to powdery mildew.
It is a further aspect of the present invention to disclose use of a
nucleotide sequence as set forth
in SEQ ID NO:873, SEQ ID NO:875, SEQ ID NO:876, SEQ ID NO:877, SEQ ID NO:879,
SEQ
ID NO:880 and SEQ ID NO:881 for identifying and/or screening for a Cannabis
plant with
comprising within its genome mutant CsML01 nucleic acid and/or polypeptide,
wherein, the
presence of at least one nucleic acid sequence selected from the group
consisting of SEQ ID
NO:873, SEQ ID NO:876, SEQ ID NO:879 and SEQ ID NO:881 indicates that the
Cannabis plant
comprises wild type CsML01 nucleic acid, and the presence of at least one
nucleic acid sequence
selected from the group consisting of SEQ ID NO:875, SEQ ID NO:877 and SEQ ID
NO:880,
optionally in combination with the absence of at least one nucleic acid
sequence selected from the
group consisting of SEQ ID NO:876, SEQ ID NO:879 and SEQ ID NO:881 indicates
that the
Cannabis plant comprises a mutant CsML01 nucleic acid.
53
Date Recue/Date Received 2022-06-30
It is a further aspect of the present invention to disclose the use as defined
in any of the above,
wherein said Cannabis plant comprising a mutant CsML01 nucleic acid is
characterized by
enhanced resistance to powdery mildew as compared to a Cannabis plant
comprising said wild
type CsML01 nucleic acid.
It is a further aspect of the present invention to disclose use of a
nucleotide sequence as set forth
in at least one of SEQ ID NO:10-870 and any combination thereof for targeted
genome
modification of at least one Cannabis MLO (CsMLO) allele.
It is a further aspect of the present invention to disclose use of a
nucleotide sequence as set forth
in at least one of SEQ ID NO:10-286 and any combination thereof for targeted
genome
modification of Cannabis CsML01 allele.
It is a further aspect of the present invention to disclose use of a
nucleotide sequence as set forth
in at least one of SEQ ID NO:17, SEQ ID NO:43 and SEQ ID NO:50 and any
combination thereof
for targeted genome modification of Cannabis CsML01 allele.
It is a further aspect of the present invention to disclose use of a
nucleotide sequence as set forth
in at least one of SEQ ID NO:287-625 and any combination thereof for targeted
genome
modification of Cannabis CsML02 allele.
It is a further aspect of the present invention to disclose use of a
nucleotide sequence as set forth
in at least one of SEQ ID NO:626-870 and any combination thereof for targeted
genome
modification of Cannabis CsML03.
It is a further aspect of the present invention to disclose a detection kit
for determining the presence
or absence of a mutant CsML01 nucleic acid nucleic acid or polypeptide in a
Cannabis
plant.comprising a primer selected from SEQ ID NO:871 and SEQ ID NO:872.
It is a further aspect of the present invention to disclose the detection kit
as defined in any of the
above, wherein said kit further comprising primers or nucleic acid fragments
for detection of a
nucleic acid sequence selected from the group consisting of SEQ ID NO:873, SEQ
ID NO:875,
SEQ ID NO:876, SEQ ID NO:877, SEQ ID NO:879, SEQ ID NO:880 and SEQ ID NO:881.
It is a further aspect of the present invention to disclose the detection kit
as defined in any of the
above, wherein said kit is useful for identifying a Cannabis plant resistant
to powdery mildew.
54
Date Recue/Date Received 2022-06-30
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
Exemplified method for production of powdery mildew resistant Cannabis plants
by genome
editing
Production of powdery mildew resistant Cannabis lines may be achieved by at
least one of the
following breeding/cultivation schemes:
Scheme I:
= line stabilization by self pollination
= Generation of F6 parental lines
= Genome editing of parental lines
= Crossing edited parental lines to generate an F I hybrid PM resistant
plant
Scheme 2:
= Identifying genes of interest
= Designing gRNA
= Transformation of plants with Cas9+gRNA constructs
= Screening and identifying editing events
= Genome editing of parental lines
It is noted that line stabilization may be performed by the following:
= Induction of male flowering on female (XX) plants
= Self pollination
According to some embodiments of the present invention, line stabilization
requires 6 self-crossing
(6 generations) and done through a single seed descent (SSD) approach.
Fl hybrid seed production: Novel hybrids are produced by crosses between
different Cannabis
strains.
Date Recue/Date Received 2022-06-30
According to a further aspect of the current invention, shortening line
stabilization is performed
by Doubled Haploids (DH). More specifically, the CRISPR-Cas9 system is
transformed into
microspores to achieve DH homozygous parental lines. A doubled haploid (DH) is
a genotype
formed when haploid cells undergo chromosome doubling. Artificial production
of doubled
haploids is important in plant breeding. It is herein acknowledged that
conventional inbreeding
procedures take six generations to achieve approximately complete
homozygosity, whereas
doubled haploidy achieves it in one generation.
It is within the scope of the current invention that genetic markers specific
for Cannabis are
developed and provided by the current invention:
= Sex markers- molecular markers are used for identification and selection
of female vs male
plants in the herein disclosed breeding program
= Genotyping markers- germplasm used in the current invention is genotyped
using molecular
markers, in order to allow a more efficient breeding process and
identification of the MLO
editing event.
It is further within the scope of the current invention that allele and
genetic variation is analysed
for the Cannabis strains used.
Reference is now made to optional stages that have been used for the
production of powdery
mildew resistant Cannabis plants by genome editing:
Stage 1: Identifying Cannabis saliva (C. saliva) MLO orthologues, Three MLO
orthologues have
herein been identified in C. saliva, namely CsML01, CsML02 and CsML03. These
homologous
genes have been sequenced and mapped. CsML01 has been found to be located on
chromosome
between position 58544241bp and position 58551241bp and has a genomic sequence
as set forth
in SEQ ID NO: 1. The CsML01 gene has a coding sequence as set forth in SEQ ID
NO:2 and it
encodes an amino acid sequence as set forth in SEQ ID NO:3.
CsML02 has been found to be located on chromosome 3 between position
92616000bp and
position 92629000bp and has a genomic sequence as set forth in SEQ ID NO:4.
The CsML02
gene has a coding sequence as set forth in SEQ ID NO:5 and it encodes an amino
acid sequence
as set forth in SEQ ID NO:6.
56
Date Recue/Date Received 2022-06-30
CsML03 has been found to be located on Chromosome 5 between position
23410000bp and
position 23420000bp and has a genomic sequence as set forth in SEQ ID NO:7.
The CsML03
gene has a coding sequence as set forth in SEQ ID NO:8 and it encodes an amino
acid sequence
as set forth in SEQ ID NO:9.
Stage 2: Designing and synthesizing gRNA molecules corresponding to the
sequence targeted for
editing, i.e. sequences of each of the genes CsML01, CsML02 and CsML03. It is
noted that the
editing event is preferably targeted to a unique restriction site sequence to
allow easier screening
for plants carrying an editing event within their genome. According to some
aspects of the
invention, the nucleotide sequence of the gRNAs should be completely
compatible with the
genomic sequence of the target gene. Therefore, for example, suitable gRNA
molecules should be
constructed for different MLO homologues of different Cannabis strains.
Reference is now made to Tables 1, 2 and 3 presenting gRNA molecules
constructed for silencing
CsML01, CsML02 and CsML03, respectively. In Tables 1, 2 and 3 the term 'PAM'
refers to
protospacer adjacent motif, which is a 2-6 base pair DNA sequence immediately
following the
DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive
immune system.
The CsMLO genomic DNA sense strand is marked as "1", and the antisense strand
is marked as
Table 1: CsMIL01 targeted gRNA sequences
Position Strand Sequence PAM SEQ ID
on SEQ NO
ID NO:1
30 1 GTGAGTGAATGAGAGCAAGA AGG 10
59 -1 ATTCCGATTTCGAATTCAGA TGG 11
67 1 AATCCATCTGAATTCGAAAT CGG 12
76 1 GAATTCGAAATCGGAATGAG TGG 13
79 1 TTCGAAATCGGAATGAGTGG CGG 14
82 1 GAAATCGGAATGAGTGGCGG TGG 15
88 1 GGAATGAGTGGCGGTGGAGA AGG 16
99 1 CGGTGGAGAAGGTGAGTCCT TGG 17
105 -1 CCATGTGGGAGTATACTCCA AGG 18
116 1 CCTTGGAGTATACTCCCACA TGG 19
119 -1 ACGACGGCGACGATCCATGT GGG 20
120 -1 GACGACGGCGACGATCCATG TGG 21
135 -1 GACGATGACAGAGCAGACGA CGG 22
57
Date Recue/Date Received 2022-06-30
159 -1 ACGCTCCGCGGCGAGAGAAA TGG 23
165 1 CGTCGCCATTTCTCTCGCCG CGG 24
171 -1 ATAGTGGAGAAGACGCTCCG CGG 25
187 1 GAGCGTCTTCTCCACTATCT CGG 26
187 -1 TCAAAACCTGACCGAGATAG TGG 27
192 1 TCTTCTCCACTATCTCGGTC AGG 28
220 -1 AGGCCTCGTATAGAGGCTTC TGG 29
227 -1 TTCTGCAAGGCCTCGTATAG AGG 30
228 1 GAACCAGAAGCCTCTATACG AGG 31
240 -1 CTCCTCCTTGATCTTCTGCA AGG 32
246 1 CGAGGCCTTGCAGAAGATCA AGG 33
249 1 GGCCTTGCAGAAGATCAAGG AGG 34
264 1 CAAGGAGGAGTTGATGCTTT TGG 35
265 1 AAGGAGGAGTTGATGCTTTT GGG 36
292 -1 TTGTGTTCTGCGAAACAGTG AGG 37
332 -1 AGATTGTCGACCAAAGAAGC AGG 38
333 1 GTTTTGCGTACCTGCTTCTT TGG 39
355 -1 GATGAGGGCGCTTACAAGGG AGG 40
358 -1 CCTGATGAGGGCGCTTACAA GGG 41
359 -1 TCCTGATGAGGGCGCTTACA AGG 42
369 1 CCCTTGTAAGCGCCCTCATC AGG 43
370 -1 AATCATTAGCTTCCTGATGA GGG 44
371 -1 GAATCATTAGCTTCCTGATG AGG 45
405 -1 AGAGCCGGAGATGTGATGAG AGG 46
412 1 TCAACCTCTCATCACATCTC CGG 47
420 -1 AAGAAGGCGTCTGAAAGAGC CGG 48
436 -1 CAGTGGAAGTTTCTTCAAGA AGG 49
453 -1 GCAATAACCCAAATGAGCAG TGG 50
456 1 AGAAACTTCCACTGCTCATT TGG 51
457 1 GAAACTTCCACTGCTCATTT GGG 52
474 1 TTTGGGTTATTGCGCTCATA AGG 53
501 -1 ACAACAACAACTAAAGATAT GGG 54
502 -1 AACAACAACAACTAAAGATA TGG 55
521 1 TTAGTTGTTGTTGTTTTTTT AGG 56
522 1 TAGTTGTTGTTGTTTTTTTA GGG 57
523 1 AGTTGTTGTTGTTTTTTTAG GGG 58
570 1 TATAAATATACTTTCCCAAA AGG 59
571 1 ATAAATATACTTTCCCAAAA GGG 60
573 -1 TAAAGCGAATAGTCCCTTTT GGG 61
574 -1 TTAAAGCGAATAGTCCCTTT TGG 62
657 -1 ATGCTTCAACGGAATAAAAG GGG 63
658 -1 AATGCTTCAACGGAATAAAA GGG 64
659 -1 CAATGCTTCAACGGAATAAA AGG 65
668 -1 CAAATGGTGCAATGCTTCAA CGG 66
684 -1 CGAAGATAAAGATATGCAAA TGG 67
58
Date Recue/Date Received 2022-06-30
708 -1 AAGTGACATGGACAATGGCT AGG 68
713 -1 ACAGAAAGTGACATGGACAA TGG 69
720 -1 TGAGAACACAGAAAGTGACA TGG 70
744 1 TGTGTTCTCACTGTTGTGTT TGG 71
747 1 GTTCTCACTGTTGTGTTTGG AGG 72
755 1 TGTTGTGTTTGGAGGTGTAA AGG 73
779 -1 GAGAGAGAAGCATATGAATT TGG 74
860 1 TACACAACTAGATACGTCAA TGG 75
869 1 AGATACGTCAATGGAAACGT TGG 76
870 1 GATACGTCAATGGAAACGTT GGG 77
873 1 ACGTCAATGGAAACGTTGGG AGG 78
910 1 GAGAGTTATGACACTGAACA AGG 79
937 -1 TTCTATAATATTGTCAAAAG TGG 80
978 -1 TAAGCGATTCATATGTTAGA AGG 81
1017 1 TGTTCTTAAGTCTAAGAAAA AGG 82
1039 -1 CCTTAATGAACGCGTGTTGA TGG 83
1050 1 CCATCAACACGCGTTCATTA AGG 84
1062 1 GTTCATTAAGGACCACTTTT TGG 85
1063 1 TTCATTAAGGACCACTTTTT GGG 86
1063 -1 CTTTACCAAAACCCAAAAAG TGG 87
1069 1 AAGGACCACTTTTTGGGTTT TGG 88
1090 1 GGTAAAGACTCAGCTCTACT AGG 89
1094 1 AAGACTCAGCTCTACTAGGC TGG 90
1098 1 CTCAGCTCTACTAGGCTGGC TGG 91
1140 -1 ATAATCCTAATTCAGAACTT TGG 92
1146 1 AGTATCCAAAGTTCTGAATT AGG 93
1159 1 CTGAATTAGGATTATTCTTA TGG 94
1174 -1 ATATCAAGAGAATAAGAAAA CGG 95
1214 -1 AACGTTCTCAAAATAGAAAG TGG 96
1267 -1 CTCCAAAGGCTCAGTATCAA TGG 97
1276 1 CTCCATTGATACTGAGCCTT TGG 98
1281 -1 GTGATGGAGAAAATCTCCAA AGG 99
1297 -1 ATGTCCTAGTTTTCATGTGA TGG 100
1304 1 TTCTCCATCACATGAAAACT AGG 101
1327 1 ACATTTTTGTGCACATGTTA AGG 102
1349 1 GAGCTAGCTAACATTAACAT TGG 103
1356 1 CTAACATTAACATTGGAAAC AGG 104
1379 -1 GGGGAAAAAGAATCAAATCA TGG 105
1398 -1 AAAAGCATGCTGTTCACAAG GGG 106
1399 -1 CAAAAGCATGCTGTTCACAA GGG 107
1400 -1 ACAAAAGCATGCTGTTCACA AGG 108
1446 -1 CATGGTCGCATAATCTGATT TGG 109
1460 1 AATCAGATTATGCGACCATG CGG 110
1464 -1 CATGATGAATCCTAACCGCA TGG 111
1465 1 GATTATGCGACCATGCGGTT AGG 112
59
Date Recue/Date Received 2022-06-30
1476 1 CATGCGGTTAGGATTCATCA TGG 113
1520 1 TTACTTATAAATTACTAGAA TGG 114
1563 1 CTTTTTTTCTTTTCACTAAA TGG 115
1603 1 TTGTATTCTAGACTCACTGC AGG 116
1604 1 TGTATTCTAGACTCACTGCA GGG 117
1605 1 GTATTCTAGACTCACTGCAG GGG 118
1674 1 GATGATTTCAAGAAAGTTGT TGG 119
1675 1 ATGATTTCAAGAAAGTTGTT GGG 120
1681 1 TCAAGAAAGTTGTTGGGATA AGG 121
1691 1 TGTTGGGATAAGGTAACCCT TGG 122
1696 -1 GAAAATAGACTGTCAACCAA GGG 123
1697 -1 AGAAAATAGACTGTCAACCA AGG 124
1777 1 TTCTTTTAAGTCTACTGTAT CGG 125
1792 -1 AAAAGCTAAAAGGTCAATCT AGG 126
1802 -1 ACTGCAGCCAAAAAGCTAAA AGG 127
1806 1 AGATTGACCTTTTAGCTTTT TGG 128
1816 1 TTTAGCTTTTTGGCTGCAGT TGG 129
1825 1 TTGGCTGCAGTTGGTACCTT TGG 130
1826 1 TGGCTGCAGTTGGTACCTTT GGG 131
1830 -1 AGATGACCACAAAAACCCAA AGG 132
1835 1 TTGGTACCTTTGGGTTTTTG TGG 133
1863 1 TTCTTGTTGCTGAATGTTAA TGG 134
1910 -1 ATCACTTAGATCTTGAGTTA TGG 135
1926 1 ACTCAAGATCTAAGTGATAT TGG 136
1958 -1 CAAAGAACCAGACTGATTAC GGG 137
1959 -1 ACAAAGAACCAGACTGATTA CGG 138
1962 1 GCAGAATCCCGTAATCAGTC TGG 139
1999 1 CTTCAAGTGTGTCATCTCTT TGG 140
2033 -1 GCTTATTTGAAACTATAATT TGG 141
2101 -1 GTGGAGGCAGAGTAAGGAAT TGG 142
2107 -1 AGAAAAGTGGAGGCAGAGTA AGG 143
2117 -1 CTCCAACCATAGAAAAGTGG AGG 144
2120 -1 TTTCTCCAACCATAGAAAAG TGG 145
2122 1 ACTCTGCCTCCACTTTTCTA TGG 146
2126 1 TGCCTCCACTTTTCTATGGT TGG 147
2148 1 GAGAAAATTATACTCCAAGT TGG 148
2151 -1 TCCAATTCCTTACACCAACT TGG 149
2155 1 TTATACTCCAAGTTGGTGTA AGG 150
2161 1 TCCAAGTTGGTGTAAGGAAT TGG 151
2203 1 TCACTAGAATGCAATCAACA AGG 152
2204 1 CACTAGAATGCAATCAACAA GGG 153
2244 -1 AATTTTTTTAATCAGAATTC TGG 154
2293 -1 TAAAAGTAAACTAAATTTCT TGG 155
2347 -1 ATGTAAATTATGTTCTATAT AGG 156
2380 -1 GATCCAATTGAATTATCTTA AGG 157
Date Recue/Date Received 2022-06-30
2388 1 ACACCTTAAGATAATTCAAT TGG 158
2406 1 ATTGGATCTTACTCCTTGTT TGG 159
2408 -1 GCCTCATTGTAGTCCAAACA AGG 160
2418 1 TCCTTGTTTGGACTACAATG AGG 161
2453 -1 ATCTTGGTTTGAGTATTGAG AGG 162
2469 -1 ATATAAATAATGAATGATCT TGG 163
2497 -1 CAAAGAAGTTTAATACACAC TGG 164
2516 1 TATTAAACTTCTTTGTTGTA TGG 165
2559 1 TTTTGTCAATGTTTTGTGAT TGG 166
2597 1 TAATAATGTGTTATATTTGC AGG 167
2601 1 AATGTGTTATATTTGCAGGC TGG 168
2616 1 CAGGCTGGCACACATaTTTC TGG 169
2640 -1 CTCAATAAACTCACAAAGAA AGG 170
2709 1 CATGTTTCATTGTTCTTGCA TGG 171
2750 1 CATTTTAAGTATCATACTGA TGG 172
2774 1 GAAAGAGATAAAATACAGAG AGG 173
2775 1 AAAGAGATAAAATACAGAGA GGG 174
2783 1 AAAATACAGAGAGGGAGAAT CGG 175
2784 1 AAATACAGAGAGGGAGAATC GGG 176
2817 1 TTTAACACAATTTTGTAAAT AGG 177
2824 1 CAATTTTGTAAATAGGCAAA TGG 178
2837 1 AGGCAAATGGACAGCTAAGA AGG 179
2852 -1 TGTTCAATTAATTCTAAATT TGG 180
2875 1 TTAATTGAACAACATGACCT AGG 181
2881 -1 AAATTGCACAATATTTACCT AGG 182
2950 1 TAAATGTAGAGTCATGAGTC AGG 183
2951 1 AAATGTAGAGTCATGAGTCA GGG 184
2973 1 GTAGAAATTTGCACCTAGAC AGG 185
2975 -1 CACCTTAAAACCACCTGTCT AGG 186
2976 1 GAAATTTGCACCTAGACAGG TGG 187
2984 1 CACCTAGACAGGTGGTTTTA AGG 188
2987 1 CTAGACAGGTGGTTTTAAGG TGG 189
3011 1 ACTTCTCATCTCCAAGTCTT AGG 190
3011 -1 CATACATATCACCTAAGACT TGG 191
3046 -1 ATGTATATCACAACAGCAAA AGG 192
3083 -1 TTAAAAGAAAAAACAACAAG TGG 193
3114 1 TAAATAGCTTCTACTTGCCG TGG 194
3115 1 AAATAGCTTCTACTTGCCGT GGG 195
3120 -1 ATGCTCCAGTTTAGTGCCCA CGG 196
3126 1 ACTTGCCGTGGGCACTAAAC TGG 197
3147 1 GGAGCATGTCATTACTCAGT TGG 198
3184 1 GAGAAACATGTAGCAATAGA AGG 199
3212 -1 AACCAAAAGTGATCATCTGA TGG 200
3221 1 AGCCATCAGATGATCACTTT TGG 201
3242 -1 ATCAGGAAGAGGACAATCTG GGG 202
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Date Recue/Date Received 2022-06-30
3243 -1 AATCAGGAAGAGGACAATCT GGG 203
3244 -1 GAATCAGGAAGAGGACAATC TGG 204
3253 -1 TGATGAAATGAATCAGGAAG AGG 205
3259 -1 AAAGGATGATGAAATGAATC AGG 206
3277 -1 TCTCAAATGAATTTTGGAAA AGG 207
3283 -1 ACGCAATCTCAAATGAATTT TGG 208
3305 1 TTGAGATTGCGTTTTTCTTC TGG 209
3312 1 TGCGTTTTTCTTCTGGATAT TGG 210
3320 1 TCTTCTGGATATTGGTAAGC TGG 211
3343 -1 TGGTAGAAGTAGAAGCAGAG TGG 212
3363 -1 AATAACAATTTGTTCTTTTT TGG 213
3411 1 ATCTTCTTTTCTGTGTATCT AGG 214
3441 1 TTCATTTAACTCCTGTATAA TGG 215
3441 -1 ACGAACGTGTCCCATTATAC AGG 216
3442 1 TCATTTAACTCCTGTATAAT GGG 217
3472 -1 TTTACCCAATGACAAGTCTT GGG 218
3473 -1 TTTTACCCAATGACAAGTCT TGG 219
3478 1 ATTGTCCCAAGACTTGTCAT TGG 220
3479 1 TTGTCCCAAGACTTGTCATT GGG 221
3541 -1 TAAAATAAAAGTTTCGTACT TGG 222
3570 -1 GAACACCCTAAAGCACAACA TGG 223
3575 1 TTTTTACCATGTTGTGCTTT AGG 224
3576 1 TTTTACCATGTTGTGCTTTA GGG 225
3588 1 GTGCTTTAGGGTGTTCATTC AGG 226
3622 -1 GTGTGACAATGGCATAGAGC GGG 227
3623 -1 TGTGTGACAATGGCATAGAG CGG 228
3633 -1 TCAACGCACCTGTGTGACAA TGG 229
3636 1 GCTCTATGCCATTGTCACAC AGG 230
3681 1 ATAATTTAATAAGTTCTAAA AGG 231
3689 1 ATAAGTTCTAAAAGGAAAGT AGG 232
3720 -1 CATTCCACAAGATTTTATTA TGG 233
3727 1 CTGACCATAATAAAATCTTG TGG 234
3743 1 CTTGTGGAATGATTTGAAGA TGG 235
3744 1 TTGTGGAATGATTTGAAGAT GGG 236
3773 1 TTACAAGAAAGCCATATTTG AGG 237
3773 -1 TTGCATGCGCTCCTCAAATA TGG 238
3789 1 TTTGAGGAGCGCATGCAAGT AGG 239
3802 1 TGCAAGTAGGAATTGTTAAT TGG 240
3803 1 GCAAGTAGGAATTGTTAATT GGG 241
3812 1 AATTGTTAATTGGGCTCAGA AGG 242
3827 1 TCAGAAGGTCAAGAAAAAGA AGG 243
3828 1 CAGAAGGTCAAGAAAAAGAA GGG 244
3849 1 GGATTTAAAGCAGCCCTCAT TGG 245
3851 -1 GCCAGCACCGGAACCAATGA GGG 246
3852 -1 AGCCAGCACCGGAACCAATG AGG 247
62
Date Recue/Date Received 2022-06-30
3855 1 AAAGCAGCCCTCATTGGTTC CGG 248
3861 1 GCCCTCATTGGTTCCGGTGC TGG 249
3863 -1 GCCTGAGCCTGAGCCAGCAC CGG 250
3867 1 ATTGGTTCCGGTGCTGGCTC AGG 251
3873 1 TCCGGTGCTGGCTCAGGCTC AGG 252
3879 1 GCTGGCTCAGGCTCAGGCTC AGG 253
3884 1 CTCAGGCTCAGGCTCAGGCT CGG 254
3885 1 TCAGGCTCAGGCTCAGGCTC GGG 255
3891 1 TCAGGCTCAGGCTCGGGATC AGG 256
3903 1 TCGGGATCAGGCTCTACTCC TGG 257
3910 -1 GTATCAGAAATTGGTTGACC AGG 258
3919 -1 GCAGAACCAGTATCAGAAAT TGG 259
3924 1 GGTCAACCAATTTCTGATAC TGG 260
3938 1 TGATACTGGTTCTGCATCTG TGG 261
3939 1 GATACTGGTTCTGCATCTGT GGG 262
3950 1 TGCATCTGTGGGAATTCAGC TGG 263
3951 1 GCATCTGTGGGAATTCAGCT GGG 264
3973 -1 TGCTCTGGCTTTGATGCTTT GGG 265
3974 -1 CTGCTCTGGCTTTGATGCTT TGG 266
3988 -1 TTAGAGTCATCACTCTGCTC TGG 267
4058 1 GAAGACATAAGTCTACCCTT AGG 268
4062 -1 CTAGTAGTAGTATTACCTAA GGG 269
4063 -1 ACTAGTAGTAGTATTACCTA AGG 270
4088 -1 ATCCCAGCACAGCTGGAAAG TGG 271
4095 -1 ATTTCTAATCCCAGCACAGC TGG 272
4096 1 TTGCCACTTTCCAGCTGTGC TGG 273
4097 1 TGCCACTTTCCAGCTGTGCT GGG 274
4132 1 AATTCTTCTGTCATATATTA TGG 275
4138 1 TCTGTCATATATTATGGCTG TGG 276
4141 1 GTCATATATTATGGCTGTGG TGG 277
4142 1 TCATATATTATGGCTGTGGT GGG 278
4160 -1 GTCTTGTCCATAAAAGACTT AGG 279
4164 1 GACTGTACCTAAGTCTTTTA TGG 280
4188 -1 TTATATAATATATTGATCAA AGG 281
4267 1 CTTCTTTCTTCTTATTATCA TGG 282
4280 1 ATTATCATGGTACATCCTTT TGG 283
4284 -1 TTCACTATTCAGTTACCAAA AGG 284
4312 1 AGTGAATACGTGTAGTCTCA TGG 285
4313 1 GTGAATACGTGTAGTCTCAT GGG 286
63
Date Recue/Date Received 2022-06-30
Table 2: CsMiL02 targeted gRNA sequences
Position Strand Sequence PAM SEQ ID
on SEQ NO
ID NO:4
1977 -1 GTATGAATATGAAATTAAGT TGG 287
2044 -1 AGAGAGAGAGAGACAGAGAG TGG 288
2117 -1 TTGAAATTGGGATGGAGATG TGG 289
2125 -1 ATTCTGTTTTGAAATTGGGA TGG 290
2129 -1 GTAAATTCTGTTTTGAAATT GGG 291
2130 -1 TGTAAATTCTGTTTTGAAAT TGG 292
2153 -1 GTTAGAATGAAAAGTTTGAT GGG 293
2154 -1 AGTTAGAATGAAAAGTTTGA TGG 294
2211 1 TATAATCAATTATTCCCAAG TGG 295
2214 -1 TAAATATAAATAGGCCACTT GGG 296
2215 -1 ATAAATATAAATAGGCCACT TGG 297
2223 -1 TAGTGATCATAAATATAAAT AGG 298
2278 1 AAAATTAAATTAAAAGAAGA TGG 299
2281 1 ATTAAATTAAAAGAAGATGG CGG 300
2284 1 AAATTAAAAGAAGATGGCGG TGG 301
2291 1 AAGAAGATGGCGGTGGCTAG CGG 302
2294 1 AAGATGGCGGTGGC TAGCGG AGG 303
2322 1 CTTTAGAACAAACAC CAAC A TGG 304
2323 1 TTTAGAACAAACACCAACAT GGG 305
2325 -1 ACTACGGCCACAGCCCATGT TGG 306
2329 1 ACAAACACCAACATGGGCTG TGG 307
2341 -1 TACCAAAACAAGACAAACTA CGG 308
2350 1 GGCCGTAGTTTGTCTTGTTT TGG 309
2371 -1 GATTATGTGCTCAATAATAA TGG 310
2393 1 GAGCACATAATCCATCTCAT TGG 311
2393 -1 GGTATACCTTGCCAATGAGA TGG 312
2398 1 CATAATC CAT CTCAT TGGCA AGG 313
2414 -1 TGAGATTAATATATATAATT GGG 314
2415 -1 GTGAGATTAATATATATAAT TGG 315
2473 1 CATTTAATTATTTAAATTAA TGG 316
2474 1 ATTTAATTATTTAAATTAAT GGG 317
64
Date Regue/Date Received 2022-06-30
2495 1 GGTATTTTTTTTTTTTTTAG TGG 318
2535 1 ACGAGCTCTTTATGAATCGT TGG 319
2551 1 TCGTTGGAAAAGATCAAATC AGG 320
2576 -1 AAAATGGGTATTCATTAATT GGG 321
2577 -1 AAAAATGGGTATTCATTAAT TGG 322
2591 -1 TTAAAAAAAAAAACAAAAAT GGG 323
2656 1 TTTGATAGAGCTTATGTTAT TGG 324
2657 1 TTGATAGAGCTTATGTTATT GGG 325
2658 1 TGATAGAGCTTATGTTATTG GGG 326
2680 1 GTTCATATCGTTGTTACTAA CGG 327
2683 1 CATATCGTTGTTACTAACGG TGG 328
2684 1 ATATCGTTGTTACTAACGGT GGG 329
2703 -1 GATATACAAATATTTGAGAT CGG 330
2726 1 ATTTGTATATCTGAGAAAAT TGG 331
2729 1 TGTATATCTGAGAAAATTGG AGG 332
2730 1 GTATATCTGAGAAAATTGGA GGG 333
2736 1 CTGAGAAAATTGGAGGGACA TGG 334
2751 -1 TCTTCTTGTTCTTTATTACA AGG 335
2777 1 CAAGAAGAGAAATTGAATAA AGG 336
2778 1 AAGAAGAGAAATTGAATAAA GGG 337
2779 1 AGAAGAGAAATTGAATAAAG GGG 338
2817 1 TCGAACATGAAAGTAACAGT CGG 339
2827 1 AAGTAACAGTCGGAGATTGC TGG 340
2839 -1 ACC GTCGCC GGACTC TAAAA AGG 341
2843 1 TTGCTGGC CT TTTTAGAGTC CGG 342
2849 1 GCC TTTTTAGAGTCC GGC GA CGG 343
2851 -1 GACACTAGCAGCACCGTCGC CGG 344
2865 1 GCGACGGTGCTGCTAGTGTC CGG 345
2873 -1 CGGC C GCC GC CAAAATTC GC CGG 346
2875 1 TGCTAGTGTCCGGCGAATTT TGG 347
2878 1 TAGTGTCCGGCGAATTTTGG CGG 348
2881 1 TGTCCGGCGAATTTTGGCGG CGG 349
2885 1 CGGCGAATTTTGGCGGCGGC CGG 350
2886 1 GGCGAATTTTGGCGGCGGCC GGG 351
2893 -1 TTCAGCACACTTATCAGTCC CGG 352
Date Recue/Date Received 2022-06-30
2908 1 GACTGATAAGTGTGCTGAAA AGG 353
2978 1 GTCTTTCTTATCCTTTTATT TGG 354
2978 -1 GACGAATATGTCCAAATAAA AGG 355
3000 -1 CTCCTATAATATTATATGTT TGG 356
3009 1 GTCCAAACATATAATATTAT AGG 357
3051 -1 AAATATATAAATTTAAAGGT TGG 358
3055 -1 AACTAAATATATAAATTTAA AGG 359
4125 1 AAATTATATACATATATGAA TGG 360
4168 1 ATATATATAATTATAATTTC AGG 361
4169 1 TATATATAATTATAATTTCA GGG 362
4187 -1 ATACCATCCGCCGAAACAAA TGG 363
4188 1 AGGGCAAGTTCCATTTGTTT CGG 364
4191 1 GCAAGTTCCATTTGTTTCGG CGG 365
4195 1 GTTCCATTTGTTTCGGCGGA TGG 366
4230 1 GCATATTTTTATCTTTGTGT TGG 367
4249 -1 TCATGATGCAGTAGAGAACA TGG 368
4272 1 CTGCATCATGACTATGTTTT TGG 369
4273 1 TGCATCATGACTATGTTTTT GGG 370
4284 1 TATGTTTTTGGGCAGACTTA AGG 371
4404 -1 AATTTATATATAATTATTTA GGG 372
4405 -1 CAATTTATATATAATTATTT AGG 373
4428 1 TATATAAATTGATTCCCAGA TGG 374
4429 1 ATATAAATTGATTCCCAGAT GGG 375
4431 -1 ATGCTTCCAACTTCCCATCT GGG 376
4432 -1 AATGCTTCCAACTTCCCATC TGG 377
4436 1 TTGATTCCCAGATGGGAAGT TGG 378
4445 1 AGATGGGAAGTTGGAAGCAT TGG 379
4446 1 GATGGGAAGTTGGAAGCATT GGG 380
4452 1 AAGTTGGAAGCATTGGGAAA AGG 381
4476 -1 ACCATGTGAGAATTGATATT CGG 382
4486 1 GCCGAATATCAATTCTCACA TGG 383
4548 1 CTTAATTTTAATTTTTCTAT AGG 384
4551 1 AATTTTAATTTTTCTATAGG TGG 385
4649 -1 CTATATGACATATTTGATGG TGG 386
4652 -1 TAACTATATGACATATTTGA TGG 387
66
Date Recue/Date Received 2022-06-30
4742 1 AATTATAAGAGCATCTTTAT TGG 388
4749 1 AGAGCATCTTTATTGGACAC CGG 389
4757 -1 TAGAAAGTGTTAAATATCAC CGG 390
4844 -1 TATTGGTATAATTAAGTATC AGG 391
4861 -1 CTTACCAATTATATTATTAT TGG 392
4868 1 TATACCAATAATAATATAAT TGG 393
4903 1 ATTTATAAGAAGTATATATA TGG 394
4904 1 TTTATAAGAAGTATATATAT GGG 395
4923 1 TGGGAGTTAGAATTAAGTAA AGG 396
4997 -1 CTC GC AAATC TGAAT CTTTC TGG 397
5009 1 CAGAAAGATTCAGATTTGCG AGG 398
5010 1 AGAAAGATTCAGATT TGC GA GGG 399
5023 1 TTTGCGAGGGACACTTCTTT TGG 400
5045 1 GAAGAAGACATTTAAGTTTC TGG 401
5058 -1 CCATATTAGGAAAGGGTGTT TGG 402
5065 -1 TTACTATCCATATTAGGAAA GGG 403
5066 -1 CTTACTATCCATATTAGGAA AGG 404
5069 1 CCAAACAC CC TTTCC TAATA TGG 405
5071 -1 GGGATCTTACTATCCATATT AGG 406
5091 -1 AAGTAAAAAGTGGGTAAAAA GGG 407
5092 -1 AAAGTAAAAAGTGGGTAAAA AGG 408
5100 -1 AATATAAAAAAGTAAAAAGT GGG 409
5101 -1 GAATATAAAAAAGTAAAAAG TGG 410
5149 -1 ATATAAGTGCATGGATATAG TGG 411
5158 -1 TATTAATAGATATAAGTGCA TGG 412
5233 -1 CATATTTATATGCATGTGAA AGG 413
5253 1 TGCATATAAATATGTTTGCA TGG 414
5269 1 TGCATGGTTTTTATACATCG TGG 415
7159 1 TATATATATAATATTTTTTT TGG 416
7213 -1 TTAATTAATAATTAAAGAGC AGG 417
7238 1 ATTAATTAATTATTTTTCGC AGG 418
7282 -1 AAAGTTAAATAATCAACTTT AGG 419
7302 1 GATTATTTAACTTTGAGACA TGG 420
7313 1 TTTGAGACATGGATTTATAA TGG 421
7373 1 ATTATAGCTGTAGAGATATT TGG 422
67
Date Recue/Date Received 2022-06-30
7387 -1 TAAGTATTATTAAAAATACA AGG 423
8017 -1 GAATGAGAATAGGAATAGAA TGG 424
8027 -1 ATAGGAATGGGAATGAGAAT AGG 425
8039 -1 ATAGGAATAGAAATAGGAAT GGG 426
8040 -1 TATAGGAATAGAAATAGGAA TGG 427
8045 -1 GAAAATATAGGAATAGAAAT AGG 428
8057 -1 GTTGAGAGGAATGAAAATAT AGG 429
8071 -1 CACAGAGGCGTTTGGTTGAG AGG 430
8079 -1 AATAGGCCCACAGAGGCGTT TGG 431
8083 1 CTCTCAACCAAACGCCTCTG TGG 432
8084 1 TCTCAACCAAACGCCTCTGT GGG 433
8086 -1 ACAAGATAATAGGCCCACAG AGG 434
8096 -1 TTAATACATAACAAGATAAT AGG 435
8150 1 ATCAATAACTAAATTAATTG AGG 436
8177 1 TTATAACAATTAATAATTTC AGG 437
8186 1 TTAATAATTTCAGGCACATT TGG 438
8198 -1 AAATTTTTGATGGCTTTGAG GGG 439
8199 -1 CAAATTTTTGATGGCTTTGA GGG 440
8200 -1 TCAAATTTTTGATGGCTTTG AGG 441
8208 -1 TTTGAAAGTCAAATTTTTGA TGG 442
8255 1 ATCTCTAGAAGAAGATTTCA AGG 443
8265 1 GAAGATTTCAAGGTCGTTGT AGG 444
8272 1 TCAAGGTCGTTGTAGGAATC AGG 445
8345 -1 ATTAAAATAAGTCATCATTT GGG 446
8346 -1 AATTAAAATAAGTCATCATT TGG 447
8399 1 TAATAATTATTATTTTGTTT TGG 448
8427 1 TCAATCTCAGTCCTCCTATT TGG 449
8427 -1 ACAGCGAAGAACCAAATAGG AGG 450
8430 -1 ACCACAGCGAAGAACCAAAT AGG 451
8440 1 TCCTATTTGGTTCTTCGCTG TGG 452
8465 1 TTCTTACTCTTCAATACCCA TGG 453
8470 -1 AATAATAAAATGCTCACCAT GGG 454
8471 -1 TAATAATAAAATGCTCACCA TGG 455
8500 -1 GGATGCATTGAAATAATTAA TGG 456
8521 -1 AATCTAAACTGTGATAATTA GGG 457
68
Date Recue/Date Received 2022-06-30
8522 -1 AAATCTAAACTGTGATAATT AGG 458
8566 -1 TTGACATATATGCACACGTT TGG 459
8605 1 TATATTTTTGTTTTTATTAT TGG 460
8618 -1 AAATGTAAACAAATTCATTA TGG 461
8631 1 ATAATGAATTTGTTTACATT TGG 462
8636 1 GAATTTGTTTACATTTGGAC AGG 463
8640 1 TTGTTTACATTTGGACAGGC TGG 464
8655 1 CAGGCTGGTATTCTTATCTT TGG 465
8670 -1 CTTACAATTAGAGGAATAAA AGG 466
8679 -1 ATATTAGTACTTACAATTAG AGG 467
8820 -1 GATTGTTTGAATTTTATTTT TGG 468
8907 -1 GTTTACAGTAAAACTTTAAA AGG 469
8932 -1 AATTAGCCCAATTTTTTTCA CGG 470
8936 1 TAAACTACCGTGAAAAAAAT TGG 471
8937 1 AAACTACCGTGAAAAAAATT GGG 472
9001 -1 CTCTTTTATTTTTTAAGAAG AGG 473
9053 1 TATTATAAATAAATTATGTT AGG 474
9065 1 ATTATGTTAGGTGATCCTAT TGG 475
9068 1 ATGTTAGGTGATCCTATTGG TGG 476
9069 1 TGTTAGGTGATCCTATTGGT GGG 477
9069 -1 GTAATTTCGTCCCCACCAAT AGG 478
9070 1 GTTAGGTGATCCTATTGGTG GGG 479
9101 1 ACAAGTGATTATAACAAAGA TGG 480
9102 1 CAAGTGATTATAACAAAGAT GGG 481
9103 1 AAGTGATTATAACAAAGATG GGG 482
9123 1 GGGCTAAGAATTCAAGAAAG AGG 483
9138 1 GAAAGAGGAGAAGTTGTAAA AGG 484
9149 1 AGTTGTAAAAGGAGTGCCTG TGG 485
9154 -1 TCGTCCCCAGGTTGGACCAC AGG 486
9159 1 GGAGTGCCTGTGGTCCAACC TGG 487
9160 1 GAGTGCCTGTGGTCCAACCT GGG 488
9161 1 AGTGCCTGTGGTCCAACCTG GGG 489
9162 -1 AGAAAAGGTCGTCCCCAGGT TGG 490
9166 -1 AACCAGAAAAGGTCGTCCCC AGG 491
9175 1 AACCTGGGGACGACCTTTTC TGG 492
69
Date Recue/Date Received 2022-06-30
9177 -1 GTGGGCGGTTGAACCAGAAA AGG 493
9192 -1 GGTAGAGAATAAGGCGTGGG CGG 494
9195 -1 TAAGGTAGAGAATAAGGCGT GGG 495
9196 -1 ATAAGGTAGAGAATAAGGCG TGG 496
9201 -1 AGTTAATAAGGTAGAGAATA AGG 497
9213 -1 GGAAGAGGACGAAGTTAATA AGG 498
9227 1 TATTAACTTCGTCCTCTTCC AGG 499
9228 -1 ATTGATTATGTACCTGGAAG AGG 500
9234 -1 ATTTTGATTGATTATGTACC TGG 501
9254 1 TAATCAATCAAAATCAGCCT TGG 502
9260 -1 GGTGCATTATAGAATTTCCA AGG 503
9281 -1 TCAATGTATTCATTTTAAGG GGG 504
9282 -1 ATCAATGTATTCATTTTAAG GGG 505
9283 -1 CATCAATGTATTCATTTTAA GGG 506
9284 -1 GCATCAATGTATTCATTTTA AGG 507
9308 -1 TTGAGTGCTAAAACAAGTAA GGG 508
9309 -1 TTTGAGTGCTAAAACAAGTA AGG 509
9350 1 TTTAGTCAAATTTTTTCTCA TGG 510
10632 -1 TCCATGCAAAGAACGCAAGC TGG 511
10642 1 TCCAGCTTGCGTTCTTTGCA TGG 512
10648 1 TTGCGTTCTTTGCATGGACT TGG 513
10649 1 TGCGTTCTTTGCATGGACTT GGG 514
10753 1 TTAATTTTTCAGTATGAATT TGG 515
10785 1 TTGCTTTCATGAACATGTTG AGG 516
10791 1 TCATGAACATGTTGAGGATG TGG 517
10809 1 TGTGGTTATCAGAATCACCA TGG 518
10810 1 GTGGTTATCAGAATCACCAT GGG 519
10811 1 TGGTTATCAGAATCACCATG GGG 520
10815 -1 ATATCTGTATACAGACCCCA TGG 521
10922 -1 AAATAAAAATTAAATATTAA TGG 522
10958 1 GTAAAAATTTCTAACACCGT TGG 523
10963 -1 CCCTGATGATCATGATCCAA CGG 524
10973 1 ACCGTTGGATCATGATCATC AGG 525
10974 1 CCGTTGGATCATGATCATCA GGG 526
10993 -1 TGACGTAGCTGCACAGAATC TGG 527
Date Recue/Date Received 2022-06-30
11016 -1 AACAAGGGCGTAGAGAGGGA GGG 528
11017 -1 TAACAAGGGCGTAGAGAGGG AGG 529
11020 -1 GTGTAACAAGGGCGTAGAGA GGG 530
11021 -1 TGTGTAACAAGGGCGTAGAG AGG 531
11031 -1 TGTAATTACTTGTGTAACAA GGG 532
11032 -1 GTGTAATTACTTGTGTAACA AGG 533
11159 -1 AGATTTTATATATTTAATTA GGG 534
11160 -1 TAGATTTTATATATTTAATT AGG 535
11524 -1 CGGACTATATTTTAATTAAA AGG 536
11544 -1 TAATTAAATAAAATTCTAAA CGG 537
11580 1 TAAAAAATATTGTCATAGTT TGG 538
11581 1 AAAAAATATTGTCATAGTTT GGG 539
11782 1 TATATATATGACACAACAGA TGG 540
11783 1 ATATATATGACACAACAGAT GGG 541
11800 -1 GTTGAATATAGTTGGTTTCA TGG 542
11808 -1 ACTTTGTCGTTGAATATAGT TGG 543
11824 1 TATATTCAACGACAAAGTAG CGG 544
11827 1 ATTCAACGACAAAGTAGCGG AGG 545
11839 -1 TGAGTGGTGCCAGTTGCGGA GGG 546
11840 -1 CTGAGTGGTGCCAGTTGCGG AGG 547
11841 1 TAGCGGAGGCCCTCCGCAAC TGG 548
11843 -1 GGGCTGAGTGGTGCCAGTTG CGG 549
11855 -1 TGATGTGCTTTCGGGCTGAG TGG 550
11863 -1 TTGGTGTTTGATGTGCTTTC GGG 551
11864 -1 TTTGGTGTTTGATGTGCTTT CGG 552
11881 1 GCACATCAAACACCAAAACA AGG 553
11882 -1 CTGACCCCGCCGCCTTGTTT TGG 554
11884 1 CATCAAACACCAAAACAAGG CGG 555
11887 1 CAAACACCAAAACAAGGCGG CGG 556
11888 1 AAACACCAAAACAAGGCGGC GGG 557
11889 1 AACACCAAAACAAGGCGGCG GGG 558
11910 -1 GTCGTCGGCCGGCTTGACAG CGG 559
11913 1 CAGTGACGCCGCTGTCAAGC CGG 560
11921 -1 GATGTGTGGGTGTCGTCGGC CGG 561
11925 -1 ATGTGATGTGTGGGTGTCGT CGG 562
71
Date Recue/Date Received 2022-06-30
11934 -1 ACCGGGGACATGTGATGTGT GGG 563
11935 -1 GACCGGGGACATGTGATGTG TGG 564
11944 1 ACCCACACATCACATGTCCC CGG 565
11950 -1 GTGGCGCAAGAGGTGGACCG GGG 566
11951 -1 AGTGGCGCAAGAGGTGGACC GGG 567
11952 -1 TAGTGGCGCAAGAGGTGGAC CGG 568
11957 -1 TGCGGTAGTGGCGCAAGAGG TGG 569
11960 -1 CACTGCGGTAGTGGCGCAAG AGG 570
11969 -1 CTGCTGCCTCACTGCGGTAG TGG 571
11974 1 CTTGCGCCACTACCGCAGTG AGG 572
11975 -1 GGCTGTCTGCTGCCTCACTG CGG 573
11996 -1 AGCGCCTTGGGGAGTTTTGG AGG 574
11999 -1 TTGAGCGCCTTGGGGAGTTT TGG 575
12003 1 ACAGCCTCCAAAACTCCCCA AGG 576
12007 -1 ATCAAAGTTTGAGCGCCTTG GGG 577
12008 -1 CATCAAAGTTTGAGCGCCTT GGG 578
12009 -1 CCATCAAAGTTTGAGCGCCT TGG 579
12020 1 CCAAGGCGCTCAAACTTTGA TGG 580
12033 1 ACTTTGATGGCGCCACTGAA CGG 581
12034 -1 ATCTGTCTCCCACCGTTCAG TGG 582
12036 1 TTGATGGCGCCACTGAACGG TGG 583
12037 1 TGATGGCGCCACTGAACGGT GGG 584
12059 -1 TGGTGGTGGTGAGATGGAGA TGG 585
12065 -1 CGGCCGTGGTGGTGGTGAGA TGG 586
12073 1 TCTCCATCTCACCACCACCA CGG 587
12073 -1 TCGCGAAGCGGCCGTGGTGG TGG 588
12076 -1 CGGTCGCGAAGCGGCCGTGG TGG 589
12079 -1 CCTCGGTCGCGAAGCGGCCG TGG 590
12085 -1 AGGAACCCTCGGTCGCGAAG CGG 591
12090 1 CCACGGCCGCTTCGCGACCG AGG 592
12091 1 CACGGCCGCTTCGCGACCGA GGG 593
12096 -1 ATGATGAGAGGAGGAACCCT CGG 594
12105 -1 ATTATTACTATGATGAGAGG AGG 595
12108 -1 ATTATTATTACTATGATGAG AGG 596
12150 1 TAAAAATCAGCAAATTGAAT TGG 597
72
Date Recue/Date Received 2022-06-30
12151 1 AAAAATCAGCAAATTGAATT GGG 598
12162 1 AATTGAATTGGGACAAATAA TGG 599
12181 1 ATGGAACAACATCATCTTCA TGG 600
12188 1 AACATCATCTTCATGGAGAT CGG 601
12204 -1 GGTTTGAGGAGGAAGCTCAT TGG 602
12215 -1 CTTAATGTAGTGGTTTGAGG AGG 603
12218 -1 TTTCTTAATGTAGTGGTTTG AGG 604
12225 -1 AGCTTGATTTCTTAATGTAG TGG 605
12267 1 TGATCAATCAGCAGCAGCAC AGG 606
12272 1 AATCAGCAGCAGCACAGGTG AGG 607
12284 -1 TTAATTTCATGGTGGGGCGG CGG 608
12287 -1 ATATTAATTTCATGGTGGGG CGG 609
12290 -1 CCAATATTAATTTCATGGTG GGG 610
12291 -1 TCCAATATTAATTTCATGGT GGG 611
12292 -1 GTCCAATATTAATTTCATGG TGG 612
12295 -1 TGTGTCCAATATTAATTTCA TGG 613
12301 1 CCCCACCATGAAATTAATAT TGG 614
12326 1 ACAGAGATTTCTCTTTTGAA CGG 615
12350 -1 CTCTCTCGTCATCAAACGCT GGG 616
12351 -1 TCTCTCTCGTCATCAAACGC TGG 617
12376 1 CGAGAGAGAATTCCGTTATT TGG 618
12377 -1 TTAACATTATAACCAAATAA CGG 619
12392 1 TATTTGGTTATAATGTTAAT CGG 620
12396 1 TGGTTATAATGTTAATCGGA CGG 621
12411 1 TCGGACGGTTCTCATTGTCT CGG 622
12423 -1 TCTAGCTCGTTGATCATCAG AGG 623
12499 -1 ATAATTAAACCGCTCATTAT TGG 624
12501 1 TAAGCAGCTCCAATAATGAG CGG 625
Table 3: CsMiL03 targeted gRNA sequences
Position Strand Sequence PAM SEQ ID
on SEQ NO
ID NO:7
777 1 TGAAACTCAAACTAAAATCA AGG 626
73
Date Regue/Date Received 2022-06-30
801 -1 TCTAACAGTTGGTATCAGAG CGG 627
812 -1 ATATATAAATGTCTAACAGT TGG 628
860 1 ATATGTTTAAGTATTAACTG CGG 629
894 1 TATATACACTATATAACTTA AGG 630
915 -1 GCTCAAGAATCAATGGCTGG AGG 631
918 -1 GAAGCTCAAGAATCAATGGC TGG 632
922 -1 GTTTGAAGCTCAAGAATCAA TGG 633
944 -1 TTGCAGATCAAAGCTTATGT GGG 634
945 -1 CTTGCAGATCAAAGCTTATG TGG 635
957 1 CACATAAGCTTTGATCTGCA AGG 636
958 1 ACATAAGCTTTGATCTGCAA GGG 637
965 1 CTTTGATCTGCAAGGGAAAC TGG 638
974 1 GCAAGGGAAACTGGTTGATG TGG 639
975 1 CAAGGGAAACTGGTTGATGT GGG 640
982 1 AACTGGTTGATGTGGGTAAT CGG 641
983 1 ACTGGTTGATGTGGGTAATC GGG 642
998 -1 TAAAGAGAGTTGAGAGAGCG AGG 643
1014 1 CTCTCTCAACTCTCTTTAGA TGG 644
1044 1 TGTTATGAACAGAATGAGTG AGG 645
1051 1 AACAGAATGAGTGAGGAGCT CGG 646
1052 1 ACAGAATGAGTGAGGAGCTC GGG 647
1053 1 CAGAATGAGTGAGGAGCTCG GGG 648
1066 -1 CACCTATAAATATAGGGTCT CGG 649
1072 -1 GTATCTCACCTATAAATATA GGG 650
1073 -1 AGTATCTCACCTATAAATAT AGG 651
1075 1 GACCGAGACCCTATATTTAT AGG 652
1096 -1 TAATGTGGCACAGATACTGA TGG 653
1111 -1 AAATATTCTGACAATTAATG TGG 654
1138 1 AATATTTTGACAATTAATTC AGG 655
1151 1 TTAATTCAGGAAATCAAATC AGG 656
1183 -1 ATTATGTAATATTCTATATA TGG 657
4585 1 GTTCTCACTATCAGTTATTA TGG 658
4595 1 TCAGTTATTATGGTTATTTA TGG 659
4615 1 TGGTTATTTATCTTTTTTAG TGG 660
4634 -1 CCTGAAGGGCTTTTTGTGTT TGG 661
74
Date Recue/Date Received 2022-06-30
4645 1 CCAAACACAAAAAGC CC TT C AGG 662
4648 -1 CTTCTCAAGCGCTTCCTGAA GGG 663
4649 -1 TCTTCTCAAGCGCTTCCTGA AGG 664
4670 1 GCGCTTGAGAAGATTAAATT AGG 665
4736 1 TTATTAGTATTTTTTTTTTT TGG 666
4751 1 TTTTTTGGTCTAATTTTAAT TGG 667
4752 1 TTTTTGGTCTAATTTTAATT GGG 668
4802 1 TGTTGCAGAGC TTAT GC TAT TGG 669
4803 1 GTTGCAGAGC TTATGCTATT GGG 670
4842 -1 ATATGTCAGCAATGTAATCT TGG 671
4870 -1 CAAGTGTTTGCTGCACTTTT TGG 672
4882 1 CAAAAAGTGCAGCAAACAC T TGG 673
4897 -1 TCTTCATTTTGGTATGGGCA AGG 674
4902 -1 TTTTCTCTTCATTTTGGTAT GGG 675
4903 -1 TTTTTCTCTTCATTTTGGTA TGG 676
4908 -1 TAGCCTTTTTCTCTTCATTT TGG 677
4916 1 ATACCAAAATGAAGAGAAAA AGG 678
4922 1 AAATGAAGAGAAAAAGGCTA AGG 679
4945 -1 TAATCAATTGTTTTTGATTT TGG 680
5012 1 TGTAATTATGTCTTAATGAT AGG 681
5033 1 GGACGTATACTAAAAGTGTG TGG 682
5078 1 AATGAGTTCTGAATTTTTGA AGG 683
5098 1 AGGACTTTTTGAATATTGTA TGG 684
5139 1 TAATATAAAATTAATATATA TGG 685
5181 1 TGATTTGTGTGTTTTGTGTG AGG 686
5187 1 GTGTGTTTTGTGTGAGGTGC AGG 687
5188 1 TGTGTTTTGTGTGAGGTGCA GGG 688
5214 1 AGTTCTTTAGTGTCTAAATA TGG 689
5215 1 GTTCTTTAGTGTCTAAATAT GGG 690
5232 -1 CAAATATGAAGATATGAAGC TGG 691
5249 1 TCATATCTTCATATTTGTCT TGG 692
5268 -1 TAGTAATGCAATATATAATA TGG 693
5285 1 TATATATTGC ATTAC TAC CT TGG 694
5291 -1 TTTGGTTCTGCCAATAGCCA AGG 695
5292 1 TGCATTACTACCTTGGCTAT TGG 696
Date Recue/Date Received 2022-06-30
5309 -1 AACTTAAAAACTACTCACTT TGG 697
5361 1 CATATTCTATAAAATTAATA TGG 698
5401 1 TTGAATTGCAGATGAGAAAA TGG 699
5410 1 AGATGAGAAAATGGAAAGTT TGG 700
5411 1 GATGAGAAAATGGAAAGTTT GGG 701
5414 1 GAGAAAATGGAAAGTTTGGG AGG 702
5450 1 ATTGAGTACATATATAGTAA CGG 703
5537 1 TTGTATAATTAATTATTTTT TGG 704
5563 1 CACTACAACTTATCTAACTC AGG 705
6711 -1 ATCTTTACATTCTTACTTTT TGG 706
6785 1 TATATAAATATTCAATCAAA TGG 707
6789 1 TAAATATTCAATCAAATGGT TGG 708
6811 -1 CTTGTAAATCTAAATCTCTC AGG 709
6837 1 TTTACAAGAGACACATCATT TGG 710
6859 1 GAAGAAGACATTTGAACATT TGG 711
6873 -1 TCCAAAGTGAAATTGGTGAT TGG 712
6880 -1 CTTACAATCCAAAGTGAAAT TGG 713
6883 1 GCCAATCACCAATTTCACTT TGG 714
6927 -1 TTGTTTTCTTCTCTATAATA AGG 715
6973 1 TCAAAAGTTTTTTATTATAT AGG 716
7030 1 TTCTTGTTTATCAAATGATC AGG 717
7055 1 TGCTTTTTCAGACAATTCTT CGG 718
7056 1 GCTTTTTCAGACAATTCTTC GGG 719
7069 1 ATTCTTCGGGTCAGTCACTA AGG 720
7089 1 AGGTTGATTACATGACACTG AGG 721
7094 1 GATTACATGACACTGAGGCA TGG 722
7105 1 ACTGAGGCATGGATTTGTAA TGG 723
7126 1 GGTATGTTGCACAATGATCT TGG 724
7137 1 CAATGATCTTGGCCTGAAAA TGG 725
7138 -1 TGTAATTTGAAGCCATTTTC AGG 726
7203 1 AGCTATGCTTTTCCCATTTC AGG 727
7204 -1 GAGCCAAATGTGCCTGAAAT GGG 728
7205 -1 GGAGCCAAATGTGCCTGAAA TGG 729
7212 1 TTTCCCATTTCAGGCACATT TGG 730
7226 -1 TCAAATCTTGTTTCACTTTC TGG 731
76
Date Recue/Date Received 2022-06-30
7272 1 CATCAGCAAATCACTTGATC AGG 732
7291 1 CAGGATTTTGTAGTAATTGT TGG 733
7292 1 AGGATTTTGTAGTAATTGTT GGG 734
7323 -1 ATATTATAAGCTGATTTCAA AGG 735
7419 -1 CGGCAACGAACCAAATTACT GGG 736
7420 1 ATATATGCAGCCCAGTAATT TGG 737
7420 -1 ACGGCAACGAACCAAATTAC TGG 738
7439 -1 GTTGGACAGTAGAAACAATA CGG 739
7457 -1 CAATAACTTACCATATGTGT TGG 740
7458 1 TTTCTACTGTCCAACACATA TGG 741
7519 -1 CAACATTTCAGTCACTGAAA TGG 742
7549 1 GTTGTTCTTTTTTAATTAAC AGG 743
7568 1 CAGGAATATACTCTTATTTG TGG 744
7583 -1 CTTACAATCAAAGGTAGAAA TGG 745
7592 -1 TGTGTTGTACTTACAATCAA AGG 746
7660 -1 TTCCACACATTAGCAAATGT GGG 747
7661 -1 TTTCCACACATTAGCAAATG TGG 748
7669 1 GTCCCACATTTGCTAATGTG TGG 749
7699 1 TTGTGATATATAAGATGAAT AGG 750
7715 1 GAATAGGCTACTCCTTTTAT AGG 751
7716 1 AATAGGCTACTCCTTTTATA GGG 752
7716 -1 CCATTTGAAAACCCTATAAA AGG 753
7727 1 CCTTTTATAGGGTTTTCAAA TGG 754
7741 -1 ATTTAGGAATAAGATGAATG GGG 755
7742 -1 AATTTAGGAATAAGATGAAT GGG 756
7743 -1 GAATTTAGGAATAAGATGAA TGG 757
7757 -1 GACATACCATGTTAGAATTT AGG 758
7762 1 CTTATTCCTAAATTCTAACA TGG 759
7788 -1 AAAAACCCAACACTGGAAAG TGG 760
7793 1 TGTGTGCCACTTTCCAGTGT TGG 761
7794 1 GTGTGCCACTTTCCAGTGTT GGG 762
7795 -1 ACAGGTCAAAAACCCAACAC TGG 763
7813 -1 AAATTTGTAGATTTTGAAAC AGG 764
7849 -1 CCAAATATCGGAAAATTTGT GGG 765
7850 -1 GCCAAATATCGGAAAATTTG TGG 766
77
Date Recue/Date Received 2022-06-30
7860 1 CCCACAAATTTTCCGATATT TGG 767
7861 -1 AATCTCACAAGGCCAAATAT CGG 768
7872 -1 ACATTTGAAAGAATCTCACA AGG 769
7892 1 ATTCTTTCAAATGTCACGTT CGG 770
7900 1 AAATGTCACGTTCGGTCCTG TGG 771
7905 -1 AACGACCTTTCAGAGACCAC AGG 772
7911 1 TCGGTCCTGTGGTCTCTGAA AGG 773
7935 -1 CGTTTGGGCCTGAAAAGTGT GGG 774
7936 -1 ACGTTTGGGCCTGAAAAGTG TGG 775
7938 1 TCGTTATACCCACACTTTTC AGG 776
7950 -1 TTAATACACTCCTCACGTTT GGG 777
7951 1 ACTTTTCAGGCCCAAACGTG AGG 778
7951 -1 CTTAATACACTCCTCACGTT TGG 779
7991 1 AGTCTCACATTGCTAATGTA TGG 780
8020 1 ATTGTGATATATAAAATGAA TGG 781
8021 1 TTGTGATATATAAAATGAAT GGG 782
8038 -1 TAAAACTAATTGGCTGTGGG AGG 783
8041 -1 TCTTAAAACTAATTGGCTGT GGG 784
8042 -1 ATCTTAAAACTAATTGGCTG TGG 785
8048 -1 GGTTTTATCTTAAAACTAAT TGG 786
8069 -1 ATTTAGGGATAAGATGAATG GGG 787
8070 -1 AATTTAGGGATAAGATGAAT GGG 788
8071 -1 GAATTTAGGGATAAGATGAA TGG 789
8084 -1 ATTAAGCATGTTAGAATTTA GGG 790
8085 -1 GATTAAGCATGTTAGAATTT AGG 791
8144 1 CAAATTGCAGATAATATTAC TGG 792
8147 1 ATTGCAGATAATATTACTGG TGG 793
8148 1 TTGCAGATAATATTACTGGT GGG 794
8180 1 TCAAGTAATCATAACAAAGA TGG 795
8181 1 CAAGTAATCATAACAAAGAT GGG 796
8202 1 GGATTAAGCATTCAAGAGAG AGG 797
8210 1 CATTCAAGAGAGAGGAGATG TGG 798
8217 1 GAGAGAGGAGATGTGGTAAA AGG 799
8228 1 TGTGGTAAAAGGTGCACCAT TGG 800
8233 -1 TCATCTCCTGGTTGAACCAA TGG 801
78
Date Recue/Date Received 2022-06-30
8238 1 GGTGCACCATTGGTTCAACC AGG 802
8245 -1 AACCAGAAGAGGTCATCTCC TGG 803
8254 1 AACCAGGAGATGACCTCTTC TGG 804
8256 -1 TAGGCCGTCCGAACCAGAAG AGG 805
8259 1 GGAGATGACCTCTTCTGGTT CGG 806
8263 1 ATGACCTCTTCTGGTTCGGA CGG 807
8275 -1 ATGAGAAAGAGCATTAATTT AGG 808
8301 -1 TAAGTACCTGAAAGAGAACA AGG 809
8306 1 CATTCACCTTGTTCTCTTTC AGG 810
8413 1 AAAATGATATCTTTTCTGCT TGG 811
8429 1 TGCTTGGTACTAATTAATGC TGG 812
8487 -1 TACTGTACTCCATGCAAAAA AGG 813
8489 1 TTCAACTTGCCTTTTTTGCA TGG 814
8531 -1 TGCCTTGAAACCAAAAATCA AGG 815
8532 1 ATTTGACTTTCCTTGATTTT TGG 816
8540 1 TTCCTTGATTTTTGGTTTCA AGG 817
8559 1 AAGGCAATAAAATTATTACA TGG 818
8624 -1 TTCGTGGAAGCAAGTGTTCA AGG 819
8640 -1 TGATATCTTCAATTTTTTCG TGG 820
8669 1 TATCATCATAAGAATTTCAA TGG 821
8670 1 ATCATCATAAGAATTTCAAT GGG 822
8671 1 TCATCATAAGAATTTCAATG GGG 823
8805 1 TTCTCTTTTTCTTTCTTACT AGG 824
8819 -1 AACTGCATAGAACTTGTATG AGG 825
8853 -1 TGTGTGACAAGAGCATATAG AGG 826
8866 1 TCTATATGCTCTTGTCACAC AGG 827
8893 -1 GATGATAATGATGATTTAGA AGG 828
8954 1 ATTTGATCATATATTACAGA TGG 829
8955 1 TTTGATCATATATTACAGAT GGG 830
8956 1 TTGATCATATATTACAGATG GGG 831
8980 -1 ACTCTGTCATTGAAAATTAC TGG 832
9013 1 TAGCAACAGCATTAAAGAAC TGG 833
9027 -1 TGTTCTTGGTTTTGGCTGAA TGG 834
9035 -1 GTGTTTTTTGTTCTTGGTTT TGG 835
9041 -1 TCGGTTGTGTTTTTTGTTCT TGG 836
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Date Recue/Date Received 2022-06-30
9059 1 CAAAAAACACAACCGAAATT CGG 837
9060 -1 GCGAGTTTGTCTCCGAATTT CGG 838
9082 -1 GTTGCAGGCCTACTTGAGAA TGG 839
9085 1 CAAACTCGCCATTCTCAAGT AGG 840
9097 -1 ATGCCATATGTTGGAGTTGC AGG 841
9105 1 AGGCCTGCAACTCCAACATA TGG 842
9106 -1 ACTGGAGACATGCCATATGT TGG 843
9124 -1 TAATTTTGCAGCAGATGAAC TGG 844
9156 1 TACAGAAGCACAGCAACTGA TGG 845
9165 1 ACAGCAACTGATGGATACTA TGG 846
9175 1 ATGGATACTATGGTTCTCCG AGG 847
9181 -1 TTTTCGACATTAGACATCCT CGG 848
9213 1 AACGATTACTATGAGCCTGA AGG 849
9214 1 ACGATTACTATGAGCCTGAA GGG 850
9217 -1 TTGGGAGATGGTGTCCCTTC AGG 851
9229 -1 GATGGTCCATTGTTGGGAGA TGG 852
9234 1 GGGACACCATCTCCCAACAA TGG 853
9235 -1 GCTGCAGATGGTCCATTGTT GGG 854
9236 -1 TGCTGCAGATGGTCCATTGT TGG 855
9247 -1 TGTATTTCACTTGCTGCAGA TGG 856
9284 1 GAATAACTATGAAGTTGAGA AGG 857
9296 1 AGTTGAGAAGGATATAAGTG AGG 858
9300 1 GAGAAGGATATAAGTGAGGA AGG 859
9311 1 AAGTGAGGAAGGACAGCCAA TGG 860
9316 -1 GAGCTTGGTTCCTGAACCAT TGG 861
9317 1 GGAAGGACAGCCAATGGTTC AGG 862
9331 -1 TTTTGCTGTGAGGAGGAGCT TGG 863
9338 -1 GACCTCATTTTGCTGTGAGG AGG 864
9341 -1 CTTGACCTCATTTTGCTGTG AGG 865
9347 1 CTCCTCCTCACAGCAAAATG AGG 866
9368 -1 CCTAAATGAGAAGTGAGATA AGG 867
9379 1 CCTTATCTCACTTCTCATTT AGG 868
9450 1 CTTTATTTCTTATTATCTTT TGG 869
9498 1 AATATGTATAAGCTTGAATT TGG 870
Date Recue/Date Received 2022-06-30
Reference is made to Table 4 summarizing sequences relating to WT CsMLO within
the scope of
the current invention.
Table 4: WT CsMLO sequence table
Sequence type CsML01 CsML02 CsML03
characterization
Genomic SEQ ID NO:1 SEQ ID NO:4 SEQ ID NO:7
sequence
Coding sequence SEQ ID NO:2 SEQ ID NO:5 SEQ ID NO:8
(CDS)
Amino acid SEQ ID NO:3 SEQ ID NO:6 SEQ ID NO:9
sequence
gRNA sequence SEQ ID NO:10- SEQ ID NO:287- SEQ ID NO:626-
SEQ ID NO:286 SEQ ID NO:625 SEQ ID NO:870
(Table 1) (Table 2) (Table 3)
The above gRNA molecules have been cloned into suitable vectors and their
sequence has been
verified. In addition different Cas9 versions have been analyzed for optimal
compatibility between
the Cas9 protein activity and the gRNA molecule in the Cannabis plant.
Stage 3: Transforming Cannabis plants using Agrobacterium or biolistics (gene
gun) methods. For
Agrobacterium and bioloistics a DNA plasmid carrying (Cas9 + gene specific
gRNA) can be used.
A vector containing a selection marker, Cas9 gene and relevant gene specific
gRNA's is
constructed. For biolistics, Ribonucleoprotein (RNP) complexes carrying (Cas9
protein + gene
specific gRNA) are used. RNP complexes are created by mixing the Cas9 protein
with relevant
gene specific gRNA's.
According to some embodiments of the present invention, transformation of
various Cannabis
tissues was performed using particle bombardment of:
= DNA vectors
= Ribonucleoprotein complex (RNP's)
81
Date Recue/Date Received 2022-06-30
According to further embodiments of the present invention, transformation of
various Cannabis
tissues was performed using Agrobacterium (Agrobacterium tumefaciens) by:
= Regeneration-based transformation
= Floral-dip transformation
= Seedling transformation
Transformation efficiency by A. tumefaciens has been compared to the
bombardment method by
transient GUS transformation experiment. After transformation, GUS staining of
the transformants
has been performed.
Reference is now made to Fig. 4A-D photographically presenting GUS staining
after transient
transformation of the following Cannabis tissues (A) axillary buds (B) leaf
(C) calli, and (D)
cotyledons.
Fig. 4 demonstrates that various Cannabis tissues have been successfully
transiently transformed
using biolistics system. Transformation has been performed into calli, leaves,
axillary buds and
cotyledons of Cannabis.
According to further embodiments of the present invention, additional
transformation tools were
used in Cannabis, including, but not limited to:
= Protoplast PEG transformation
= Extend RNP use
= Directed editing screening using fluorescent tags
= El ectroporati on
Stage 4: Regeneration in tissue-culture. When transforming DNA constructs into
the plant,
antibiotics is used for selection of positive transformed plants. An improved
regeneration protocol
was herein established for the Cannabis plant.
Reference is now made to Fig. 5 presenting regeneration of Cannabis tissue. In
this figure, arrows
indicate new meristem emergence.
Stage 5: Selection of positive transformants. Once regenerated plants appear
in tissue culture, DNA
is extracted from leaf sample of the transformed plant and PCR is performed
using primers
flanking the edited region. PCR products are then digested with enzymes
recognizing the
82
Date Recue/Date Received 2022-06-30
restriction site near the original gRNA sequence. If editing event occurred,
the restriction site will
be disrupted and the PCR product will not be cleaved. No editing event will
result in a cleaved
PCR product.
Reference is now made to Fig. 6 showing PCR detection of Cas9 DNA in shoots of
transformed
Cannabis plants. DNA extracted from shoots of plants transformed with Cas9
using biolistics. This
figure shows that three weeks post transformation, Cas9 DNA was detected in
shoots of
transformed plants.
Screening for CRISPR/Cas9 gene editing events has been performed by at least
one of the
following analysis methods:
= Restriction Fragment Length Polymorphism (RFLP)
= Next Generation Sequencing (NGS)
= PCR fragment analysis
= Fluorescent-tag based screening
= High resolution melting curve analysis (HRMA)
Reference is now made to Fig. 7 presenting results of in vitro analysis of
CRISPR/Cas9 cleavage
activity. Fig. 7A schematically shows the genomic area targeted for editing
(PAM is marked in
red) and amplified by the reverse and forward designed primers Fig. 7B
photographically presents
a gel showing successful digestion of the resulted PCR amplicon containing the
gene specific
gRNA sequence, by RNP complex containing Cas9. The analysis included the
following steps:
1) Amplicon was isolated from two exemplified Cannabis strains by primers
flanking the
sequence of the gene of interest targeted by the predesigned sgRNA.
2) RNP complex was incubated with the isolated amplicon.
= 3) The reaction mix was then loaded on agarose gel to evaluate Cas9
cleavage activity at the
target site.
Stage 6: Selection of transformed Cannabis plants presenting resistance to PM
by establishing a
protocol adapted for Cannabis. It is within the scope that different gRNA
promoters were tested
in order to maximize editing efficiency.
83
Date Recue/Date Received 2022-06-30
EXAMPLE 2
Identifying powdery mildew (PM) pathogen specific for Cannabis
Powdery Mildew is one of the most destructive fungal pathogens infecting
Cannabis. It is an
obligate biotroph that can vascularize into the plant tissue and remain
invisible to a grower. Under
ideal conditions, powdery mildew has a 4-7 days post inoculation (dpi) window
where it remains
invisible as it builds a network internally in the plant. It is herein
acknowledged that the powdery
mildew vascularized network in Cannabis is detectable with a PCR DNA based
test prior to
conidiospore generation. At later stages, powdery mildew infection and
conidiospore generation
results in rapid spreading of the fungus to other plants. This tends to emerge
and sporulate within
2 weeks into flowering thus destroying very mature crops with severe economic
consequences.
DNA based tools could facilitate early detection and rapid removal of infected
plant materials or
screening of incoming clones.
To date, there are no fungal disease resistant Cannabis varieties on the
market. Golovinomyces
cichoracearum is known for causing PM on several Cucurbits and on Cannabis
(Pepin et al., 2018).
In order to identify the specific fungi type affecting Cannabis, a molecular
analysis has been
performed. Internal Transcribed Spacer (ITS) DNA of PM samples obtained from
Cannabis strains
growing in our greenhouse has been isolated and sequenced. The term Internal
transcribed spacer
(ITS) as used hereinafter refers to the spacer DNA region situated between the
small-subunit
ribosomal RNA (rRNA) and large-subunit rRNA genes in the chromosome or the
corresponding
transcribed region in the polycistronic rRNA precursor transcript. It is
herein acknowledged that
the internal transcribed spacer (ITS) region is considered to have the highest
probability of
successful identification for the broadest range of fungi, with the most
clearly defined barcode gap
between inter- and intraspecific variation. Thus ITS is proposed for adoption
as the primary fungal
barcode marker, namely as potential DNA marker or finger print for fungi
(Schoch C.L. et al,
PNAS, 2012 109 (16) 6241-6246). The results of the molecular analysis of PM
isolated from
Cannabis revealed that Golovinomyces ambrosiae or Golovinomyces cichoracearum
are the cause
of the disease.
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Date Recue/Date Received 2022-06-30
A further achievement of the present invention is the establishment of an
inoculation assay and
index for Cannabis, or in other words establishment of bio-assay for powdery
mildew inoculation
in Cannabis. Such an assay establishment may include:
= Development of susceptibility index
= Designing a protocol by testing different inoculation approaches at
several plant developmental
stages
EXAMPLE 3
Production of genome-edited Cannabis MLO (CsMLO) genes
Three single guide RNAs (sgRNA) targeting the first exon (exon 1) of the
CsML01 gene were
designed and synthesized. These sgRNAs include sgRNA having nucleotide
sequence as set forth
in SEQ ID NO:17 (first guide), SEQ ID NO:43 (second guide) and SEQ ID NO:50
(third guide)
starting at position 99, 369 and 453 of SEQ ID NO: 1. The predicted Cas9
cleavage sites directed
by these guide RNAs were designed to overlap with the nucleic acid recognition
site of the
restriction enzymes: Hinfl, BseLI and BtsI for the first, second and third
gRNA, respectively (see
Fig. 9). Transformation was performed using a DNA plasmid such as a plant
codon optimized
Streptococcus pyogenes Cas9 (pcoSpCas9) plasmid presented in Fig. 8. The
plasmid contained the
plant codon optimized SpCas9 and the above mentioned at least one sgRNA.
About two months post transformation, leaves from mature plants were sampled,
and their DNA
was extracted and digested with the suitable enzymes. Digested genomic DNA was
used as a
template for PCR using a primer pair flanking the 5' and 3' ends of the first
exon of CsML01. The
forward primer (fwd) (5-GAGTGGAACTAGAAGAAATGC-3) comprises a nucleotide
sequence
as set forth in SEQ ID NO:871, and the reverse primer (rev) (5-
CCCTCCAAACACAACAGTGA-
3) comprises a nucleotide sequence as set forth in SEQ ID NO:872 (see Fig. 9
and Fig. 10). As
shown in Fig. 10, the aforementioned primer pair (marked with arrows)
generates a 778 bp
amplicon comprising the entire exon 1 of CsML01, having a nucleotide sequence
as set forth in
SEQ ID NO:873 (nucleotide positions 4-782 of SEQ ID NO:1). In Fig. 10 the
three gRNA
sequences used to target exon 1 of CsML01 genomic sequence are underlined. The
translation
initiation codon ATG (encoding Methionine amino acid) is marked with a square.
Fig. 11 presents
the amino acid sequence of CsML01 first exon as set forth in SEQ ID NO:874.
Date Recue/Date Received 2022-06-30
Reference is now made to Fig. 12 photographically presenting detection of
CsML01 PCR
products showing length variation (i.e. truncated fragments) as a result of
Cas9- mediated genome
editing. DNA from plants two months post transformation was used as a template
for the PCR
using primers having nucleic acid sequence as set forth in SEQ ID NO:871 and
SEQ ID NO:872.
DNA fragments shorter than the expected WT 780 bp amplicon were obtained by
the PCR reaction
and subcloned into a sequencing plasmid and sequenced. The sequencing results
are described
below.
It can be seen in Fig. 12 that WT or non-edited PCR products result in a 780
bp band, while DNA
extracted from edited plants exhibit a shorter band than the expected 780 bp
WT exon 1 length,
i.e. samples 1 and 2 show a 450 bp fragment and samples 3 and 4 show a 350 bp
fragment.
Fig. 13 schematically presents sequences of WT and genome edited CsML01 DNA
fragments
obtained for the first time by the present invention. In this figure, sgRNA
sequences are underlined.
sgRNA having nucleotide sequence as set forth in SEQ ID NO:17 (first guide)
with Hinfl
restriction site appears on the left hand of exon 1, and sgRNA having
nucleotide sequence as set
forth in SEQ ID NO:50 (third guide) with BtsI restriction site appears on the
right hand of exon 1
fragments. PAM sequences (NGG) are in marked italics and bold and are circled.
ATG codon
position is marked with a square.
The sequencing results show that three CsML01 exon 1 genome edited fragments
were achieved
by the present invention.
Reference is now made to Table 5 summarizing sequences relating to mutated
(genome edited)
exon 1 fragments of CsML01 achieved by the current invention.
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Date Recue/Date Received 2022-06-30
Table 5: Sequences of mutated CsMIL01 exon 1
Sequence type Exon 1 of WT 65-L4 (A447) 65-L5 (A373) 85-4
(A456)
CsML01 fragment of fragment of fragment of
CsMIL01 CsML01 CsML01
Genomic SEQ ID NO:873 SEQ ID NO:875 SEQ ID NO:877 SEQ ID NO:880
sequence
(nucleic acid 4- (deletion of (deletion of (deletion of
(Position in SEQ 782 in SEQ ID nucleic acid 109- nucleic acid 128- nucleic
acid 96-
ID NO:1) NO:1) (Fig. 10) 556 in SEQ ID 501 in SEQ ID 552 in SEQ
ID
NO:1) (Fig. 13) NO:1) (Fig. 13) NO:1) (Fig. 13)
Deleted nucleic SEQ ID NO:876 SEQ ID NO:879 SEQ ID NO:881
acid sequence
Amino acid SEQ ID NO: 874 SEQ ID NO:887 SEQ ID NO: 878 No amino- acid
sequence sequence is
(Fig. 11) MS MSGGGEGE
produced
gRNA sequence SEQ ID NO:17,
targeted to Exon SEQ ID NO:43
1 of CsMIL01 and SEQ ID
NO:50 (Table 1)
The resulted mutated CsML01 fragments include the following:
(1) Fragment 1: CsML01 fragment marked as 65-L4 A447 comprises a nucleotide
sequence
as set forth in SEQ ID NO:875 (about 330 bp). This fragment contains a
deletion of 447
bp (position 109-556 of SEQ ID NO:1) haying a nucleotide sequence as set forth
in SEQ
ID NO:876. It should be noted that this fragment encodes a two amino acid
peptide (SEQ
ID NO:887, as shown in Table 5). The short CsML01 exon 1 peptide generated by
the
targeted genome editing is expected to result is a non-functional, silenced
CsML01 gene
or allele.
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Date Recue/Date Received 2022-06-30
(2) Fragment 2: CsML01 fragment marked as 65-L5 A373 comprises a nucleotide
sequence
as set forth in SEQ ID NO:877 (about 405 bp). This fragment contains a
deletion of 373
bp (position 128-501 of SEQ ID NO:1) having a nucleotide sequence as set forth
in SEQ
ID NO:879. It should be noted that this fragment encodes a short peptide of
eight amino
acids (SEQ ID NO:878, as shown in Table 5). Such a short exon 1 fragment is
expected to
result in a non-functional CsML01 allele.
(3) Fragment 3: CsML01 fragment marked as 85-4 A456 comprises a nucleotide
sequence as
set forth in SEQ ID NO:880 (about 320 bp). This fragment contains a deletion
of 456 bp
(position 96-552 of SEQ ID NO:1) having a nucleotide sequence as set forth in
SEQ ID
NO:881. It is emphasized that fragment 3 was edited such that it lacks the ATG
translation
start codon, therefore no translated protein is generated. The resulted
truncated CsML01
gene/protein is expected to be non-functional.
The genome-edited CsML01 truncated fragments of the present invention are
characterized by
deletion of significant parts of the first exon sequence of CsML01 gene. Thus
these genome edited
fragments produce truncated CsML01 proteins. The truncated proteins lack
significant part of the
Open Reading Frame (ORF), e.g. absent of the translation start codon or
significant part of exon-
1 protein encoding sequence, and therefore would be non-functional.
EXAMPLE 4
Production of mutated Csmlol gene by genome editing events
This example presents the production of new genome editing events within
CsML01 gene. A
mutated Csmlol allele has been generated encompassing at least one of the
following genome
editing events within CsML01 gene:
1. indel d14 - 14pb deletion (bp389-402 of SEQ ID NO:1) having SEQ ID NO:883
2. i/- lbp insertion of A (bp 482-483 of SEQ ID NO:1)
As compared to the WT CsML01 378-500 fragment of SEQ ID NO:1 having a nucleic
acid
sequence as set forth in SEQ ID NO:882, the mutated Csmlol allele may comprise
one or more of
the following mutated DNA fragments:
-Csmlol dl4i1 encompassing the above identified deletion and insertion events,
comprising a
nucleic acid sequence corresponding to the sequence as set forth in SEQ ID
NO:886.
88
Date Recue/Date Received 2022-06-30
-Csmlol d14 encompassing the above identified deletion event, comprising a
nucleic acid
sequence corresponding to the sequence as set forth in SEQ ID NO:884.
-Csmlo / i/ encompassing the above identified insertion event, comprising a
nucleic acid sequence
corresponding to the sequence as set forth in SEQ ID NO:885.
The sequence of the mutated DNA fragments Csmlol d141, Csmlol d14, Csmlol ii,
as well as
the presence of a deletion indel d14 and/or insertion il provided by the
present invention, is useful
to identify and generate Cannabis plants with mutated alleles of ML01 gene,
desirable for the
production of Cannabis plants with PM resistance.
Reference is now made to Fig. 14, schematically presenting sequence comparison
(homology)
between WT CsML01 378-500 fragment of SE ID NO:1, having a nucleic acid
sequence as set
forth in SEQ ID NO:882, and genome edited Csmlol d1411 fragment, having a
nucleic acid
sequence as set forth in SEQ ID NO:886. In this figure, the deleted nucleic
acids within
Csm/o/ dl4i1 fragment are marked with dashed line, and the inserted nucleotide
(A) is underlined
and marked in bold. The gRNA designed to target the deletion region (having a
sequence as set
forth in SEQ ID NO:43) comprises BslI restriction site and is marked with a
square having a
continuous line. The gRNA designed to target the insertion region (having a
nucleotide sequence
complementary to the nucleotide sequence as set forth in SEQ ID NO:50)
comprises BtsI
restriction site and is marked with a square having a dashed line. PAM
sequence regions are
marked in bold and italics.
As shown in this figure, specific genome editing events within CsML01 gene are
generated by the
present invention resulting in mutated Csmlol alleles (such as alleles
comprising Csmlol d14,
Csmlo 1 il and Csmlol d1411 fragments). According to one embodiment, a mutated
Csmlo allele
comprising a deletion of 14 bp (having SEQ ID NO:883) at position
corresponding to position 12
of SEQ ID NO: 882 is produced (Csmlo allele containing Csmlol d14 fragment).
According to a
further embodiment, a mutated Csmlo allele comprising a nucleic acid insertion
of A at position
corresponding to position 104-105 of SEQ ID NO: 882 is produced (Csmlo allele
containing
Csmlo / i/ fragment). According to yet another embodiment, a mutated Csmlo
allele comprising
a deletion of 14 bp (having SEQ ID NO:883) at position corresponding to
position 12 of SEQ ID
NO: 882 in combination with a nucleic acid insertion of A at position
corresponding to position
104-105 of SEQ ID NO: 882 (Csmlo allele containing Csmlol d1411 fragment) is
produced.
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Date Recue/Date Received 2022-06-30
The genome editing events herein described introduce mutations that silence or
significantly
reduce CsML01 gene expression or function in the plant.
By silencing genes encoding MLO proteins (e.g. CsML01, CsML02 and/or CsML03)
in
Cannabis, plants with enhanced resistance to Powdery Mildew disease, can be
produced. These
PM resistant plants are highly desirable for the medical Cannabis industry
since usage of chemical
agents to control pathogen diseases is significantly reduced or avoided.
Date Recue/Date Received 2022-06-30
