Cannabis plants having modified expression of THCA synthase
The invention relates to novel genetically modified plants and methods or materials, such as polynucleotides, expression cassettes, or vectors for producing the same. Moreover, the invention relates to altering the content of cannabinoids in plants and to medical compositions derived from such plants. In particular embodiments, the present invention relates to cannabis plants having modified expression of tetrahydrocannabinolic acid (THCA) synthase and methods of modifying the amount of delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in cannabis by modifying expression of THCA synthase.
1. A method for altering a content of one or more cannabinoids in a cannabis plant, the method comprising down-regulating activity of tetrahydrocannabinolic acid (THCA) synthase in the cannabis plant, wherein the THCA synthase is expressed from a THCA synthase gene comprising a polynucleotide sequence selected from SEQ ID NOs: 1-28, 38, 40, 44-45, and 47;
wherein said down-regulating comprises introducing into the plant a selected DNA encoding an antisense RNA or a siRNA effective to suppress expression of the THCA synthase, the selected DNA operably linked to a heterologous promoter.
2. The method of claim 1 , wherein altering the content of cannabinoids comprises decreasing delta-9-tetrahydrocannabinol (THC) content or increasing cannabidiol (CBD) content in the cannabis plant.
3. The method of claim 2 , further comprising selecting a cannabis plant with a desired reduced THC content, increased CBD content, or reduced THC content and increased CBD content relative to a cannabis plant of a similar genotype that does not comprise the alteration.
4. The method of claim 1 , wherein introducing the selected DNA comprises genetic transformation.
5. The method of claim 1 , wherein the siRNA is encoded by a polynucleotide having a sequence selected from the group consisting of SEQ ID NO: 61, 63, 64, 69, 71, 72, 68, and a complement thereof.
6. The method of claim 1 , wherein the antisense RNA is encoded by a polynucleotide sequence comprising SEQ ID NO: 81 or a fragment thereof capable of reducing the expression of the THCA synthase.
7. A method for altering a content of one or more cannabinoids in a cannabis plant, the method comprising down-regulating activity of tetrahydrocannabinolic acid (THCA) synthase in the cannabis plant, wherein the THCA synthase is expressed from a THCA synthase gene comprising a polynucleotide sequence selected from SEQ ID NOs: 1-28, 38, 40, 44-45, and 47; wherein said down-regulating comprises:
(a) introducing into a cannabis plant or a cell thereof (i) at least one RNA-guided endonuclease comprising at least one nuclear localization signal or nucleic acid encoding at least one RNA-guided endonuclease comprising at least one nuclear localization signal, (ii) at least one guide RNA or DNA encoding at least one guide RNA, and, optionally, (iii) at least one donor polynucleotide; and
(b) culturing the cannabis plant or cell thereof such that each guide RNA directs an RNA-guided endonuclease to a targeted site in the chromosomal sequence where the RNA-guided endonuclease introduces a double-stranded break in the targeted site, and the double-stranded break is repaired by a DNA repair process such that the chromosomal sequence is modified, wherein the targeted site is located in the THCA synthase gene and the chromosomal modification interrupts or interferes with transcription and/or translation of the THCA synthase gene.
8. The method of claim 7 , wherein the RNA-guided endonuclease is derived from a clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system.
9. The method of claim 7 , wherein down-regulating the activity of the THCA synthase does not insert exogenous genetic material and produces a non-naturally occurring cannabis plant or cell thereof.
10. A method for altering a content of one or more cannabinoids in a cannabis plant, the method comprising down-regulating activity of tetrahydrocannabinolic acid (THCA) synthase in the cannabis plant, wherein the THCA synthase is expressed from a THCA synthase gene comprising a polynucleotide sequence selected from SEQ ID NOs: 1-28, 38, 40, 44-45, and 47; wherein said down-regulating comprises:
(a) identifying at least one THCA synthase gene locus within a DNA sequence in a cannabis plant or a cell thereof;
(b) identifying at least one custom endonuclease recognition sequence within the at least one THCA synthase gene locus;
(c) introducing into the cannabis plant or a cell thereof at least a first custom endonuclease, wherein the cannabis plant or a cell thereof comprises the recognition sequence for the custom endonuclease in or proximal to the THCA synthase gene locus, and the custom endonuclease is expressed transiently or stably;
(d) assaying the cannabis plant or a cell thereof for a custom endonuclease-mediated modification in the DNA making up or flanking the THCA synthase gene locus; and
(e) identifying the cannabis plant, a cell thereof, or a progeny cell thereof as comprising a modification in the THCA synthase gene locus.
11. A transgenic cannabis plant produced by the method of claim 1 .
12. A seed of the transgenic plant of claim 11 , wherein the seed comprises the selected DNA.
13. A method for producing a medical cannabis composition, the method comprising:
(a) obtaining the transgenic cannabis plant of claim 11 ;
(b) growing the transgenic cannabis plant under plant growth conditions to produce transgenic plant tissue from the transgenic cannabis plant; and
(c) preparing the medical cannabis composition from the transgenic plant tissue.