Genetic engineering of fungi to modulate tryptamine expression

Abstract

To provide methods for modulating the psilocybin biosynthesis pathway in fungi or other organisms.SOLUTION: Also provided are genetically modified fungi and organisms with induced and / or increased expression of psilocybin and psilocin and psilocybin and / or psilocin compositions generated by the provided methods. In embodiments described herein, the compound of Formula I is Dimethyltryptamine (DMT), the compound of Formula II is psilocybin, the compound of Formula III is psilocin, and the compound of Formula IV is tryptamine.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 909,159, filed October 1, 2019, which incorporates by reference in its entirety. [Background technology]

[0002] Tryptamine-derived substances such as psilocybin and psilocin in fungi are natural drugs with known hallucinogenic and other medicinal effects. The pharmacological effects are caused by modified tryptamines, with psilocybin being the main chemical constituent of these fungi. This prodrug-like natural product is rapidly dephosphorylated after oral ingestion to yield psilocin, the actual psychoactive drug also produced in small amounts by fungi. Tryptamine-derived substances have attracted pharmaceutical attention because clinical trials have shown favorable trends in the treatment of existential anxiety and nicotine addiction in advanced cancer patients. Recently, research is underway to investigate the use of psilocybin for the treatment of depression. Fungi with modified profiles of therapeutic component(s) may be useful in the production of tryptamine-derived substances and / or in the production of genetically modified fungi that result in desired drug profiles. Summary of the Invention

[0003] As used herein, a genetically modified organism or a cell or tissue thereof comprising a genetic modification, wherein the genetic modification is in a comparable control organism that does not have the genetic modification: [ka] Provided herein is a genetically modified organism, or a cell or tissue thereof, that results in increased production of a compound selected from a derivative or analog thereof, compared to the production of the same compound. Provided herein is a genetically modified organism, comprising an endonuclease-mediated genetic modification, wherein the genetic modification results in increased production of the compound in a comparable control organism that does not have the genetic modification. [ka] Also provided is a genetically modified organism that increases the amount of the same compound compared to the amount of its derivative or analogue.In some cases, the organism is a fungus, yeast, bacteria, animal, or insect.In the embodiment described herein, the compound of formula I is dimethyltryptamine (DMT), the compound of formula II is psilocybin, the compound of formula III is psilocin, and the compound of formula IV is tryptamine.

[0004] In the present specification, in a living organism, [ka] or a derivative or analog thereof, the method comprising introducing a genetic modification into the organism, the genetic modification resulting in increased production of the same compound compared to a comparable control organism lacking the modification. [ka] or a derivative or analog thereof, the method comprising introducing a genetic modification in the organism, the genetic modification resulting in increased production of the same compound compared to an equivalent control organism lacking the modification, the organism being a fungus, and the fungus being of the phylum Basidiomycota.

[0005] In some cases, the genetically modified organisms described herein are plants. In some cases, the genetically modified organisms described herein are bacteria. In some cases, the bacteria is Agrobacterium. In some cases, the genetically modified organisms provided herein are fungi. In some cases, the fungi are basidiomycetes. In some cases, the basidiomycetes can be selected from the group consisting of Psilocybe, Conocybe, Gymnopilus, Panaeolus, Pluteus, and Stropharia. In some cases, the fungus is Panaeolus cyanescecens. In some cases, the fungus is Panaeolus cubensis. In some cases, the fungus is Pleurotus nebrodensis.

[0006] In some embodiments, the genetically modified organisms described herein include a genetic modification that is an alteration within or adjacent to a gene or a promoter or enhancer of a gene, the gene encoding a PLP-independent phosphatidylserine decarboxylase, a tryptophan decarboxylase (TDC), a 5-methylthione ribose family small molecule kinase, a 4-hydroxytryptamine kinase, a class I methyltransferase, a facilitator-type transporter PsiT1, or a facilitator-type transporter PsiT2.

[0007] In some embodiments, the genetic modification in the organism described herein results in at least one of the following in the genetically modified organism, compared to a comparable control organism without the genetic modification: (a) increased tryptophan decarboxylation, (b) increased tryptamine 4-hydroxylation, (c) increased 4-hydroxytryptine O-phosphorylation, and (d) increased psilocybin through sequential N-methylation with reduced expression of psilocin intermediates. In some cases, the genetic modification results in (i) upregulation of the expression of a tryptophan decarboxylase gene, a psilocybin-related hydroxylase gene, a psilocybin-related N-methyltransferase gene, or a psilocybin-related phosphotransferase gene, (ii) reduced synthesis of tryptamine other than psilocybin, or (iii) increased production of tryptophan in the genetically modified organism, compared to a comparable control organism without the genetic modification.

[0008] In some embodiments, the genetic modification may be in the promoter or enhancer region of the gene of interest or associated with the gene of interest. In some cases, the genetic modification results in upregulation of the expression of the gene. In some embodiments, the gene of interest described herein encodes PLP-independent phosphatidylserine decarboxylase, tryptophan decarboxylase (TDC), 5-methylthione ribose family small molecule kinase, 4-hydroxytryptamine kinase, or class I methyltransferase. In some cases, the gene of interest described herein comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:1. In some cases, the gene of interest described herein comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:2. In some cases, the gene of interest described herein comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:3. In some cases, the gene of interest described herein encodes a class I methyltransferase. In some cases, the class I methyltransferase comprises a Rossmann fold. In some cases, the class I methyltransferase can be norbaeocystin methyltransferase. In some cases, the gene of interest described herein comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:4. In some cases, the gene of interest described herein comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:5. In some cases, the gene of interest described herein comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:6.In some cases, the gene of interest described herein comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:7. In some cases, the gene of interest described herein comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:8. In some cases, the gene of interest described herein comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:9. In some cases, the gene of interest described herein comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:10. In some cases, the gene of interest described herein comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:11. In some cases, the genes of interest described herein comprise at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 12. In some cases, the genes of interest described herein comprise at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 13. In some cases, the genes of interest described herein comprise at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 14.

[0009] In some cases, the gene may be a PsiD gene, a PsiM gene, a PsiH gene, a PsiK gene, a PsiR gene, a PsiT1 gene, or a PsiT2 gene, or any part thereof. In some cases, the expression of the gene is upregulated by at least 1.1, at least 1.2, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, or at least 5 times in the genetically modified organism compared to a comparable control organism without the genetic modification. In some cases, the genetic modification in the genetically modified organism described herein comprises a change in a gene selected from the group consisting of indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), and TrpM. In some cases, the genetic modification may be within the coding region of the gene. In some cases, the genetic modification comprises an alteration in a gene selected from the group consisting of phospho-2-dehydro-3-deoxyheptonate aldolase, 3-dehydroquinate synthase, 3-dehydroquinate dehydratase, shikimate dehydrogenase, 3-phosphoshikimate 1-carboxyvinyltransferase, shikimate kinase 1, shikimate kinase 2, chorismate synthase, tryptophan synthase alpha chain, tryptophan synthase beta chain, anthranilate phosphoribosyltransferase, and anthranilate synthase.

[0010] In some embodiments, the genetic modification may be in the promoter region of the gene. In some cases, the genetically modified organism may have 25% more virulence, as measured by dry weight, compared to a comparable control organism that does not have the genetic modification. [ka] In some cases, the genetically modified organism contains 25% more psilocybin, as measured by dry weight, compared to a comparable control organism without the genetic modification. In some cases, the genetically modified organism contains 10% more psilocin, as measured by dry weight, compared to a comparable control organism without the genetic modification.

[0011] In some cases, the genetic modification may be performed by contacting a cell of the organism with an endonuclease system. In some embodiments, the endonuclease system comprises a CRISPR enzyme, a TALE-nuclease, a transposon-based nuclease, a zinc finger nuclease, a meganuclease, an Argonaute, a Mega-TAL, or a DNA-guided nuclease. In some embodiments, the DNA-guided nuclease comprises an Argonaute. In some cases, the endonuclease system comprises a CRISPR enzyme and a guide polynucleotide that hybridizes to a target sequence within or adjacent to a gene or its associated promoter or enhancer. In some cases, the target sequence may be at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, or at least 22 nucleotides in length. In some cases, the target sequence is up to 17 nucleotides in length. In some cases, the target sequence may hybridize to at least one of SEQ ID NOs: 1-14, or a complementary sequence thereof. In some cases, the guide polynucleotide may be chemically modified. In some embodiments, the guide polynucleotide is a single guide RNA (sgRNA). In some embodiments, the guide polynucleotide can be a chimeric single guide comprising RNA and DNA. In some cases, the guide polynucleotide can hybridize to at least one of SEQ ID NOs: 1-14 or their complements.

[0012] In some cases, the CRISPR enzyme can be a Cas protein or a variant or derivative thereof. In some cases, the Cas protein can be Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, The Cas protein may comprise Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, C2c1, C2c2, C2c3, Cpf1, CARF, DinG, homologs thereof, or modified versions thereof. In some cases, the Cas protein may be Cas9. In some cases, the Cas9 is a modified Cas9 that binds to a canonical PAM. In some cases, the Cas9 recognizes a non-canonical PAM. In some cases, the guide polynucleotide binds to the target sequence 3-10 nucleotides away from the PAM. In some cases, the CRISPR enzyme coupled to the guide polynucleotide may be delivered to the genetically modified organism as an RNP. In some cases, the CRISPR enzyme coupled to the guide polynucleotide may be delivered to the genetically modified organism by an mRNA encoding the CRISPR enzyme and the guide polynucleotide.

[0013] In some cases, the CRISPR enzyme coupled with the guide polynucleotide may be delivered to the genetically modified organism by a vector that includes a nucleic acid encoding the CRISPR enzyme and the guide polynucleotide. In some embodiments, the vector may be a binary vector or a Ti plasmid. In some embodiments, the vector further includes a selection marker or a reporter gene. In some cases, the RNP, complex, or vector may be delivered via electroporation, microinjection, mechanical deformation of cells, lipid nanoparticles, AAV, lentivirus, Agrobacterium-mediated transformation, biolistic particle bombardment, or protoplast transformation. In some cases, the RNP, mRNA, or vector further includes a donor polynucleotide or a nucleic acid encoding the donor polynucleotide. In some embodiments, the donor polynucleotide includes homology with sequences flanking the target sequence. In some embodiments, the donor polynucleotide further includes a barcode, a reporter gene, or a selection marker.

[0014] In another embodiment, the genetically modified organism comprises an exogenous nucleotide. Optionally, the exogenous nucleotide comprises a cis-acting promoter sequence. Optionally, the exogenous nucleotide causes an increase in the production of psilocybin in the genetically modified organism, compared to an equivalent control organism without the exogenous nucleotide, through tryptophan decarboxylation, tryptamine 4-hydroxylation, 4-hydroxytryptaine O-phosphorylation, or sequential N-methylation without psilocin intermediate. Optionally, the exogenous nucleotide causes an increase in the production of tryptophan decarboxylase gene, psilocybin-related hydroxylase gene, psilocybin-related N-methyltransferase gene, or psilocybin-related phosphotransferase gene, compared to an equivalent control organism without the exogenous nucleotide, (i) in the genetically modified organism, compared to an equivalent control organism without the exogenous nucleotide, (ii) in the synthesis of tryptamine that is not psilocybin, or (iii) in the production of tryptophan. In some cases, the exogenous nucleotide encodes a PLP-independent phosphatidylserine decarboxylase, a tryptophan decarboxylase (TDC), a putative monooxygenase, a 5-methylthio ribose family small molecule kinase, or a 4-hydroxytryptamine kinase.

[0015] In some cases, the nucleotide is incorporated into a plasmid. In some cases, the plasmid is pGWB5 or pGHGWY. In some cases, the plasmid is delivered to said genetically modified organism via electroporation, microinjection, mechanical deformation of cells, lipid nanoparticles, AAV, lentivirus, Agrobacterium-mediated transformation, biolistic particle bombardment, or protoplast transformation. In some cases, the plasmid further comprises a barcode, a reporter gene, or a selection marker. In some cases, the plasmid further comprises a promoter. In some cases, the promoter is 35S, GPD, EF1a, actin, or CcDED1.

[0016] In the embodiments described herein, the genetically modified organism may be a multicellular or unicellular organism. In certain embodiments, the organism may be a single plant or fungal cell. The embodiments described herein also include cell populations, such as cell populations of the fungal species described herein.

[0017] Provided herein is a kit for genome editing comprising the composition provided herein. Also provided herein is a cell comprising the composition provided herein. The cell may be a plant cell. In some cases, the cell is a fungal cell. In some cases, the cell is a bacterial cell. In some cases, the cell is an animal cell. In some cases, the cell is an insect cell. Provided herein is a pharmaceutical composition comprising an extract of a genetically modified organism, a genetically modified cell, a composition, or a cell. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, diluent, or carrier. In some cases, the pharmaceutically acceptable excipient is a lipid.

[0018] Provided herein is a nutraceutical composition comprising an extract of a genetically modified organism, a genetically modified cell, a composition, or a cell. Provided herein is a nutraceutical composition comprising an extract of a genetically modified organism, a genetically modified cell, a composition, or a cell.In some embodiments, the nutraceutical composition or nutraceutical can be an oral form, a transdermal form, an oil formulation, an edible product, a food matrix, an aqueous dispersion, an emulsion, a solution, a suspension, an elixir, a gel, a syrup, an aerosol, a mist, a powder, a tablet, a lozenge, a gel, a lotion, a paste, a formulation stick, a balm, a cream, or an ointment.

[0019] Provided herein is a method for treating a disease or condition, comprising administering a pharmaceutical composition, a nutraceutical composition, or a dietary supplement to a subject. In some embodiments, the disease or condition is selected from the group consisting of depression, anxiety, post-traumatic stress disorder, addiction or withdrawal-related side effects, psychological distress, and psychiatric disorders and conditions.

[0020] In certain embodiments, the genetically modified organisms described herein may be fungi, yeast, plants, animals, or bacteria. In some cases, the fungi are mushrooms. In some cases, the mushrooms may produce at least one of dimethyltryptamine (DMT), psilocybin, psilocin, and / or any combination thereof.

[0021] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0022] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. In certain embodiments, for example, the following are provided: (Item 1) A genetically modified organism comprising a genetic modification, the genetic modification being capable of detecting a genetic alteration in a comparable control organism that does not have the genetic modification. [ka] and derivatives or analogs thereof, resulting in increased production of the same compound compared to production of the same compound selected from the group consisting of: (Item 2) A genetically modified organism comprising a genetic modification, the genetic modification being capable of detecting a genetic alteration in a comparable control organism that does not have the genetic modification. [ka] and derivatives or analogs thereof, wherein the genetically modified organism is a fungus, and the fungus is of the Basidiomycota phylum. (Item 3) 2. The genetically modified organism of item 1, wherein the organism is a fungus. (Item 4) 4. The genetically modified organism according to item 2 or 3, wherein the fungus is selected from the group consisting of Psilocybe, Conocybe, Gymnopilus, Panaeolus, Pluteus, and Stropharia. (Item 5) 5. The genetically modified organism according to item 4, wherein the fungus is Panaeolus cyanescecens. (Item 6) 5. The genetically modified organism according to item 4, wherein the fungus is Panaeolus cubensis. (Item 7) 5. The genetically modified organism according to item 4, wherein the fungus is Pleurotus nebrodensis. (Item 8) The genetic modification is Increased tryptophan decarboxylation b. increased tryptamine 4-hydroxylation c. Increased 4-hydroxytryptophan O-phosphorylation, and d. Increased psilocybin production via sequential N-methylation 3. The genetically modified organism according to item 1 or 2, which results in at least one of the following: (Item 9) 9. The genetically modified organism of item 8, wherein the genetic modification results in upregulation of expression of a gene. (Item 10) 10. The genetically modified organism of item 9, wherein the gene is a tryptophan decarboxylase gene, a psilocybin-related hydroxylase gene, a psilocybin-related N-methyltransferase gene, or a psilocybin-related phosphotransferase gene. (Item 11) 10. The genetically modified organism of item 9, wherein the gene is PsiD, PsiM, PsiH, or PsiK. (Item 12) 10. The genetically modified organism of item 9, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:1. (Item 13) 10. The genetically modified organism of item 9, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:2. (Item 14) 10. The genetically modified organism of item 9, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:3. (Item 15) 10. The genetically modified organism of item 9, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:4. (Item 16) 10. The genetically modified organism of item 9, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:5. (Item 17) 10. The genetically modified organism of item 9, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:6. (Item 18) 10. The genetically modified organism of item 9, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:7. (Item 19) 10. The genetically modified organism of item 9, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:8. (Item 20) 10. The genetically modified organism of item 9, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:9. (Item 21) 10. The genetically modified organism of item 9, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:10. (Item 22) 10. The genetically modified organism of item 9, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:11. (Item 23) 10. The genetically modified organism of item 9, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:12. (Item 24) 10. The genetically modified organism of item 9, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:13. (Item 25) 10. The genetically modified organism of item 9, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:14. (Item 26) 26. The genetically modified organism according to any one of items 9 to 25, wherein the expression of the gene is upregulated by at least 1.1, at least 1.2, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, or at least 5 fold in the genetically modified organism compared to a comparable control organism lacking the genetic modification. (Item 27) 3. The genetically modified organism according to item 1 or 2, wherein the genetically modified organism comprises an exogenous nucleotide. (Item 28) 28. The genetically modified organism of item 27, wherein the exogenous nucleotide comprises a cis-acting promoter sequence. (Item 29) 28. The genetically modified organism of item 27, wherein the exogenous nucleotide results in increased production of psilocybin via tryptophan decarboxylation, tryptamine 4-hydroxylation, 4-hydroxytryptine O-phosphorylation, or sequential N-methylation without a psilocin intermediate in the genetically modified organism compared to an equivalent control organism lacking the exogenous nucleotide. (Item 30) 28. The genetically modified organism of claim 27, wherein the exogenous nucleotide results in (i) upregulation of expression of a tryptophan decarboxylase gene, a psilocybin-related hydroxylase gene, a psilocybin-related N-methyltransferase gene, or a psilocybin-related phosphotransferase gene, (ii) a reduction in synthesis of non-psilocybin tryptamine, or (iii) an increase in production of tryptophan in the genetically modified organism compared to an equivalent control organism lacking the exogenous nucleotide. (Item 31) 28. The genetically modified organism of item 27, wherein the exogenous nucleotide encodes a PLP-independent phosphatidylserine decarboxylase, a tryptophan decarboxylase (TDC), a putative monooxygenase, a 5-methylthio ribose family small molecule kinase, or a 4-hydroxytryptamine kinase. (Item 32) 32. The genetically modified organism according to any one of items 27 to 31, wherein the nucleotide is incorporated into a plasmid. (Item 33) 33. The genetically modified organism of item 32, wherein the plasmid is pGWB5 or pGHGWY. (Item 34) 33. The genetically modified organism of item 32, wherein the plasmid is delivered to the genetically modified organism via electroporation, microinjection, mechanical deformation of cells, lipid nanoparticles, AAV, lentivirus, Agrobacterium-mediated transformation, biolistic particle bombardment, or protoplast transformation. (Item 35) 33. The genetically modified organism of item 32, wherein the plasmid further comprises a barcode, a reporter gene, or a selection marker. (Item 36) 33. The genetically modified organism of item 32, wherein the plasmid further comprises a promoter. (Item 37) 37. The genetically modified organism of item 36, wherein the promoter is 35S, GPD, EF1a, actin, or CcDED1. (Item 38) 38. A pharmaceutical composition comprising an extract of a genetically modified organism according to any one of items 1 to 37. (Item 39) 39. The pharmaceutical composition according to item 38, further comprising a pharma- ceutically acceptable excipient, diluent, or carrier. (Item 40) A nutritional supplement composition comprising an extract of a genetically modified organism according to any one of items 1 to 37. (Item 41) 38. A dietary supplement composition comprising an extract of a genetically modified organism according to any one of items 1 to 37. (Item 42) In living organisms, [ka] or a derivative or analog thereof, the method comprising introducing a genetic modification into the organism, the genetic modification resulting in increased production of the same compound compared to an equivalent control organism lacking the modification. (Item 43) In living organisms, [ka] or a derivative or analog thereof, the method comprising introducing a genetic modification into the organism, the genetic modification resulting in increased production of the same compound compared to an equivalent control organism lacking the modification, the organism being a fungus, and the fungus being of the phylum Basidiomycota. (Item 44) 43. The method of claim 42, wherein the organism is a fungus. (Item 45) 45. The method according to claim 43 or 44, wherein the fungus is selected from the group consisting of Psilocybe, Conocybe, Gymnopilus, Panaeolus, Pluteus, and Stropharia. (Item 46) 46. ​​The method of claim 45, wherein the fungus is Panaeolus cyanescecens. (Item 47) Item 48. The method according to item 45, wherein the fungus is Panaeolus cubensis. 46. ​​The method of claim 45, wherein the fungus is Pleurotus nebrodensis. (Item 49) The genetic modification is Increased tryptophan decarboxylation b. increased tryptamine 4-hydroxylation c. Increased 4-hydroxytryptophan O-phosphorylation, and d. Increased psilocybin production via sequential N-methylation 44. The method according to item 42 or 43, which results in at least one of the following: (Item 50) 50. The method of claim 49, wherein the genetic modification results in upregulation of expression of a gene. (Item 51) 51. The method of claim 50, wherein the gene is a tryptophan decarboxylase gene, a psilocybin-related hydroxylase gene, a psilocybin-related N-methyltransferase gene, or a psilocybin-related phosphotransferase gene. (Item 52) 51. The method of claim 50, wherein the gene is PsiD, PsiM, PsiH, or PsiK. (Item 53) 51. The method of claim 50, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:1. (Item 54) 51. The method of claim 50, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:2. (Item 55) 51. The method of claim 50, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:3. (Item 56) 51. The method of claim 50, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:4. (Item 57) 51. The method of claim 50, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:5. (Item 58) 51. The method of claim 50, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:6. (Item 59) 51. The method of claim 50, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:7. (Item 60) 51. The method of claim 50, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:8. (Item 61) 51. The method of claim 50, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:9. (Item 62) 51. The method of claim 50, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:10. (Item 63) 51. The method of claim 50, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:11. (Item 64) 51. The method of claim 50, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:12. (Item 65) 51. The method of claim 50, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:13. (Item 66) 51. The method of claim 50, wherein the gene comprises at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:14. (Item 67) 67. The method of any one of items 50 to 66, wherein expression of the gene is upregulated by at least 1.1, at least 1.2, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, or at least 5 fold in the genetically modified organism compared to a comparable control organism lacking the genetic modification. (Item 68) 44. The method of claim 42 or 43, wherein the introduction of the genetic modification comprises introducing an exogenous nucleotide. (Item 69) 69. The method of claim 68, wherein the exogenous nucleotide comprises a cis-acting promoter sequence. (Item 70) 69. The method of claim 68, wherein the exogenous nucleotide results in increased production of psilocybin via tryptophan decarboxylation, tryptamine 4-hydroxylation, 4-hydroxytryptaine O-phosphorylation, or sequential N-methylation without a psilocin intermediate in the genetically modified organism compared to an equivalent control organism lacking the exogenous nucleotide. (Item 71) 69. The method of claim 68, wherein the exogenous nucleotide results in (i) upregulation of expression of a tryptophan decarboxylase gene, a psilocybin-related hydroxylase gene, a psilocybin-related N-methyltransferase gene, or a psilocybin-related phosphotransferase gene, (ii) a reduction in the synthesis of non-psilocybin tryptamine, or (iii) an increase in the production of tryptophan in the genetically modified organism compared to an equivalent control organism lacking the exogenous nucleotide. (Item 72) 69. The method of claim 68, wherein the exogenous nucleotide encodes a PLP-independent phosphatidylserine decarboxylase, a tryptophan decarboxylase (TDC), a putative monooxygenase, a 5-methylthio ribose family small molecule kinase, or a 4-hydroxytryptamine kinase. (Item 73) 73. The method according to any one of items 68 to 72, wherein the nucleotide is incorporated into a plasmid. (Item 74) 74. The method of claim 73, wherein the plasmid is pGWB5 or pGHGWY. (Item 75) 74. The method of claim 73, wherein the plasmid is delivered to the genetically modified organism via electroporation, microinjection, mechanical deformation of cells, lipid nanoparticles, AAV, lentivirus, Agrobacterium-mediated transformation, biolistic particle bombardment, or protoplast transformation. (Item 76) 74. The method of claim 73, wherein the plasmid further comprises a barcode, a reporter gene, or a selection marker. (Item 77) 74. The method of claim 73, wherein the plasmid further comprises a promoter. (Item 78) 78. The method of claim 77, wherein the promoter is 35S, GPD, EF1a, actin, or CcDED1. (Item 79) 27. The genetically modified organism according to any one of items 1 to 26, wherein the genetic modification is carried out by contacting cells of the organism with an endonuclease system. (Item 80) 80. The genetically modified organism of item 79, wherein the endonuclease system comprises a CRISPR enzyme, a TALE-nuclease, a transposon-based nuclease, a zinc finger nuclease, a meganuclease, a Mega-TAL or a DNA-guided nuclease. (Item 81) 81. The genetically modified organism of item 80, wherein the DNA-guided nuclease comprises Argonaute. (Item 82) 80. The genetically modified organism of claim 79, wherein the endonuclease system comprises a CRISPR enzyme and a guide polynucleotide that hybridizes to a target sequence within or adjacent to the gene or the promoter or enhancer associated therewith. (Item 83) 83. The genetically modified organism of item 82, wherein the target sequence is at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, or at least 22 nucleotides in length. (Item 84) 83. The genetically modified organism of item 82, wherein the target sequence is at most 17 nucleotides in length. (Item 85) 85. The genetically modified organism according to any one of items 82 to 84, wherein the target sequence is capable of hybridizing to at least one of SEQ ID NOs: 1 to 14 or a complementary sequence thereof. (Item 86) 86. The genetically modified organism according to any one of items 82 to 85, wherein the guide polynucleotide is chemically modified. (Item 87) 86. The genetically modified organism according to any one of items 82 to 85, wherein the guide polynucleotide is a single guide RNA (sgRNA). (Item 88) 86. The genetically modified organism according to any one of items 82 to 85, wherein the guide polynucleotide is a chimeric single guide comprising RNA and DNA. (Item 89) 86. The genetically modified organism according to any one of items 82 to 85, wherein the guide polynucleotide is capable of hybridizing to at least one of SEQ ID NOs: 1 to 14 or a complementary sequence thereof. (Item 90) 91. The genetically modified organism according to any one of items 82 to 90, wherein the CRISPR enzyme is a Cas protein or a variant or derivative thereof. (Item 91) The Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Csm5, Csm6, Csm1, Csm2, Csm3, Csm ... 91. The genetically modified organism of item 90, comprising mr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, C2c1, C2c2, C2c3, Cpf1, CARF, DinG, homologs thereof, or modified versions thereof. (Item 92) 92. The genetically modified organism of claim 90 or 91, wherein the Cas protein is Cas9. (Item 93) 93. The genetically modified organism of claim 92, wherein the Cas9 is a modified Cas9 that binds to a canonical PAM. (Item 94) 93. The genetically modified organism of claim 92, wherein the Cas9 recognizes a non-canonical PAM. (Item 95) 95. The genetically modified organism according to any one of claims 82 to 94, wherein the guide polynucleotide binds to the target sequence 3 to 10 nucleotides away from the PAM. (Item 96) 96. The genetically modified organism of claim 95, wherein the CRISPR enzyme coupled to the guide polynucleotide is delivered to the genetically modified organism by an RNP. (Item 97) 96. The genetically modified organism of claim 95, wherein the CRISPR enzyme coupled to the guide polynucleotide is delivered to the genetically modified organism by an mRNA encoding the CRISPR enzyme and the guide polynucleotide. (Item 98) 96. The genetically modified organism of claim 95, wherein the CRISPR enzyme coupled to the guide polynucleotide is delivered to the genetically modified organism by a vector comprising a nucleic acid encoding the CRISPR enzyme and the guide polynucleotide. (Item 99) 99. The genetically modified organism of item 98, wherein the vector is a binary vector or a Ti plasmid. (Item 100) 99. The genetically modified organism of item 98, wherein the vector further comprises a selection marker or reporter gene. (Item 101) 101. The genetically modified organism of any one of items 96 to 100, wherein the RNP, complex, or vector is delivered via electroporation, microinjection, mechanical deformation of cells, lipid nanoparticles, AAV, lentivirus, Agrobacterium-mediated transformation, biolistic particle bombardment, or protoplast transformation. (Item 102) 101. The genetically modified organism according to any one of items 96 to 100, wherein the RNP, mRNA, or vector further comprises a donor polynucleotide or a nucleic acid encoding the donor polynucleotide. (Item 103) 103. The genetically modified organism of claim 102, wherein the donor polynucleotide comprises homology to sequences flanking the target sequence. (Item 104) 104. The genetically modified organism of claim 102 or 103, wherein the donor polynucleotide further comprises a barcode, a reporter gene, or a selection marker. (Item 105) 27. An isolated cell derived from a genetically modified organism according to any one of items 1 to 26, wherein the genetically modified organism is a multicellular organism. (Item 106) A genetically modified basidiomycete cell comprising a genetic modification, the genetic modification being capable of inhibiting the growth of a basidiomycete cell in a comparable control basidiomycete cell lacking the genetic modification. [ka] and derivatives or analogs thereof, resulting in increased production of the same compound as compared to the production of the same compound selected from the group consisting of basidiomycete cells. (Item 107) 107. The genetically modified basidiomycete cell of item 106, wherein the cell is of a fungus selected from the group consisting of Psilocybe, Conocybe, Gymnopilus, Panaeolus, Pluteus, and Stropharia. [Brief description of the drawings]

[0023] [Figure 1]A schematic of the syntenic locus (Psi) for biosynthesis in P. cubensis (I) and P. cyanescens (II) is shown. Genes involved in enzyme synthesis are labeled in bold font. The cluster includes genes for a kinase (PsiK), a methyltransferase (PsiM), a tryptophan decarboxylase (PsiD), and a P450 monooxygenase (PsiH). In addition, two facilitator-type transporters (PsiT1 and PsiT2) and a putative transcriptional regulator (PsiR) are encoded and shown. Hypothetical genes are shown in light grey. Introns are not shown. [Diagram 2] A representative in vitro psilocybin biosynthetic pathway is shown. [Figure 3A] 1 shows representative vector constructs for the genetically modified organisms and cells described herein that overexpress the Psi gene under the control of the 35S promoter. 2 shows a representative vector that overexpresses the PsiD gene under the control of the 35S promoter. [Figure 3B] 1 shows representative vector constructs for the genetically modified organisms and cells described herein that overexpress the Psi gene under the control of the 35S promoter. 2 shows a representative vector that overexpresses the PsiH gene under the control of the 35S promoter. [Figure 3C] 1 shows representative vector constructs for the genetically modified organisms and cells described herein that overexpress the Psi gene under the control of the 35S promoter. 2 shows a representative vector that overexpresses the PsiK gene under the control of the 35S promoter. [Figure 3D] 1 shows representative vector constructs for the genetically modified organisms and cells described herein that overexpress the Psi gene under the control of the 35S promoter. 2 shows a representative vector that overexpresses the PsiM gene under the control of the 35S promoter. [Figure 4A] 1 shows representative vector constructs for the genetically modified organisms and cells described herein that overexpress genes under the control of a fungal-specific overexpression promoter. A representative vector with the CcDED1 promoter is shown. [Figure 4B] 1 shows representative vector constructs for the genetically modified organisms and cells described herein that overexpress genes under the control of a fungal-specific overexpression promoter. A representative vector with a GPD promoter is shown. [Figure 5A] Figure 1 shows the scheme and workflow for Psi gene overexpression in Psliocybe cubensis. A panel of expression vectors with various promoters of different strengths is shown. [Figure 5B] Figure 1 shows the scheme and workflow of Psi gene overexpression in Psliocybe cubensis.Isolated protoplasts and extract fold tissue are shown. [Figure 5C] Figure 1 shows the scheme and workflow of Psi gene overexpression in Psliocybe cubensis. Selection of plasmid DNA or Agrobacterium-integrated transformation is shown. [Figure 5D] Figure 1 shows the scheme and workflow of Psi gene overexpression in Psliocybe cubensis. [Figure 5E] Figure 1 shows the method and workflow of Psi gene overexpression in Psliocybe cubensis. Figure 2 shows the analysis of psilocybin content in genetically modified mushrooms. [Figure 6] Growth of Psilocybe cubensis for tissue extraction and transformation. Psilocybe cubensis was grown in PDA agar (A and B) and in barley-perlite compost (C) at room temperature for 7 days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] As used in the specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "chimeric transmembrane receptor polypeptide" includes a plurality of chimeric transmembrane receptor polypeptides.

[0025] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which may depend in part on how the value can be measured or determined, i.e., on the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation, as is customary in the art. Alternatively, "about" may mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, more preferably within 2-fold, of a value. When a particular value is described in the present application and claims, unless otherwise specified, it should be assumed that the term "about" means within an acceptable error range of the particular value.

[0026] As used herein, a "cell" may generally refer to a biological cell. A cell may be the basic structural, functional, and / or biological unit of an organism. A cell may originate from any organism having one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, unicellular eukaryotic cells, protozoan cells, plant cells, algal cells, seaweed, fungal cells, animal cells, invertebrate cells, vertebrate cells, mammalian cells, and the like. In some cases, a cell does not originate from a natural organism (e.g., a cell may be synthetically produced and may be referred to as an artificial cell).

[0027] The term "gene" as used herein refers to a nucleic acid (e.g., DNA, such as genomic DNA and cDNA) and its corresponding nucleotide sequence that may be involved in encoding an RNA transcript. As used herein with respect to genomic DNA, the term includes intervening non-coding regions as well as regulatory regions and may include 5' and 3' ends. In some usages, the term encompasses the transcribed sequence, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons and introns. In some genes, the transcribed region may include an "open reading frame" that encodes a polypeptide. In some usages of the term, a "gene" includes only coding sequences (e.g., "open reading frame" or "coding region") necessary to encode a polypeptide. In some cases, genes, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes, do not encode a polypeptide. In some cases, the term "gene" includes not only the transcribed sequence, but also non-transcribed regions, such as upstream and downstream regulatory regions, enhancers, and promoters. A gene may refer to an "endogenous gene," i.e., a native gene in its natural location in the genome of an organism. A gene may refer to an "exogenous gene," or a non-native gene. A non-native gene may refer to a gene that is not normally found in a host organism but that can be introduced into a host organism by gene transfer. A non-native gene may also refer to a gene that is not in its natural location in the genome of an organism. A non-native gene may also refer to a nucleic acid or polypeptide sequence of natural origin that contains mutations, insertions, and / or deletions (e.g., a non-native sequence).

[0028] The term "nucleotide" as used herein generally refers to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. Nucleotides may be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include the ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives may include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules that contain them. The term nucleotide as used herein may refer to dideoxyribonucleoside triphosphates (ddNTPs) and derivatives thereof. Illustrative examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or detectably labeled by known techniques. Labeling may also be performed using quantum dots. Detectable labels may include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides may include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City, Calif.; FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, Ill.; Fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2'-dATP available from Mannheim, Indianapolis, Ind.; and Molecular Chromosomal labeling nucleotides available from Probes, Eugene, Oreg. may include BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, Fluorescein-12-UTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP. Nucleotides may also be labeled or marked by chemical modification. The chemically modified single nucleotide can be a biotin-dNTP.Some non-limiting examples of biotinylated dNTPs can include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0029] Reference to a percentage of sequence identity between two nucleotide sequences means that in comparing the two sequences, when aligned, that percentage of nucleotides are the same. This alignment and the percentage of homology or sequence identity can be determined using software programs known in the art, such as those described in Section 7.7.18 of Current Protocols in Molecular Biology (FMAusubel et al., eds., 1987) Supplement 30 (incorporated by reference). A preferred alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap opening penalty of 12 and a gap extension penalty of 2, and a BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is disclosed in Smith & Waterman (1981) Adv. Appl. Math. 2:482-489 (incorporated by reference).

[0030] As used herein, the term "plant" includes the whole plant and any descendants, cells, tissues, or parts of the plant. The classes of plants that may be used in this disclosure may be broad, such as the classes of higher and lower plants suitable for mutagenesis, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae in general. Thus, "plant" includes dicotyledonous and monocotyledonous plants. The term "plant part" includes any part or parts of a plant, including, but not limited to, seeds (including mature and immature seeds), plant slices, plant cells; plant cell cultures; plant organs (e.g., pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, and explants). Plant tissues or plant organs may be seeds, protoplasts, calli, or any other group of plant cells that can be organized into a structural or functional unit. Plant cell or tissue cultures may be capable of regenerating plants having physiological and morphological characteristics of the plant from which the cells or tissues were derived, as well as plants having substantially the same genotype as the plant. In contrast, some plant cells cannot be regenerated to produce plants. The regenerable cells in plant cell or tissue cultures may be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, silks, flowers, grains, ears, cobs, husks, or stalks.

[0031] As used herein, the term "transgene" refers to a segment of DNA that is integrated into a host genome or capable of autonomous replication in a host cell and capable of causing expression of one or more coding sequences. Exemplary transgenes confer a novel phenotype to a host cell, or a plant regenerated therefrom, compared to the corresponding non-transformed cell or plant. Transgenes may be directly introduced into a plant by genetic transformation, or may be inherited from any previous generation plant that has been transformed with a DNA segment. In some cases, a transgene may be a barcode. In some cases, a transgene may be a marker.

[0032] As used herein, a transgenic organism generally refers to a recombinant organism in which a desired DNA sequence or locus within the genome of the organism has been modified by the insertion, deletion, substitution, or other manipulation of nucleotide sequences.

[0033] As used herein, the term "transgenic plant" refers to a plant, or a progeny of a subsequent generation derived therefrom, in which the DNA of the plant or its progeny contains an introduced foreign DNA segment that is not naturally present in a non-transgenic plant of the same lineage. A transgenic plant further contains sequences native to the plant being transformed, but the "foreign" gene may be altered, for example, by the use of one or more heterologous regulatory or other elements to alter the level or pattern of expression of the gene.

[0034] A vector can be a polynucleotide (e.g., DNA or RNA) used as a vehicle to artificially carry genetic material into a cell and allow its replication and / or expression. In some embodiments, the vector is a binary vector or a Ti plasmid. Such a polynucleotide can be in the form of, for example, a plasmid, a YAC, a cosmid, a phagemid, a BAC, a virus, or a linear DNA (e.g., a linear PCR product), or any other type of construct useful for transferring a polynucleotide sequence into another cell. The vector (or a portion thereof) can be present in the target cell either transiently (i.e., not integrated into the genome) or stably (i.e., integrated into the genome). In some embodiments, the vector can further comprise a selection marker or reporter.

[0035] The practice of some of the methods disclosed herein will employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA that are within the skill of the art. See, e.g., Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012), Current Protocols in Molecular Cloning, Vol. 1, No. 1, 2012, pp. 1171-1175, 2012. in Molecular Biology (FMAusubel, et al. eds.), Series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GRTaylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture See, for example, Microbiology of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (RI Freshney, ed. (2010)).

[0036] The present disclosure provides genetically modified organisms that produce increased amounts of tryptamine-derived substances, such as psilocybin and psilocin, as well as expression cassettes, vectors, compositions, and materials and methods for producing the same. Regularly Interspaced Short Palindromic Also provided are methods of producing genetically modified organisms using nucleotide sequences from ...

[0037] Psilocybe mushrooms contain trace amounts (0.1-1.7%) of psilocybin (Table 1). Production of psilocybin is expensive due to its rarity in mushrooms and an expensive synthetic production process. Research costs for psilocybin range from $7,000 to $10,000 per gram. [Table 1]

[0038] Although the structure of psilocybin has been known for 60 years, only recently have psilocybin biosynthetic enzymes been identified. This now provides an opportunity to enhance the production of this psychoactive compound in mushrooms and advance research into the medical use of psilocybin. The yield, potency, and efficacy of psilocybin production can be improved by state-of-the-art plant CRISPR engineering platforms. The demonstrated 10-fold increase in psilocybin production in mushrooms from 1 to 10% (% of dry mycelium mass) would be of significant benefit to the industry.

[0039] Genetically Modified Organisms Provided herein are methods and compositions for modifying biosynthetic pathways in an organism to increase production of psilocybin and psilocin in the organism. In embodiments provided herein, gene editing is used to increase production of early, intermediate, and / or late precursor compounds, such as tryptamine and tryptamine derivatives, such as dimethyltryptamine, to produce desired end products, such as psilocybin and psilocin.

[0040] Additionally, methods and compositions are provided for using gene editing to switch off specific pathways of tryptophan consumption to generate genetically modified organisms with increased expression levels of tryptamine and / or tryptamine-related substances such as psilocybin and psilocin.

[0041] The genetically modified organisms described herein can be plants, animals, bacteria, yeast, or fungi. In some cases, the fungi are mushrooms. Certain mushrooms of the genera Psilocybe, Conocybe, Gymnopilus, Panaeolus, Pluteus, and Stropharia produce psychoactive tryptamine-derived substances, such as psilocybin or psilocin, which are enhanced by the genetic modifications described herein. In some cases, the genetically modified organisms described herein are mushrooms selected from Panaeolus cyanescecens, Panaeolus cubensis, and Pleurotus nebrodensis.

[0042] In the embodiment described herein, the genetically modified cell or organism enhances the conversion of L-tryptophan or 4-hydroxy-L-tryptophan to tryptamine.In some cases, the genetically modified cell or organism comprises a genetic modification that enhances the formation of tryptamine and optionally downstream derivatives of tryptamine, such as psilocybin and psilocin, by suppressing or minimizing the alternative pathway of consumption of either 4-hydroxy-L-tryptophan or tryptophan.In some cases, this enhancement is achieved by introducing or upregulating the gene related to the expression or activity of tryptophan decarboxylase PsiD.

[0043] In some cases, the genetically modified cell or organism is characterized in that the enhanced production of psilocin or psilocybin is achieved by introducing or upregulating the gene associated with the conversion of tryptamine to 4-hydroxytryptamine, such as P450 monooxygenase PsiH.In some cases, the genetically modified cell or organism is characterized in that the enhanced production of norbaeocystin is achieved by upregulating the gene associated with the conversion of tryptamine, tryptophan, or 4-hydroxytryptamine to norbaeocystin.In some cases, such upregulation is achieved by upregulating or introducing 4-hydroxytryptamine kinase, PsiK, by modifying the promoter or enhancer sequence associated with the gene, or by knocking the gene into the cell or organism.

[0044] In some cases, the enhanced production of psilocin or psilocybin is achieved by introducing or upregulating a gene associated with the conversion of norbaeocystin to baeocystin or by increasing the production of baeocystin. In some cases, the upregulation is achieved by increasing the synthesis of the norbaeocystin methyltransferase gene by modifying the promoter or enhancer sequence associated with the gene or by knocking the gene into the cell or organism.

[0045] In certain embodiments, the tryptophan decarboxylase gene described herein can be PsiD (representative mRNA sequence is provided in Table 3). In some cases, the gene encoding tryptophan decarboxylase can comprise about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or up to about 100% sequence identity to SEQ ID NO:1. The enzyme PsiD can be a 49.6 kDa enzyme and belongs to the PLP-independent phosphatidylserine decarboxylase family. In certain embodiments, PsiD is upregulated in a cell or organism by gene editing a promoter or enhancer sequence in or associated with a gene. In certain embodiments, PsiD is upregulated or synthesized in a genetically modified cell or organism by introducing the PsiD gene into said cell or organism by using gene editing techniques described herein.

[0046] In some cases, the genetically modified cells or organisms described herein include upregulation of expression of the P450 monooxygenase PsiH gene (representative mRNA sequences are provided in Table 3). In some cases, the gene encoding the monooxygenase may include about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or up to about 100% sequence identity to SEQ ID NO:2. In certain embodiments, PsiH is upregulated in the cell or organism by gene editing a promoter or enhancer sequence in or associated with the gene. In certain embodiments, PsiH is upregulated or synthesized in the genetically modified cell or organism by introducing the PsiH gene into the cell or organism by using gene editing techniques described herein.

[0047] In some cases, the genetically modified cell or organism described herein comprises upregulation of expression of the 4-hydroxytryptamine kinase PsiK gene (representative mRNA sequences are provided in Table 3). In some cases, the gene encoding 4-hydroxytryptamine kinase may comprise about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or up to about 100% sequence identity to SEQ ID NO: 3. In certain embodiments, PsiK is upregulated in a cell or organism by gene editing a promoter or enhancer sequence in or associated with a gene. In certain embodiments, PsiK is upregulated or synthesized in a genetically modified cell or organism by introducing a PsiK gene, such as the gene of SEQ ID NO: 3, into said cell or organism by using gene editing techniques described herein.

[0048] In some cases, the genetically modified cells or organisms described herein include up-regulation of the expression of norbaeocystin methyltransferase PsiM gene (representative mRNA sequences are provided in Table 3). In some cases, the gene encoding the methyltransferase may include about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or up to about 100% sequence identity to any one of SEQ ID NO: 4. In certain embodiments, PsiM is up-regulated in a cell or organism by gene editing a promoter or enhancer sequence in or associated with a gene. In certain embodiments, PsiM is up-regulated or synthesized in a genetically modified cell or organism by introducing a PsiM gene, such as the gene of SEQ ID NO: 4, into said cell or organism by using gene editing techniques described herein. In certain cases, a Rossmann fold-containing class I methyltransferase gene having the amino acid sequence GVDIGTGAS, or a derivative thereof, is introduced into a cell or organism to increase psilocybin production.

[0049] Other putative transcriptional regulators and transporters that affect the production and accumulation of psilocybin produced in fungi or other organisms may be engineered in the organisms and cells described herein. In some cases, the putative transcriptional regulator may promote transcription or translation of a methyltransferase, hydroxylase, monooxygenase, kinase, or decarboxylase described herein, such as PsiD, PsiH, PsiK, or PsiM. In some cases, the putative transcriptional regulator may promote downregulation of transcription or translation of an enzyme, such as a methyltransferase, hydroxylase, monooxygenase, kinase, or decarboxylase, such as PsiD, PsiH, PsiK, or PsiM, described herein.

[0050] In certain embodiments, the genetic modification techniques disclosed herein can be used to enhance expression of the facilitator family transporters (PsiT1 and PsiT2, or the helix-loop-helix (HLH) domain transcription regulator (PsiR) by genetically editing a promoter or enhancer sequence within or associated with the gene, or by introducing additional copies of one or more of said genes or homologs thereof. This may also be involved in ensuring that the synthesized psilocybin is correctly transported and localized in fungi and other organisms. In certain embodiments, PsiR, PsiT1, or PsiT2 is upregulated in a cell or organism by genetically editing a promoter or enhancer sequence within or associated with the gene. In certain embodiments, PsiR, PsiT1, or PsiT2 is upregulated or synthesized in a genetically modified cell or organism by using the genetic editing techniques described herein to introduce a PsiR, PsiT1, or PsiT2 gene, e.g., the gene of SEQ ID NO:5, into said cell or organism.

[0051] Representative sequences of genes encoding PsiT2 are listed in Table 3. In some cases, the gene encoding PsiT2 may comprise from about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or up to about 100% sequence identity to any one of SEQ ID NO:5.

[0052] The above-mentioned genes can be modified by the genetic modification techniques disclosed herein to increase the production of enzymes, putative regulators, and putative transporters involved in the psilocybin biosynthetic pathway, or such enzymes, regulators, and transporters can be de novo produced in the genetically modified cells or organisms described herein.For example, the expression level of certain enzymes along the psilocybin biosynthetic pathway can be increased to increase the production of one or more of tryptamine, 4-hydroxytryptamine, baeocystin, norbaeocystin, and psilocybin.In some cases, the genetic modification is within the promoter or enhancer region of or associated with one or more of the genes described herein.

[0053] In certain embodiments, genes related to pathways that also utilize tryptophan and / or 4-hydroxy-L-tryptophan are modified by the genetic modification techniques described herein to reduce tryptophan and / or 4-hydroxy-L-tryptophan consumption by these pathways by downregulating or knocking out these genes. Downregulating or knocking out genes can include, for example, indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), and TrpM. TrpM is a methyltransferase that has monomethylation and dimethylation activity on tryptophan but is not part of the psilocybin biosynthetic pathway. Downregulating or knocking out genes such as IDO, TDO, and TrpM in the genetically modified organisms or cells described herein increases the availability of tryptophan and / or 4-hydroxy-L-tryptophan for psilocybin production.

[0054] In certain embodiments, the genetically modified cells or organisms contain modifications that result in increased production of tryptophan and / or 4-hydroxy-L-tryptophan. These modifications include upregulation of genes encoding phospho-2-dehydro-3-deoxyheptonate aldolase, 3-dehydroquinate synthase, 3-dehydroquinate dehydratase, shikimate dehydrogenase, 3-phosphoshikimate 1-carboxyvinyltransferase, shikimate kinase 1, shikimate kinase 2, chorismate synthase, tryptophan synthase alpha chain, tryptophan synthase beta chain, anthranilate phosphoribosyltransferase, or anthranilate synthase components. Upregulation of these genes is achieved by increasing production of the genes by modifying promoters or enhancers within or associated with the genes, or by increasing the copy number of the genes in the organism or cell.

[0055] Increasing the expression of these enzymes results in the production of more of the substrates tryptophan and / or 4-hydroxy-L-tryptophan, leading to increased production of psilocybin and / or psilocin.

[0056] Provided herein are methods and compositions for characterizing the psilocybin biosynthetic pathway and enzymes. In embodiments provided herein, candidate psilocybin genes are identified in three diverse psilocybin-positive (PS + ) Identified by sequencing the mushroom homokaryon genomes: Ps. cyanescens, Pa. (=Copelandia) cyanescens, and Gy. Dilepis. In certain embodiments, all are PS +In the genome, five genes are clustered: tryptophan decarboxylase (PsiD); psilocybin-related N-methyltransferase (PsiM); psilocybin-related hydroxylase (PsiH); psilocybin-related phosphotransferase (PsiK); psilocybin-related transporter (PsiT). In certain embodiments, PsiD, which is the first committed step in the reaction and is the only one that does not produce drug-scheduled compounds, has specific decarboxylase activity for tryptophan and produces tryptamine. In certain embodiments, gene overlap between clusters is associated with alternative or reticulated pathways of genetic modification.

[0057] In the embodiments described herein, PS + The coding sequences of the genes in the cluster have been identified from several mushrooms and are provided herein. In certain embodiments, information is also present about the intron or exon organization of these genes (a representative list of genes is provided in Table 2). [Table 2] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13]

[0058] In some cases, the efficiency of genome disruption of fungi, or any other organism, including but not limited to cells, by any of the nucleic acid delivery platforms described herein may result in about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to about 100% disruption of genes or portions thereof as measured by nucleic acid or protein analysis.

[0059] In some cases, the genetically modified fungi and other organisms contain about 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% more of a compound of any one of Formulas I-IV, as measured by dry weight of the fungus, compared to a comparable control lacking the genetic modification.

[0060] In some cases, the genetically modified fungi and other organisms contain about 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% more dimethyltryptamine (DMT) as measured by dry weight of the fungus compared to comparable controls without the genetic modification.

[0061] In some cases, the genetically modified fungi and other organisms contain about 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% more psilocybin, as measured by dry weight of the fungus, compared to comparable controls without the genetic modification.

[0062] In some cases, the genetically modified fungi and other organisms contain about 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% more psilocin, measured by dry weight of the fungus, compared to comparable controls without the genetic modification.

[0063] Various methods can be utilized to identify potential targets for gene editing in the psilocybin and / or psilocin biosynthetic pathway. In some cases, any one of bioinformatics, gRNA design, CRISPR reagent construction, plant transformation, plant regeneration, and / or genotyping can be utilized. Bioinformatics can include gene mapping, gene alignment and copy number analysis, and gene annotation. gRNA design can include grouping of gRNAs to design clusters of guides for their intended function, rank, and selection based on target gene specificity and off-targets within the cannabis genome. CRISPR reagent construction can include the generation of infection-ready AGRO reagents to co-deliver cannabis codon-optimized Cas9 and gRNA. Plant transformation and regeneration can include the development of techniques to infect plant tissue with CRISPR AGRO (e.g., callus), isolate cannabis protoplasts and transform RNP reagents, and / or obtain growing plantlets from transformed tissue. Genotyping may involve isolating plant DNA and analyzing target sequences. Functional analysis may involve analyzing the cannabinoid content in plant tissues and quantifying the relevant cannabinoids.

[0064] The various techniques of genetic modification disclosed above may also be used in other organisms, such as different plants, E. coli and other suitable bacteria, or yeast, to produce the end products psilocybin and / or psilocin. In the genetically engineered fungi and other organisms of the present disclosure, the amount of psilocybin and / or psilocin is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 300%, or up to 400% more compared to a comparable control fungus or organism lacking such genetic modifications of the present disclosure.

[0065] Genetic manipulation A system of genome manipulation can be provided herein. The system of genome manipulation can include any one of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) enzyme, transcription activator-like effector (TALE)-nuclease, transposon-based nuclease, zinc finger nuclease, meganuclease, Argonaute, or Mega-TAL. In some embodiments, the genome editing system can utilize guiding polynucleic acid, which includes DNA, RNA, or a combination thereof. In some cases, the guide can be guide DNA or guide RNA.

[0066] I. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) In some cases, genetic manipulation can be performed using CRISPR system or part thereof.CRISPR system can be a multi-component system that includes guide polynucleotide or nucleic acid that encodes guide polynucleotide and CRISPR enzyme or nucleic acid that encodes CRISPR enzyme.CRISPR system can include any modification of CRISPR component or any part of any of CRISPR components.

[0067] The methods described herein can utilize the CRISPR system. There are at least five types of CRISPR systems, all of which incorporate guide RNA and Cas proteins and encoding polynucleic acids. The general mechanism and recent advances of the CRISPR system are described in Cong, L. et al., "Multiplex genome engineering using CRISPR systems," Science, 339(6121):819-823(2013); Fu, Y. et al., "High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells,”Nature Biotechnology,31,822-826(2013)、Chu,VT et al. “Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells,”Nature Biotechnology 33,543-548(2015), Shmakov,S.et al.,“Discovery and functional characterization of diverse Class 2 CRISPR-Cas systems,”Molecular Cell,60,1-13(2015),Makarova,KS et al.,“An updated evolutionary classification of CRISPR-Cas systems,”,Nature Reviews Microbiology, 13, 1-15 (2015). Site-specific cleavage of the target DNA occurs at a position determined by both 1) base pairing complementarity between the guide RNA and the target DNA (also called the protospacer) and 2) a short motif in the target DNA called the protospacer adjacent motif (PAM). The PAM may be a canonical or non-canonical PAM. For example, engineered cells, such as plant cells, can be generated using a CRISPR system, e.g., a type II CRISPR system. The Cas enzyme used in the methods disclosed herein can be Cas9, which catalyzes DNA cleavage. Streptococcus Enzymatic action by Cas9 from P. pyogenes or any closely related Cas9 can generate a double-stranded break at a target site sequence that hybridizes to about 20 nucleotides of a guide sequence and has a protospacer adjacent motif (PAM) after about 20 nucleotides of the target sequence. In some embodiments, fewer than 20 nucleotides can be hybridized. In some embodiments, more than 20 nucleotides can be hybridized.Genomic disruption of the activity of THCA synthase can be provided herein, comprising introducing at least one RNA-guided endonuclease comprising at least one nuclear localization signal, or a nucleic acid encoding at least one RNA-guided endonuclease comprising at least one nuclear localization signal, into a cannabis and / or hemp plant or a cell thereof, wherein the at least one guiding nucleic acid encodes at least one guide RNA. In some embodiments, the modified plant or part thereof can be cultivated.

[0068] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) enzyme The CRISPR enzyme may include or be a Cas enzyme. In some aspects, a nucleic acid encoding a Cas protein or a portion thereof may be utilized in the embodiments provided herein. Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Csm4, Csm5, Csm7, Csm8, Csm9, Csm10, Csm11, Csm12, Csm13, Csm14, Csm15, Csm16, Csm17, Csm18, Csm19, Csm11, Csm11, Csm11, Csm12, Csm13, Csm14, Csm15, Csm16, Csm17, Csm18, Csm19 ... CRISPR enzyme may include Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, C2c1, C2c2, C2c3, Cpf1, CARF, DinG, their homologues, or their modified versions. In some cases, catalytically inactive Cas protein may be used, such as dCas9. Unmodified CRISPR enzyme, such as Cas9, may have DNA cleavage activity. CRISPR enzymes can induce cleavage of one or both strands in a target sequence, for example, within the target sequence and / or within the complement of the target sequence. In some embodiments, the target sequence is at least about 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, or at least 22 nucleotides in length. In some cases, the target sequence is up to 17 nucleotides in length. In some embodiments, the target can be selected from sequences that include about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or up to about 100% homology with any one of SEQ ID NO:1-SEQ ID NO:7.

[0069] In some embodiments, the target sequence may be found within an intron or exon of a gene. In some cases, the CRISPR system may target an exon of a gene involved in the cannabinoid biosynthetic pathway. For example, the CRISPR enzyme may induce cleavage of one or both strands within the target sequence or within a distance of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence. For example, the CRISPR enzyme may induce cleavage of one or both strands within the PAM sequence or within a distance of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the PAM sequence. In some cases, the guide polynucleotide binds to the target sequence 3-10 nucleotides away from the PAM. A vector can be used that encodes a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme, so that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide that contains the target sequence. The Cas protein can be a high-fidelity Cas protein, such as Cas9HiFi. In some cases, the Cas protein can be modified. For example, the modification of the Cas protein can include N7-methyl-Gppp (2'-O-methyl-A).

[0070] Cas9 may refer to a polypeptide having at least about 50%, 60%, 70%, 80%, 90%, 100% sequence identity and / or sequence similarity to a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from S. pyogenes). Cas9 may refer to a polypeptide having at most about 50%, 60%, 70%, 80%, 90%, 100% sequence identity and / or sequence similarity to a wild-type exemplary Cas9 polypeptide (e.g., from S. pyogenes). Cas9 may refer to a wild-type or modified form of Cas9 protein that may include amino acid changes such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof. In some cases, CRISPR enzymes such as Cas may be codon-optimized for expression in plants.

[0071] A polynucleotide encoding an endonuclease (e.g., a Cas protein such as Cas9) can be codon-optimized for expression in a particular cell, such as a plant cell. This type of optimization can involve mutations in the foreign (e.g., recombinant) DNA that mimic the codon preferences of the intended host organism or cell while still encoding the same protein.

[0072] The endonuclease may comprise an amino acid sequence having at least, or at least about, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid sequence identity to the nuclease domain of a wild-type exemplary site-directed polypeptide (e.g., Cas9 from S. pyogenes).

[0073] S. pyogenes Cas9 (SpCas9) can be used as a CRISPR endonuclease for genome manipulation. In some cases, different endonucleases can be used to target specific genome targets. In some cases, synthetic SpCas9-derived variants containing non-NGG PAM sequences can be used. In addition, other Cas9 orthologs from various species have been identified, and these "non-SpCas9" bind to different PAM sequences that can also be useful in the present invention. For example, the relatively large size of SpCas9 (approximately 4 kb coding sequence) means that a plasmid carrying SpCas9 cDNA may not be efficiently expressed in cells. Conversely, the coding sequence of Staphylococcus aureus Cas9 (SaCas9) is approximately 1 kilobase shorter than SpCas9 and may be efficiently expressed in cells.

[0074] Alternatives to S.pyogenes Cas9 may include RNA-guided endonucleases of the Cpf1 family. Unlike Cas9 nuclease, the result of Cpf1-mediated DNA cleavage is a double-stranded break with a short 3' overhang. The alternating cleavage pattern of Cpf1 may open up the possibility of directional gene transfer, similar to conventional restriction enzyme cloning, which may increase the efficiency of gene editing. Similar to the above-mentioned Cas9 variants and orthologs, Cpf1 may also increase the number of sites that can be targeted by CRISPR into AT-rich regions or AT-rich genomes that lack the NGG PAM sites preferred by SpCas9.

[0075] In some embodiments, the Cas sequence may include a nuclear localization sequence (NLS). The nuclear localization sequence may be derived from SV40. The NLS may be derived from at least one of SV40, nucleoplasmin, importin alpha, C-myc, EGL-13, TUS, hnRNPA1, Mata2, or PY-NLS. The NLS may be at the C-terminus or N-terminus of the Cas protein. In some cases, the Cas protein may include one to five NLS sequences. The Cas protein may include one, two, three, four, five, six, seven, eight, nine, or up to ten NLS sequences. The Cas protein, such as Cas9, may include two NLS sequences. The Cas protein may include SV40 and nucleoplasmin NLS sequences. The Cas protein may include at least one untranslated region.

[0076] In some embodiments, the vector encoding the CRISPR enzyme may include a nuclear localization sequence (NLS) sequence. In some cases, the vector may include one or more NLSs. In some cases, the vector may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 NLSs. For example, the CRISPR enzyme may include more than or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs at or near the amino terminus, or more than or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs at or near the carboxyl terminus, or any combination thereof (e.g., one or more NLSs at the amino terminus and one or more NLSs at the carboxyl terminus). When more than one NLS is present, each may be selected independently from the others, such that a single NLS may be present in more than one copy and / or in combination with one or more other NLSs present in one or more copies.

[0077] NLS may be mono- or bi-knot type. In some cases, bi-knot type NLS may have a spacer sequence different from mono-knot type NLS. NLS may be derived from at least one of SV40, nucleoplasmin, importin alpha, C-myc, EGL-13, TUS, hnRNPA1, Mata2, or PY-NLS. NLS may be located anywhere in the polypeptide chain, for example, near the N-terminus or C-terminus. For example, NLS may be located within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 amino acids from the N-terminus or C-terminus, or within about that distance along the polypeptide chain. In some cases, NLS may be located within 50 amino acids or more, for example, within 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 amino acids from the N-terminus or C-terminus, or within about that distance.

[0078] Any functional concentration of Cas protein can be introduced into cells. For example, 15 micrograms of Cas mRNA can be introduced into cells. In other cases, Cas mRNA can be introduced from 0.5 micrograms to 100 micrograms. Cas mRNA can be introduced from 0.5 to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 micrograms.

[0079] In some cases, a double nickase approach can be used to introduce double-stranded or genomic breaks. Cas proteins can be mutated at known amino acids in either nuclease domain, thereby eliminating the activity of one nuclease domain and generating a nickase Cas protein that can generate single-stranded breaks. Utilizing a nickase with two specific guide RNAs that target opposite strands can generate double-stranded breaks (DSBs) within the target site (often referred to as a "double nick" or "double nickase" CRISPR system). This approach can dramatically increase target specificity, since two off-target nicks are unlikely to be generated close enough to cause a DSB.

[0080] Nucleases such as Cas9 can be tested for identity and potency prior to use. For example, identity and potency can be determined using at least one of spectrophotometric analysis, RNA agarose gel analysis, LC-MS, endotoxin analysis, and sterility testing. In some cases, nuclease sequences such as Cas9 sequences can be sequenced to confirm their identity. In some cases, Cas proteins such as Cas9 proteins can be sequenced prior to clinical or therapeutic use. For example, purified in vitro transcription products can be evaluated by polyacrylamide gel electrophoresis to verify the absence of other mRNA species other than Cas9, or the substantial absence of other mRNA species, in the clinical product. Additionally, purified mRNAs encoding Cas proteins such as Cas9 can be subjected to validation by reverse transcription, followed by a sequencing step to verify identity at the nucleotide level. Purified in vitro transcripts can be assessed by polyacrylamide gel electrophoresis (PAGE) to verify that the mRNA is of the expected size for Cas9 and that other mRNA species are substantially absent in the clinical or therapeutic product.

[0081] In some cases, the endotoxin level of the nuclease, such as Cas9, can be determined. The clinically / therapeutically acceptable level of endotoxin can be less than 3EU / mL. The clinically / therapeutically acceptable level of endotoxin can be less than 2EU / mL. The clinically / therapeutically acceptable level of endotoxin can be less than 1EU / mL. The clinically / therapeutically acceptable level of endotoxin can be less than 0.5EU / mL.

[0082] In some cases, nucleases such as Cas9 may be subjected to sterility testing. The clinically / therapeutically acceptable level of sterility testing may be represented by 0 or no growth in culture. The clinically / therapeutically acceptable level of sterility testing may be less than 0.5%, 0.3%, 0.1%, or 0.05% growth.

[0083] Guiding Polynucleic Acid The guiding polynucleic acid may be DNA or RNA. The guiding polynucleic acid may be single-stranded or double-stranded. In some cases, the guiding polynucleic acid may include regions of single-stranded and double-stranded regions. The guiding polynucleic acid may also form secondary structures. As used herein, the term "guide RNA (gRNA)" and its grammatical equivalents may refer to an RNA that can be specific to a target DNA and can form a complex with a Cas protein. The guide RNA may include a guide sequence, or a spacer sequence, which identifies a target site and directs the RNA / Cas complex to the target DNA identified for cleavage. For example, the guide RNA may target a CRISPR complex to a target gene or a portion thereof to perform targeted double-stranded cleavage. Site-specific cleavage of the target DNA occurs at a location determined by both 1) the base-pairing complementarity between the guide RNA and the target DNA (also called a protospacer) and 2) a short motif within the target DNA, referred to as a protospacer adjacent motif (PAM). In some cases, gRNAs can be designed using algorithms that can identify gRNAs that map to early exons in commonly expressed transcripts.

[0084] In some cases, the guide polynucleotide can be complementary to a target sequence of a gene encoding a methyltransferase, hydroxylase, monooxygenase, kinase, decarboxylase, transcription regulator, transporter, indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), TrpM, phospho-2-dehydro-3-deoxyheptonate aldolase, 3-dehydroquinate synthase, 3-dehydroquinate dehydratase, shikimate dehydrogenase, 3-phosphoshikimate 1-carboxyvinyltransferase, shikimate kinase 1, shikimate kinase 2, chorismate synthase, tryptophan synthase alpha chain, tryptophan synthase beta chain, anthranilate phosphoribosyltransferase, and anthranilate synthase component. In some cases, the gRNA or gDNA can bind to a target sequence homologous or complementary to SEQ ID NOs: 1-5 or any of the genes described above.

[0085] Functional gene copies, gene variants, and pseudogenes are mapped and aligned to create sequence templates for CRISPR design. In some cases, multiple guide RNAs targeting conserved sequences across aligned copies of THCA synthase are designed to disrupt initial coding sequences and introduce mutations such as frameshift mutation indels into coding sequences. In some cases, guide RNAs with low incidence of off-target sites elsewhere in the cannabis and hemp genome can be selected.

[0086] In some embodiments, CRISPR gRNA libraries can be generated and used to screen variant plants by DNA analysis. Multiplex CRISPR manipulation can generate diverse genotypes of novel cannabinoid-producing cannabis plants. In some cases, these plants produce elevated levels of minor, rare, and / or understudied cannabinoids.

[0087] In some cases, gRNAs can be designed to target exons of genes involved in the cannabinoid biosynthesis pathway. In some cases, gRNAs can be designed to disrupt initial coding sequences. In some embodiments, the subject guide RNAs can be clustered into two categories: those that target coding sequences with initial locations in these genes and aim to disrupt the production of functional proteins by introducing frameshift mutation indels (KO guides) and those that target sequences that span gene regulatory regions (expression-regulating guides). In addition, guide RNAs can be selected that have the lowest incidence of off-target sites elsewhere in the cannabis and hemp genome.

[0088] In some cases, a gRNA may be selected based on the pattern of indels it inserts into a target gene. Candidate gRNAs may be ranked by off-target potential using a scoring system that may take into account: (a) the total number of mismatches between the gRNA sequence and any genomic sequence that it closely matches; (b) the mismatch location(s) relative to the PAM site that correlates with the negative impact on activity of mismatches near the PAM site; (c) the distance between mismatches that accounts for the cumulative effect of adjacent mismatches in disrupting guide-DNA interactions; and any combination of these. In some cases, the greater the number of mismatches between the gRNA and the genomic target site, the lower the likelihood of CRISPR-mediated cleavage of that site. In some cases, the mismatch location is directly adjacent to the PAM site. In other cases, the mismatch location may be one nucleotide up to 100 kilobases away from the PAM site. A candidate gRNA containing mismatches may not be adjacent to a PAM in some cases. In other cases, at least two candidate gRNAs containing mismatches may bind to the genome one nucleotide up to 100 kilobases away from each other. The mismatch may be a substitution of a nucleotide. For example, in some cases, G is substituted for T. Mismatches between the gRNA and the genome may allow for reduced fidelity of CRISPR gene editing. In some cases, a gRNA with a positive score may be about 110 nucleotides in length and may not contain a mismatch with a complementary genome sequence. In other cases, a gRNA with a positive score may be about 110 nucleotides in length and may contain up to 3 mismatches with a complementary genome sequence. In other cases, a gRNA with a positive score may be about 110 nucleotides in length and may contain up to 20 mismatches with a complementary genome sequence. In some cases, the guiding polynucleic acid may contain internucleotide linkages that may be phosphorothioates. Any number of phosphorothioates may be present. For example, 1 to about 100 phosphorothioates may be present in the guiding polynucleic acid sequence. In some cases, 1 to 10 phosphorothioates are present.Optionally, eight phosphorothioates are present in the guiding polynucleic acid sequence.

[0089] In some cases, gRNAs with top scores may be designed and selected, and the on-target editing efficiency of each may be experimentally evaluated in plant cells. In some cases, the editing efficiency determined by TiDE analysis may be at least greater than about 20%. In other cases, the editing efficiency may be about 20% to about 50%, about 50% to about 80%, about 80% to about 100%. In some cases, the percentage of indels may be determined in a pilot GMP run. For example, the final cell product may be analyzed for on-target indel formation by Sanger sequencing and TIDE analysis. In both control and experimental samples, about 1×10 6 Genomic DNA may be extracted from the cells and subjected to PCR using primers flanking the disrupted gene, such as a gene involved in the cannabinoid biosynthetic pathway. Sanger sequencing chromatograms may be analyzed using the TIDE software program, which can quantify the frequency of indels and the size distribution of indels by comparing control and knockout samples.

[0090] The method disclosed herein may include introducing at least one guide RNA or nucleic acid, for example, DNA encoding at least one guide RNA, into a cell or plant embryo. The guide RNA can interact with an RNA-guided endonuclease to direct the endonuclease to a specific target site, where the 5' end of the guide RNA base pairs with a specific protospacer sequence in a chromosomal sequence.

[0091] The guide RNA may comprise two RNAs, for example, a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA). The guide RNA may optionally comprise a single guide RNA (sgRNA) formed by fusion of a portion (e.g., a functional portion) of the crRNA and the tracrRNA. The guide RNA may be a duplex RNA comprising a crRNA and a tracrRNA. The guide RNA may comprise the crRNA and lack the tracrRNA. Additionally, the crRNA may hybridize with the target DNA or the protospacer sequence.

[0092] As mentioned above, the guide RNA can be an expression product. For example, the DNA encoding the guide RNA can be a vector comprising a sequence encoding the guide RNA. The guide RNA can be transferred to a cell or an organism by transfecting the cell or plant embryo with an isolated guide RNA or a plasmid DNA comprising a sequence encoding the guide RNA and a promoter. In some embodiments, the promoter can be selected from the group consisting of a leaf-specific promoter, a flower-specific promoter, a THCA synthase promoter, a CaMV35S promoter, an FMV35S promoter, and a tCUP promoter. The guide RNA can also be transferred to a cell or a plant embryo by other methods, such as using particle bombardment.

[0093] The guide RNA may be isolated. For example, the guide RNA may be transfected into a cell or plant embryo in the form of isolated RNA. The guide RNA may be any in The guide RNA may be prepared by in vitro transcription using a vitro transcription system. The guide RNA may be introduced into the cell in the form of isolated RNA, rather than in the form of a plasmid containing the coding sequence of the guide RNA.

[0094] A guide RNA may include a DNA-targeting segment and a protein-binding segment. The DNA-targeting segment (or DNA-targeting sequence, or spacer sequence) includes a nucleotide sequence that can be complementary to a specific sequence (e.g., a protospacer) in the target DNA. The protein-binding segment (or protein-binding sequence) can interact with a site-directed modifying polypeptide, such as an RNA-guided endonuclease, such as a Cas protein. By "segment" is meant a segment / section / region of a molecule, such as a continuous stretch of nucleotides in an RNA. A segment may also mean a region / section of a complex, such that a segment may include a region of more than one molecule. For example, in some cases, the protein-binding segment of a DNA-targeting RNA is one RNA molecule, and thus the protein-binding segment includes a region of that RNA molecule. In other cases, the protein-binding segment of a DNA-targeting RNA includes two separate molecules hybridized along a complementary region.

[0095] A guide RNA can comprise two separate RNA molecules or a single RNA molecule. An exemplary unimolecular guide RNA comprises both a DNA targeting segment and a protein binding segment.

[0096] An exemplary two-molecule DNA-targeting RNA may include a crRNA-like ("CRISPR RNA" or "targeter RNA" or "crRNA" or "crRNA repeat") molecule and a corresponding tracrRNA-like ("trans-acting CRISPR RNA" or "activator RNA" or "tracrRNA") molecule. The first RNA molecule may be a crRNA-like molecule (targeter RNA) that may include a DNA-targeting segment (e.g., a spacer) and a stretch of nucleotides that can form one half of a double-stranded RNA (dsRNA) duplex with the protein-binding segment of the guide RNA. The second RNA molecule may be a corresponding tracrRNA-like molecule (activator RNA) that may include a stretch of nucleotides that can form the other half of a dsRNA duplex of the protein-binding segment of the guide RNA. In other words, the stretch of nucleotides of the crRNA-like molecule may be complementary to the stretch of nucleotides of the tracrRNA-like molecule and may hybridize with it to form a dsRNA duplex of the protein-binding domain of the guide RNA. Thus, each crRNA-like molecule can be said to have a corresponding tracrRNA-like molecule. The crRNA-like molecule can further provide a single-stranded DNA targeting segment, or a spacer sequence. Thus, the crRNA-like molecule and the tracrRNA-like molecule (as a corresponding pair) can hybridize to form a guide RNA. The subject bimolecular guide RNA can include any corresponding crRNA and tracrRNA pair.

[0097] The DNA targeting segment or spacer sequence of the guide RNA can be complementary to the sequence of the target site in the chromosomal sequence, for example, the protospacer sequence, so that the DNA targeting segment of the guide RNA can base-pair with the target site or the protospacer.In some cases, the DNA targeting segment of the guide RNA can comprise 10 or about 10 nucleotides to 25 or about 10 or more nucleotides.For example, the region of base-pairing between the first region of the guide RNA and the target site in the chromosomal sequence can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25 nucleotides in length, or about that length.In some cases, the first region of the guide RNA can be 19, 20, or 21 nucleotides in length, or about that length.

[0098] The guide RNA may target a nucleic acid sequence of 20 or about that number of nucleotides. The target nucleic acid may be less than 20 or less than about that number of nucleotides. The target nucleic acid may be at least, or at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides. The target nucleic acid may be up to, or up to about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides. The target nucleic acid sequence may be 20 bases immediately 5' of the first nucleotide in the PAM, or about that number of bases. The guide RNA may target a nucleic acid sequence of a gene encoding a protein involved in the cannabinoid biosynthetic pathway. In some cases, the guiding polynucleic acid, such as an RNA, may bind to a genomic region about 1 base pair to about 20 base pairs away from the PAM. The guide can bind to a genomic region about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to about 20 base pairs away from the PAM.

[0099] Guide nucleic acid, e.g., guide RNA, may refer to a nucleic acid that can hybridize to another nucleic acid, e.g., a target nucleic acid or a protospacer in the genome of a cell. Guide nucleic acid may be RNA. Guide nucleic acid may be DNA. Guide nucleic acid may be programmed or designed to site-specifically bind to a sequence of a nucleic acid. Guide nucleic acid may include a polynucleotide strand and may be referred to as single guide nucleic acid. Guide nucleic acid may include two polynucleotide strands and may be referred to as double guide nucleic acid.

[0100] The guide nucleic acid may include one or more modifications that result in a nucleic acid with new or enhanced characteristics. The guide nucleic acid may include a nucleic acid affinity tag. The guide nucleic acid may include synthetic nucleotides, synthetic nucleotide analogs, nucleotide derivatives, and / or modified nucleotides. The guide nucleic acid may include a nucleotide sequence (e.g., a spacer) that can hybridize to a sequence in the target nucleic acid (e.g., a protospacer), for example, at or near the 5' or 3' end. The spacer of the guide nucleic acid can interact with the target nucleic acid in a sequence-specific manner by hybridization (i.e., base pairing). The spacer sequence can hybridize to the target nucleic acid located 5' or 3' of the protospacer adjacent motif (PAM). The length of the spacer sequence can be at least, or at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. The length of the spacer sequence can be up to, or up to about, 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides.

[0101] The guide RNA may include a dsRNA duplex region that forms a secondary structure. For example, the secondary structure formed by the guide RNA may include a stem (or hairpin) and a loop. The length of the loop and stem may vary. For example, the loop may be in the range of about 3 to about 10 nucleotides in length and the stem may be in the range of about 6 to about 20 base pairs in length. The stem may include one or more bulges of 1 to about 10 nucleotides in length. The total length of the second region may be in the range of about 16 to about 60 nucleotides in length. For example, the loop may be 4 nucleotides or about that long and the stem may be 12 base pairs or about that long. The dsRNA duplex region may include a protein-binding segment that can form a complex with an RNA-binding protein, such as an RNA-guided endonuclease, e.g., a Cas protein.

[0102] The guide RNA may include a tail region at the 5' or 3' end, which may be essentially single stranded. For example, the tail region may not be complementary to any chromosomal sequence in the cell of interest, and may not be complementary to the remainder of the guide RNA. Furthermore, the length of the tail region may vary. The tail region may be more than or about four nucleotides in length. For example, the length of the tail region may range from about 5 to about 60 nucleotides in length.

[0103] The guide RNA may be introduced into a cell or embryo as an RNA molecule. For example, the RNA molecule may be in vitro transcribed and / or chemically synthesized. The guide RNA may then be introduced into a cell or embryo as an RNA molecule. The guide RNA may be introduced into a cell or embryo in the form of a non-RNA nucleic acid molecule, such as a DNA molecule. For example, the DNA encoding the guide RNA may be operably linked to a promoter control sequence for expression of the guide RNA in the cell or embryo of interest. The RNA coding sequence may be operably linked to a promoter sequence recognized by RNA polymerase III (Pol III).

[0104] The DNA molecule encoding the guide RNA may be linear. The DNA molecule encoding the guide RNA may be circular. The DNA sequence encoding the guide RNA may be part of a vector. Some examples of vectors may include plasmid vectors, phagemids, cosmids, artificial / minichromosomes, transposons, and viral vectors. For example, the DNA encoding the RNA-guided endonuclease is present in a plasmid vector. Other non-limiting examples of suitable plasmid vectors include pUC, pBR322, pET, pBluescript, and variants thereof. In addition, the vector may include additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), selectable marker sequences (e.g., antibiotic resistance genes), origins of replication, etc.

[0105] When both the RNA-guided endonuclease and the guide RNA are introduced into a cell as DNA molecules, each can be part of a separate molecule (e.g., one vector containing the fusion protein coding sequence, and a second vector containing the guide RNA coding sequence), or both can be part of the same molecule (e.g., one vector containing the coding (and regulatory) sequences for both the fusion protein and the guide RNA).

[0106] Cas protein, such as Cas9 protein, or any derivative thereof, may be pre-complexed with guide RNA to form a ribonucleoprotein (RNP) complex. RNP complex may be introduced into plant cells. Introduction of RNP complex may be timed. Cells may be synchronized with other cells in the G1, S, and / or M phases of the cell cycle. RNP complex may be delivered at a cell cycle that enhances HDR. RNP complex may promote homologous recombination repair.

[0107] The guide RNA may be modified. The modification may include chemical changes, synthetic modifications, addition of nucleotides, and / or subtraction of nucleotides. The modification may also enhance CRISPR genome engineering. The modification may change the chirality of the gRNA. In some cases, the chirality may be homogeneous or stereochemically pure after modification. The guide RNA may be synthetic. The synthetic guide RNA may enhance CRISPR genome engineering. The guide RNA may be truncated. The truncation may be used to reduce undesired off-target mutagenesis. The truncation may include any number of nucleotide deletions. For example, the truncation may include 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more nucleotides. The guide RNA may include a target complementarity region of any length. For example, the target complementarity region may be less than 20 nucleotides in length. The target complementarity region may be more than 20 nucleotides in length. The target complementarity region may target about 5 bp to about 20 bp immediately adjacent to the PAM sequence. The target complementarity region may target about 13 bp immediately adjacent to the PAM sequence. The polynucleic acids described herein may be modified. Modifications may be made at any position of the polynucleic acid. More than one modification may be made to a single polynucleic acid. The polynucleic acid may be subjected to quality control after modification. In some cases, quality control may include PAGE, HPLC, MS, or any combination thereof. Modifications may be substitutions, insertions, deletions, chemical modifications, physical modifications, stabilization, purification, or any combination thereof. The polynucleic acid may be a 5' adenylate, a 5' guanosine-triphosphate cap, a 5' N-terminated nucleotide ... 7-Methylguanosine-triphosphate cap, 5'triphosphate cap, 3'phosphate, 3'thiophosphate, 5'phosphate, 5'thiophosphate, Cis-Syn thymidine dimer, trimer, C12 spacer, C3 spacer, C6 spacer, d spacer, PC spacer, r spacer, spacer 18, spacer 9, 3'-3' modified, 5'-5' modified, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-biotin, double biotin, PC biotin, psoralen C2, psoralen C6, TINA, 3'DABCYL, black hole quencher 1, black hole quencher 2, DABCYL SE, dT-DABCYL, IRDye It may also be modified with QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linkers, thiol linkers, 2' deoxyribonucleoside analogs purines, 2' deoxyribonucleoside analogs pyrimidines, ribonucleoside analogs, 2'-0-methylribonucleoside analogs, sugar modified analogs, wobble / universal bases, fluorescent dye labels, 2' fluoro RNA, 2' O-methyl RNA, methyl phosphonates, phosphodiester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, or any combination thereof. In some cases, the modifications may be permanent. In other cases, the modifications may be transient. In some cases, multiple modifications are made to the polynucleic acid. The modification of polynucleic acid may change the physiochemical properties of nucleotides, such as their higher order structure, polarity, hydrophobicity, chemical reactivity, base pairing interaction, or any combination thereof. In some embodiments, gRNA may be modified. In some cases, the modification is 5' end, 3' end, 5' end to 3' end, single base modification, 2'-ribose modification, or any combination thereof. The modification may be selected from the group consisting of base substitution, insertion, deletion, chemical modification, physical modification, stabilization, purification, and any combination thereof. In some cases, the modification is a chemical modification.

[0108] In some cases, the modification is the addition of 2-O-methyl 3 phosphorothioate, represented as "m". The phosphothioate backbone may be represented as "(ps)". The addition of 2-O-methyl 3 phosphorothioate may be made to 1 base to 150 bases. The addition of 2-O-methyl 3 phosphorothioate may be made to 1 base to 4 bases. The addition of 2-O-methyl 3 phosphorothioate may be made to 2 bases. The addition of 2-O-methyl 3 phosphorothioate may be made to 4 bases. The modification may be a truncation. The truncation may be a truncation of 5 bases. In some cases, the modification may be at the C-terminal and N-terminal nucleotides.

[0109] The modification may be a phosphorothioate substitution. In some cases, natural phosphodiester bonds may be subject to rapid degradation by cellular nucleases, and modification of internucleotide linkages using phosphorothioate (PS) bond substitutions may be more stable to hydrolysis by cellular degradation. The modification may increase stability in polynucleic acids. The modification may also enhance biological activity. In some cases, phosphorothioate-enhanced RNA polynucleic acids may inhibit RNase A, RNase T1, calf serum nuclease, or any combination thereof. These properties may allow for the use of PS-RNA polynucleic acids to be used in applications where there is a high probability of exposure to nucleases in vivo or in vitro. For example, phosphorothioate (PS) bonds may be introduced between the last 3-5 nucleotides at the 5' or 3' end of the polynucleic acid, which may inhibit exonuclease degradation. In some cases, phosphorothioate bonds may be added throughout the polynucleic acid to reduce attack by endonucleases.

[0110] In another embodiment, genetically modifying a fungus includes introducing into the fungus, or a cell thereof, (i) at least one RNA-guided endonuclease comprising at least one nuclear localization signal, or a 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, such as a barcode, to increase tryptamine-derived substances, such as dimethyltryptamine, psilocybin, or psilocin, and and culturing the bacterium or cells thereof, wherein each guide RNA directs an RNA-guided endonuclease to a target site within a chromosomal sequence, where the RNA-guided endonuclease introduces a double-stranded break at the target site and the double-stranded break is repaired by a DNA repair process such that the chromosomal sequence is modified, where the target site is located in any of genes encoding methyltransferases, hydroxylases, monooxygenases, kinases, decarboxylases, putative transcriptional regulators, and putative transporters, and where the chromosomal modification disrupts or prevents transcription and / or translation of said genes.

[0111] In some cases, GUIDE-Seq analysis can be performed to determine the specificity of the engineered guide RNA. The general mechanism and protocol of GUIDE-Seq profiling of off-target cleavage by CRISPR system nucleases is described in Tsai, S. et al., “GUIDE-Seq enables genome-wide profiling of off-target cleavage by CRISPR system nucleases,” Nature, 33:187-197 (2015). To assess off-target frequency by next generation sequencing, cells can be transfected with Cas9 mRNA and guiding RNA. Approximately 72 hours after transfection, genomic DNA can be isolated from transfected cells and PCR amplified at potential off-target sites. Potential off-target sites can be predicted using the Wellcome Trust Sanger Institute Genome Editing database (WGE) algorithm. Candidate off-target sites can be selected based on sequence homology with the on-target site. In some cases, sites with about 4 or less mismatches between the gRNA and the genomic site can be utilized. For each candidate off-target site, two primer pairs can be designed. PCR amplicons can be obtained from both untreated (control) and Cas9 / gRNA-treated cells. The PCR amplicons can be pooled. NGS libraries can be prepared using the TruSeq Nano DNA Library Preparation Kit (Illumina). Samples can be analyzed on an Illumina HiSeq machine using a 250 bp paired-end workflow. In some cases, approximately 40 million mappable NGS reads can be obtained per gRNA library. This can be considered as equivalent to an average number of approximately 450,000 reads for each candidate off-target site of the gRNA. In some cases, detection of CRISPR-mediated disruptions can be as low as 0.1% frequency at any genomic locus.

[0112] Using computational prediction, candidate gRNAs that are likely to be the safest choice for the gene to be targeted can be selected. Candidate gRNAs can then be empirically tested using a focused approach driven by computational prediction of potential off-target sites. In some cases, gRNA off-target safety assessment can use next-generation deep sequencing approaches to analyze potential off-target sites predicted by CRISPR design tools for each gRNA. In some cases, gRNAs with less than 3 mismatches with any sequence in the genome (except for the perfectly matched intended target) can be selected. In some cases, gRNAs with less than 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 mismatch(es) with any sequence in the genome can be selected. In some cases, computer systems or software can be utilized that provide recommendations for candidate gRNAs with predictions of low off-target potential.

[0113] In some cases, potential off-target sites may be identified by at least one of GUIDE-Seq and targeted PCR amplification, and next-generation sequencing. Additionally, modified cells, such as cells treated with Cas9 / gRNA, may be subjected to karyotyping to identify chromosomal rearrangements or translocations.

[0114] The gRNA may be introduced at any functional concentration. For example, the gRNA may be introduced into the cell at 10 micrograms. In other cases, the gRNA may be introduced at 0.5 micrograms to 100 micrograms. The gRNA may be introduced at 0.5 to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 micrograms.

[0115] The guiding polynucleic acid may have any base frequency. For example, the guiding polynucleic acid may have 29 A, 17 C, 23 G, 23 U, 3 mG, 1 mC, and 4 mU. The guiding polynucleic acid may have about 1 to about 100 nucleotides. The guiding polynucleic acid may have about 1 to 30 single polynucleotides. The guiding polynucleic acid may have about 1 to 10, 10 to 20, or 20 to 30 single nucleotides.

[0116] The guiding polynucleic acid may be tested for identity and potency prior to use. For example, identity and potency may be determined using at least one of spectrophotometric analysis, RNA agarose gel analysis, LC-MS, endotoxin analysis, and sterility testing. In some cases, identity testing may determine a level acceptable for clinical / therapeutic use. For example, an acceptable spectrophotometric analysis result may be 14±2 μL / vial at 5.0±0.5 mg / mL. An acceptable spectrophotometric analysis result may be about 10-20±2 μL / vial at 5.0±0.5 mg / mL or about 10-20±2 μL / vial at about 3.0-7.0±0.5 mg / mL. An acceptable clinical / therapeutic size of the guiding polynucleic acid may be about 100 bases. A clinical / therapeutic size of the guiding polynucleic acid may be about 5 bases to about 150 bases. The clinical / therapeutic size of the guiding polynucleic acid may be from about 20 bases to about 150 bases. The clinical / therapeutic size of the guiding polynucleic acid may be from about 40 bases to about 150 bases. The clinical / therapeutic size of the guiding polynucleic acid may be from about 60 bases to about 150 bases. The clinical / therapeutic size of the guiding polynucleic acid may be from about 80 bases to about 150 bases. The clinical / therapeutic size of the guiding polynucleic acid may be from about 100 bases to about 150 bases. The clinical / therapeutic size of the guiding polynucleic acid may be from about 110 bases to about 150 bases. The clinical / therapeutic size of the guiding polynucleic acid may be from about 120 bases to about 150 bases.

[0117] In some cases, the mass of the guiding polynucleic acid may be determined. The mass may be determined by LC-MS assay. The mass may be about 32,461.0 amu. The guiding polynucleic acid may have a mass of about 30,000 amu to about 50,000 amu. The guiding polynucleic acid may have a mass of about 30,000 amu to 40,000 amu, about 40,000 amu to about 50,000 amu. The mass may be that of a sodium salt of the guiding polynucleic acid.

[0118] In some cases, the endotoxin level of the guiding polynucleic acid may be determined. The clinically / therapeutically acceptable level of endotoxin may be less than 3 EU / mL. The clinically / therapeutically acceptable level of endotoxin may be less than 2 EU / mL. The clinically / therapeutically acceptable level of endotoxin may be less than 1 EU / mL. The clinically / therapeutically acceptable level of endotoxin may be less than 0.5 EU / mL.

[0119] In some cases, the guiding polynucleic acid may be subjected to a sterility test. The clinically / therapeutically acceptable level of sterility test may be represented by 0 or no growth in culture. The clinically / therapeutically acceptable level of sterility test may be less than 0.5% growth.

[0120] The guiding polynucleic acid can be assembled by various methods, for example, by automated solid-phase synthesis. The polynucleic acid can be constructed using standard solid-phase DNA / RNA synthesis. The polynucleic acid can also be constructed using synthetic procedures. The polynucleic acid can also be synthesized either manually or in a fully automated manner. In some cases, the synthetic procedure can include first converting the 5'-hydroxyl oligonucleotide to the corresponding 5'-H-phosphonate monoester, followed by oxidation in the presence of imidazole to the activated 5'-phosphorimidazolidate, and finally reacting with pyrophosphate on a solid support. This procedure can include a purification step after synthesis, such as PAGE, HPLC, MS, or any combination thereof.

[0121] Donor sequence In some cases, the donor sequence can be introduced into the genome of a fungus, yeast, plant, or part thereof. In some cases, the donor is inserted into a genome break. In some embodiments, the donor contains homology with a sequence flanking the target sequence. Methods of introducing the donor sequence are known to those skilled in the art, but can include the use of homology arms. For example, the donor sequence can contain homology arms with at least a portion of the genome that contains the genome break. In some cases, the donor sequence is randomly inserted into the genome of cannabis or the genome of a cannabis plant cell.

[0122] In some cases, the donor sequence can be introduced in a site-directed manner using homologous recombination. Homologous recombination allows for site-specific modification of endogenous genes, thus allowing inherited or acquired mutations to be corrected and / or new changes to be engineered into the genome. Homologous recombination and site-directed integration in plants are discussed, for example, in U.S. Patent Nos. 5,451,513, 5,501,967, and 5,527,695.

[0123] In some embodiments, the donor sequence comprises a promoter sequence. Increased expression of the designed gene product can be achieved by adjusting the promoter region or by inserting a stronger promoter upstream of the desired gene sequence to synthetically increase expression. In some embodiments, promoters such as 35s and Ubi10 that are highly functional in Arabidopsis and other plants can be introduced. In some cases, promoters that are highly functional in cannabis and / or hemp are introduced.

[0124] In some cases, the barcode may include a non-native sequence. In some aspects, the barcode includes a natural sequence. In some aspects, the barcode may be utilized to allow identification of the transgenic organism via genotyping. In some aspects, the donor sequence may be a marker. Selectable marker genes may include, for example, photosynthesis (atpB, tscA, psaA / B, petB, petA, ycf3, rpoA, rbcL), antibiotic resistance (rrnS, rrnL, aadA, nptII, aphA-6), herbicide resistance (psbA, bar, AHAS(ALS), EPSPS, HPPD, sul) and metabolism (BADH, codA, ARG8, ASA2) genes. The bacterial sul gene has herbicidal sulfonamide-insensitive dihydropteroate synthase activity and may be used as a selectable marker when the protein product is targeted to plant mitochondria (U.S. Pat. No. 6,121,513). In some embodiments, sequences encoding markers can be integrated into genetically modified cells or organisms, such as fungi, yeast, or plants described herein. In some embodiments, the integrated sequences encoding markers can then be removed from the transformed genome. Removal of the sequences encoding markers can be facilitated by the presence of direct repeats before and after the region encoding the marker. Removal of the sequences encoding markers can occur via the organelle's endogenous homologous recombination system or by the use of site-specific recombinase systems such as cre-lox or FLP / FRT.

[0125] In some cases, a marker may refer to a detectable label, such as, for example, a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme. Examples of detectable markers include, but are not limited to, fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotinyl groups, and predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).

[0126] Selectable or detectable markers typically comprise a DNA segment that allows a cell, or a molecule marked with a "tag" within a cell of interest, to be identified, often under specific conditions. Such markers may code for an activity selected from, but not limited to, the production of RNA, peptides, or proteins, or the marker may provide binding sites for RNA, peptides, proteins, inorganic and organic compounds or complexes, and the like. By way of example, selectable markers include DNA segments containing restriction enzyme cut sites, DNA segments containing fluorescent probes, DNA segments encoding products that confer resistance to otherwise toxic compounds, including antibiotics such as spectinomycin, ampicillin, kanamycin, tetracycline, BASTA, neomycin-phosphotransferase II (NEO) and hygromycin-phosphotransferase (HPT), DNA segments encoding products that the desired plant target cells do not have under native conditions, such as tRNA genes, auxotrophic markers, etc., products that can be easily identified, particularly optically observable markers, such as phenotypic markers, e.g., -galactosidase, GUS, fluorescent proteins such as green fluorescent protein (GFP) and other fluorescent proteins such as blue (CFP), yellow (YFP) or red (RFP) fluorescent proteins. , and DNA segments encoding surface proteins (wherein fluorescent proteins exhibiting high fluorescence intensity are of particular interest when complex plant target structures or plant materials or plants comprising multiple types of tissues or cells, instead of a single cell, are to be analyzed, as these proteins can be identified in deeper tissue layers as well), new primer sites for PCR, records of DNA sequences that cannot be modified in accordance with the present disclosure by restriction endonucleases or other DNA modifying enzymes or effector domains, DNA sequences used for specific modifications, such as epigenetic modifications, for example methylation, as well as DNA sequences with PAM motifs that can be identified by a suitable CRISPR system in accordance with the present disclosure, and also DNA sequences without PAM motifs as may naturally occur in endogenous plant genomic sequences.

[0127] In one embodiment, the donor comprises a selectable, screenable, or scorable marker gene, or a portion thereof. In some cases, the marker serves as a selection or screening device that can function in the regenerable genetically modified organism to produce a compound that confers resistance to an otherwise toxic compound to the organism's tissues. Genes of interest used as selectable, screenable, or scorable markers include, among others, gus, green fluorescent protein (gfp), luciferase (lux), kanamycin (Dekeyser et al., 2003). Genes conferring resistance to antibiotics such as spectinomycin (e.g., spectinomycin aminoglycoside adenyltransferase (aadA)), glyphosate (e.g., 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS); glyphosate oxidoreductase (GOX); glyphosate decarboxylase; or glyphosate N-acetyltransferase (GAT)), dalapon (e.g., dehI encoding a 2,2-dichloropropionic acid dehalogenase conferring resistance to 2,2-dichloropropionic acid), bromoxynil (haloarylnitrilase (Bxn) conferring resistance to bromoxynil), sulfonyl herbicides (e.g., sulfonylureas, imidazolinones, triazolopyrimidines, pyrim ... These include, but are not limited to, genes encoding enzymes that confer tolerance to herbicides, such as acetohydroxyacid synthase or acetolactate synthase; ALS, encoding a GST-II, conferring tolerance to acetolactate synthase inhibitors such as midyloxybenzoic acid and phthalide, bialaphos or phosphinothricin or derivatives (e.g., phosphinothricin acetyltransferase (bar), conferring tolerance to phosphinothricin or glufosinate, atrazine (encoding a GST-III), dicamba (dicamba monooxygenase), or sethoxydim (a modified acetyl-coenzyme A carboxylase, conferring tolerance to cyclohexanedione (sethoxydim) and aryloxyphenoxypropionates (haloxyfop)).Other selection procedures can also be implemented, including positive selection mechanisms (e.g., use of the manA gene of E. coli, which allows growth in the presence of mannose), and double selection (e.g., simultaneous use of 75-100 ppm spectinomycin and 3-10 ppm glufosinate, or 75 ppm spectinomycin and 0.2-0.25 ppm dicamba). The use of spectinomycin at a concentration of about 25-1000 ppm, e.g., about 150 ppm, can also be envisioned. In certain embodiments, the detectable marker can be attached by spacer arms of various lengths to reduce potential steric hindrance.

[0128] In some cases, the donor polynucleotide comprises homology with a sequence flanking the target sequence. In some cases, the donor polynucleotide introduces a stop codon into a gene provided herein, for example, to block synthesis of tryptamine other than psilocybin. In some cases, the donor polynucleotide comprises a barcode, a reporter, or a selection marker.

[0129] Transformation Suitable transformation techniques may include, but are not limited to, electroporation of fungal protoplasts, liposome-mediated transformation, polyethylene glycol (PEG)-mediated transformation, transformation using viruses, microinjection of cells, biolistic bombardment of cells, vacuum infiltration, and Agrobacterium tumeficiens-mediated transformation. Transformation refers to the introduction of a nucleotide sequence into a cell in a manner that results in stable or transient expression of the sequence.

[0130] After transformation, the fungus or other organism can be selected using a dominant selectable marker incorporated into the transformation vector. In certain embodiments, such a marker confers resistance to an antibiotic or herbicide to the transformed fungus or other organism, and selection of the transformants can be achieved by exposing the fungus and other organism to an appropriate concentration of the antibiotic or herbicide. After selection and growth to maturity of the transformed fungus or other organism, fungus and other organisms that exhibit the altered trait are identified. The altered trait can be any of the traits described above. Furthermore, the expression level or activity of the polypeptide or polynucleotide of the present invention can be determined by analysis of mRNA expression using Northern blot, RT-PCR, RNA seq or microarray, or analysis of protein expression using immunoblot or Western blot or gel shift assay.

[0131] Methods suitable for transformation of fungi or other cells for use in the present invention are believed to include virtually any method by which DNA can be introduced into cells, such as PEG-mediated transformation of protoplasts, desiccation / inhibition-mediated DNA uptake, electroporation, agitation with silicon carbide fibers, Agrobacterium-mediated transformation, and direct delivery of DNA by acceleration of DNA-coated particles, etc. By application of techniques such as these, cells of virtually any fungal species can be stably transformed and these cells can be developed into transgenic fungi.

[0132] Agrobacterium-mediated transformation Agrobacterium-mediated transfer is a widely applicable system for introducing genes into fungal cells, since it can introduce DNA into the entire fungal tissue, thereby avoiding the need to regenerate intact fungi from protoplasts.The use of Agrobacterium-mediated fungal integration vectors for introducing DNA into fungal cells, including, for example, CRISPR systems or donor sequences, is well known in the art.

[0133] In addition, Agrobacterium-mediated transformation can be efficient in other organisms, such as dicotyledonous plants, and can be used to transform dicotyledonous plants, including Arabidopsis, tobacco, tomato, alfalfa, and potato. In fact, Agrobacterium-mediated transformation has been routinely used in dicotyledonous plants for many years. In some cases, Agrobacterium-mediated transformation can be used in monocotyledonous plants. For example, Agrobacterium-mediated transformation techniques are currently being applied to rice, wheat, barley, alfalfa, and maize.

[0134] Modern Agrobacterium transformation vectors are capable of replicating in E. coli as well as Agrobacterium, allowing for convenient manipulation as described above. Furthermore, recent technological advances in vectors for Agrobacterium-mediated gene transfer have improved the placement of genes and restriction sites in the vector, facilitating the construction of vectors capable of expressing a variety of polypeptide-encoding genes. In some embodiments, the vectors can have convenient multilinker regions flanked by promoters and polyadenylation sites for direct expression of inserted polypeptide-encoding genes, making them suitable for the purposes described herein. Furthermore, Agrobacterium containing both armed and disarmed Ti genes can be used for transformation.

[0135] Electroporation In some embodiments, fungi, yeast, plants, or cells thereof can be modified using electroporation. To carry out transformation by electroporation, fragile tissues such as suspension cultures of cells or embryogenic callus can be used, or alternatively, immature embryos or other organized tissues can be directly transformed. In this technique, the cell walls of selected cells are partially degraded by exposure to pectin-degrading enzymes (pectolyases) or mechanically wounded in a controlled manner.

[0136] Any transfection system may be utilized. In some cases, the Neon transfection system may be utilized. The Neon system may be a three-component electroporation device that includes a central control module, an electroporation chamber that may be connected to the central control module by a 3-foot long electrical cord, and a dedicated pipette. In some cases, the dedicated pipette may include a replaceable and / or disposable sterile tip. In some cases, the electroporation chamber may include a replaceable / disposable sterile electroporation cuvette. In some cases, the standard electroporation buffer provided by the manufacturer of the system, such as the Neon system, may be replaced with GMP certified solutions and buffers. In some cases, the standard electroporation buffer may be replaced with GMP grade phosphate buffered saline (PBS). Prior to the start of electroporation of the sample, a self-diagnostic system check may be performed on the control module to ensure that the Neon system is functioning properly. In some cases, the transfection may be performed in a class 1,000 biosafety cabinet in a class 10,000 clean room in a cGMP facility. In some cases, the electroporation pulse voltage may be altered to optimize transfection efficiency and / or cell viability. In some cases, the electroporation pulse width can be altered to optimize transfection efficiency and / or cell viability. In some cases, the number of electroporation pulses can be altered to optimize transfection efficiency and / or cell viability. In some cases, the electroporation can include a single pulse. In some cases, the electroporation can include more than one pulse. In some cases, the electroporation can include 2 pulses, 3 pulses, 4 pulses, 5 pulses 6 pulses, 7 pulses, 8 pulses, 9 pulses, or 10 or more pulses.

[0137] In some embodiments, fungal and / or plant protoplasts may be used for electroporation transformation.

[0138] Microprojectile bombardment Another method of delivering transforming DNA segments to fungal cells and cells derived from other organisms according to the present invention is biolistic bombardment. In this method, particles can be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those made of tungsten, platinum, and preferably gold. It is contemplated that in some cases DNA deposition on metal particles is not necessary to deliver DNA to recipient cells using biolistic bombardment. However, it is contemplated that particles may contain DNA rather than be coated with DNA. In some embodiments, DNA-coated particles may increase the level of DNA delivery via particle bombardment. For bombardment, cells in suspension are concentrated on a filter or solid culture medium. Alternatively, immature embryos or other target cells may be placed on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate.

[0139] An illustrative embodiment of a method for delivering DNA into fungal cells by acceleration is a biolistic particle delivery system that can propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, to a filter surface covered with suspension-cultured monocotyledonous plant cells. The screen disperses the particles so that they are not delivered to the recipient cells as large aggregates.

[0140] Other transformation methods Additional transformation methods include, but are not limited to, calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments.

[0141] To transform fungi that cannot be successfully regenerated from protoplasts, other methods of introducing DNA into intact cells or tissues can be utilized. For example, regeneration of plants from immature embryos or explants can be performed as described above. Silicon carbide fiber-mediated transformation can also be used with or without protoplasting. Transformation using this technique can be achieved by stirring silicon carbide fibers with cells in a DNA solution. When the cells are punctured, the DNA passively enters.

[0142] In some cases, the starting cell density for genome editing can be altered to optimize editing efficiency and / or cell viability. In some cases, the starting cell density for genome editing is about 1×10 5 In some cases, the starting cell density for electroporation is at least about 1×10 5 Cells, at least about 2 x 10 5 Cells, at least about 3 x 10 5 Cells, at least about 4 x 10 5 Cells, at least about 5 x 10 5 Cells, at least about 6 x 10 5 Cells, at least about 7 x 10 5 Cells, at least about 8 x 10 5 Cells, at least about 9 x 10 5 Cells, at least about 1 x 10 6 Cells, at least about 1.5 x 10 6 Cells, at least about 2 x 10 6 Cells, at least about 2.5 x 10 6 Cells, at least about 3 x 10 6 Cells, at least about 3.5 x 10 6 Cells, at least about 4 x 10 6 Cells, at least about 4.5 x 10 6 Cells, at least about 5 x 10 6 Cells, at least about 5.5 x 10 6 Cells, at least about 6 x 10 6 Cells, at least about 6.5 x 10 6 Cells, at least about 7 x 10 6 Cells, at least about 7.5 x 106 Cells, at least about 8 x 10 6 Cells, at least about 8.5 x 10 6 Cells, at least about 9 x 10 6 Cells, at least about 9.5 x 10 6 Cells, at least about 1 x 10 7 Cells, at least about 1.2 x 10 7 Cells, at least about 1.4 x 10 7 Cells, at least about 1.6 x 10 7 Cells, at least about 1.8 x 10 7 Cells, at least about 2 x 10 7 Cells, at least about 2.2 x 10 7 Cells, at least about 2.4 x 10 7 Cells, at least about 2.6 x 10 7 Cells, at least about 2.8 x 10 7 Cells, at least about 3 x 10 7 Cells, at least about 3.2 x 10 7 Cells, at least about 3.4 x 10 7 Cells, at least about 3.6 x 10 7 Cells, at least about 3.8 x 10 7 Cells, at least about 4 x 10 7 Cells, at least about 4.2 x 10 7 Cells, at least about 4.4 x 10 7 Cells, at least about 4.6 x 10 7 Cells, at least about 4.8 x 10 7 cells, or at least about 5×10 7 It may be a cell.

[0143] The efficiency of genome disruption of a plant, or any portion thereof, including but not limited to cells, by any of the nucleic acid delivery platforms described herein can result in about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or up to about 100% disruption of genes or portions thereof as measured by nucleic acid or protein analysis.

[0144] Biological breeding In some embodiments, the fungi, yeast, or plants of the present disclosure can be used to create new plant varieties. In some embodiments, the plants are used to generate new, unique, and superior varieties or hybrids with desired phenotypes. In some embodiments, selection methods, such as molecular marker-assisted selection, can be combined with breeding methods to accelerate the process. In some embodiments, the method includes (i) crossing any organism provided herein containing an expression cassette as a donor with a recipient organism system to generate a FI population, and (ii) selecting progeny that have the expression cassette. Optionally, the progeny can be further selected by testing the expression of the gene of interest. In some embodiments, the complete chromosome of the donor organism is transmitted. For example, a transgenic organism with an expression cassette can act as a male or female parent in cross-pollination to receive a transgene from a donor to generate progeny, which generates progeny that have the expression cassette. The method of generating an organism with an expression cassette can also use protoplast fusion to transfer the transgene from the donor to the recipient. Protoplast fusion is the creation of a single binucleate or multinucleate cell by induced or spontaneous combination, such as somatic cell hybridization, of two or more protoplasts (cells whose cell walls have been removed by enzymatic treatment). The fused cells, which can be obtained from species that cannot naturally interbreed, are tissue cultured into hybrid organisms that exhibit the desired combination of traits. More specifically, a first protoplast can be obtained from an organism that carries an expression cassette. A second protoplast can be obtained from a second organism, optionally from another species or variety, or from the same species or variety, that contains commercially desirable traits, such as, but not limited to, disease resistance, insect resistance, etc. The protoplasts are then fused using conventional protoplast fusion procedures known in the art to create hybrids. Alternatively, embryo rescue can be used to transfer the expression cassette from the donor to the recipient. Embryo rescue can be used as a procedure to isolate the embryo and tissue culture it.

[0145] In some cases, population improvement methods may be utilized. Population improvement methods fall naturally into two groups: those based purely on phenotypic selection, usually referred to as population selection, and those based on selection by progeny testing. Interpopulation improvement utilizes the concept of open breeding populations, which transfer genes from one population to another. Selection can be applied to improve one (or possibly both) population(s) by isolating plants containing desirable traits from both origins.

[0146] In another embodiment, mass selection can be used. In mass selection, desirable individual plants are selected, collected, and the seeds are combined without progeny testing to produce the next generation. Since selection is based only on the mother and pollination is not controlled, mass selection is essentially a form of random breeding with selection. As described herein, the purpose of mass selection is to increase the proportion of superior genotypes in the population. Although mass selection may be used, progeny testing is generally preferred in multi-crossing because it is operationally simple and has a clear relevance to its purpose, i.e., utilizing the overall combined ability in synthesis.

[0147] In some embodiments, breeding may utilize molecular markers. The molecular markers are designed and created based on the genome of the plant of the present application. In some embodiments, the molecular markers are selected from isozyme electrophoresis, restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), arbitrarily primed polymerase chain reaction (AP-PCR), DNA amplification fingerprinting (DAF), sequence specific amplified region (SCAR), amplified fragment length polymorphism (AFLP), and simple sequence repeats (SSR), also called microsatellites, and the like. Methods for developing molecular markers and their uses are described in Avise (Molecular markers, natural history, and evolution, Publisher: Sinauer Associates, 2004, ISBN 0878930418, 9780878930418), Snvastava et al. (Plant biotechnology and molecular markers, Publisher: Springer, 2004, ISBN 1402019114, 9781402019111), and Vienne (Molecular markers in plant genetics and biotechnology, Publisher: Science Publishers, 2003), each of which is incorporated herein by reference in its entirety for all purposes. Molecular markers can be used in molecular marker-assisted breeding. Methods of generating transgenic fungi may also be provided herein. In some embodiments, the methods provided herein may include (a) contacting a fungal cell with an endonuclease or a polypeptide encoding an endonuclease. In some cases, the endonuclease introduces a genetic modification into the genome of the fungal cell, increasing the amount of one of Formulas I-IV, their derivatives or analogs, compared to the amount of the same compound in a comparable control without the genetic modification. In some embodiments, the method may further include culturing the fungal cell that has been genetically modified as described above to generate a transgenic fungus. Methods of making transgenic fungi may include electroporation, Agrobacterium-mediated transformation, biolistic particle bombardment, or protoplast transformation. In some embodiments, the method may further include culturing the fungal cell to generate a fungus.

[0148] In some embodiments, also provided herein is a method for producing a transgenic plant, comprising contacting a plant cell with an endonuclease or a polypeptide encoding an endonuclease, the endonuclease may introduce a genetic modification that increases the amount of psilocybin, psilocin, or dimethyltryptamine (DMT), derivatives, or analogs thereof, compared to the amount of the same compound in a comparable control without the genetic modification.

[0149] In some aspects, also provided herein are methods of producing transgenic animals comprising contacting an animal cell with an endonuclease or a polypeptide encoding an endonuclease, the endonuclease may introduce a genetic modification that increases the amount of psilocybin, psilocin, or dimethyltryptamine (DMT), derivatives, or analogs thereof, compared to the amount of the same compound in a comparable control without the genetic modification.

[0150] In some embodiments, also provided herein are methods of producing transgenic insects, comprising contacting an insect cell with an endonuclease or a polypeptide encoding an endonuclease, the endonuclease may introduce a genetic modification that increases the amount of psilocybin, psilocin, or dimethyltryptamine (DMT), derivatives, or analogs thereof, compared to the amount of the same compound in a comparable control without the genetic modification.

[0151] In some aspects, also provided herein are methods of producing transgenic yeast, comprising contacting a yeast cell with an endonuclease or a polypeptide encoding an endonuclease, the endonuclease may introduce a genetic modification that increases the amount of psilocybin, psilocin, or dimethyltryptamine (DMT), a derivative, or analog thereof, compared to the amount of the same compound in a comparable control without the genetic modification.

[0152] In some embodiments, also provided herein is a method of producing a transgenic E. coli comprising contacting an E. coli cell with an endonuclease or a polypeptide encoding an endonuclease, the endonuclease may introduce a genetic modification that increases the amount of psilocybin, psilocin, or dimethyltryptamine (DMT), derivatives, or analogs thereof, compared to the amount of the same compound in a comparable control without the genetic modification.

[0153] Methods involving modification of the fungal cell genome may result in 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or up to about 80% more genomic DNA, as measured by dry weight, in transgenic fungi compared to comparable controls without genome modification. [ka] Additionally, methods that include modifications can result in about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 100%, or up to about 200% more cell proliferation, as measured by dry weight in the transgenic compared to comparable controls without the modifications. [ka] Additionally, methods involving modifications can also result in about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 100%, or up to about 200% more psilocybin or psilocin, as measured by dry weight in the transgenics, compared to comparable controls without the modifications.

[0154] As used herein, a genetically modified cell comprising a disruption of a gene, the disruption of the gene being a signal to a compound in an equivalent control cell lacking said genetic modification. [ka] Also provided herein are genetically modified cells that increase the amount of the same compound compared to the amount of a derivative or analog thereof. Further provided herein are genetically modified cells that include a gene disruption, where the gene disruption increases the amount of the compound in a comparable control cell that does not have the genetic modification. [ka] Genetically modified cells may also be provided that increase the amount of the same compound compared to the amount of its derivative or analogue.Furthermore, genetically modified cells may also be provided herein that include gene disruption, which increases the amount of the same compound compared to the amount of psilocybin and / or psilocin, its derivative or analogue in a comparable control cell that does not have the genetic modification.Alternatively, the genetically modified cell is a plant cell, a fungal cell, a bacterial cell, an animal cell, or an insect cell.

[0155] Further provided herein is a composition comprising an endonuclease or a polynucleotide encoding said endonuclease capable of introducing a genetic modification that increases the amount of psilocybin or psilocin, a derivative or analogue thereof, compared to an equivalent control cell lacking said genetic modification.

[0156] Psilocybin synthesis transgene methods and compositions Methods for transforming mushrooms with psilocybin synthesis genes can be provided herein. In some embodiments, the coding sequences of the four main psilocybin synthesis genes are synthesized and cloned into an overexpression vector system pGWB5 under the control of the 35S promoter. In some embodiments, additional vectors are also created that contain different promoters to drive the expression of these genes (including Gpd, EF1a, and actin).

[0157] In some cases, pGWB5 will be used to transform basidiomycetes to test transformation efficiency and develop protocols. In some cases, transformations will include different Psi genes individually and in combination to observe the possibility of increased psilocybin production. In some cases, an all-in-one expression vector of the four Psi genes in tandem in a polycistronic vector will be generated and tested.

[0158] In some embodiments, Psilocybe cubensis is allowed to propagate and grow on different substrates to produce both mature mushroom fruiting bodies and mycelium. In some embodiments, tissue is extracted from the mushroom folds and transformed with the Psi gene by Agrobacterium-mediated transformation. In some embodiments, protoplasts are generated from the mycelium and PEG-mediated transformation of the Psi gene is performed along with Agrobacterium-mediated transformation of the mycelium. In some embodiments, Psilocybe cubensis is grown in PDA agar or barley-perlite compost at room temperature for 7 days. In some cases, mycelium and fruiting bodies are harvested and tissue extraction and cell isolation are performed prior to transformation.

[0159] In some embodiments, Psi gene overexpression is under the control of two unique promoters: the 35S promoter, a widely used plant overexpression promoter, and two fungal-specific overexpression promoters, GPD and CcDED1 (Table 4, Figure 3, Figure 4). [Table 4]

[0160] In some embodiments, the PsiD gene overexpression comprises a vector expressing the PsiD gene under the control of the 35S promoter (Table 5: SEQ ID NO: 18, 17,647 bp; Figure 3A). In some embodiments, the PsiH gene overexpression comprises a vector expressing the PsiH gene under the control of the 35S promoter (Table 5: SEQ ID NO: 17, 18,494 bp; Figure 3B). In some embodiments, the PsiK gene overexpression comprises a vector expressing the PsiK gene under the control of the 35S promoter (Table 5: SEQ ID NO: 16, 17,420 bp; Figure 3C). In some embodiments, the PsiM gene overexpression comprises a vector expressing the PsiM gene under the control of the 35S promoter (Table 5: SEQ ID NO: 15, 17,267 bp; Figure 3D).

[0161] In some embodiments, Psi gene overexpression comprises a vector expressing the Psi gene under the control of the GcDED1 promoter (Table 5: SEQ ID NO: 19, 9,462 bp; Figure 4A). In some embodiments, Psi gene overexpression comprises a vector expressing the Psi gene under the control of the GPD promoter (Table 5: SEQ ID NO: 20, 8,067 bp; Figure 4B).

[0162] Pharmaceutical and Nutraceutical Compositions and Methods Provided herein may be a pharmaceutical or nutraceutical composition comprising the genetically modified cells, organisms, fungi, or plants described herein, or an extract, derivative, or product thereof. Also provided herein may be a pharmaceutical or nutraceutical agent, a method of using the same, and a method of making a pharmaceutical or nutraceutical composition comprising the genetically modified cells, organisms, fungi, or plants described herein, or an extract or product thereof. Also provided herein are cells, organisms, or plants suitable for medicine and nutraceuticals, or an extract, derivative, or product thereof, described herein.

[0163] In some cases, the genetically modified cells, organisms, fungi, or plants described herein, or their extracts or products, can be used as pharmaceuticals or nutritional supplements. In some cases, compositions containing such pharmaceuticals or nutritional supplements can be used to treat or stabilize symptoms associated with conditions or conditions, such as depression, anxiety, post-traumatic stress, addiction, or side effects associated with cessation, such as smoking cessation, and psychological distress, including psychological distress associated with cancer. The specifically genetically modified cells, organisms, fungi, or plants described herein, or their extracts, derivatives, or products, can be used to alleviate various symptoms associated with mental disorders and conditions.

[0164] In some aspects, the cells, organisms, or plants described herein, or their extracts or products, can be used to treat certain conditions. For example, pain, nausea, weight loss, wasting, multiple sclerosis, allergies, infections, vasoconstriction, depression, migraine, hypertension, neuroprotection after stroke, as well as inhibition of tumor growth, inhibition of angiogenesis, and inhibition of metastasis, antioxidants, and neuroprotective agents. In some aspects, the cells, organisms, or plants described herein, or their extracts or products, can be used to treat additional conditions. For example, persistent muscle spasms including those characteristic of multiple sclerosis, severe arthritis, peripheral neuropathy, intractable pain, migraine headaches, terminal illness requiring end-of-life care, hydrocephalus with intractable headache, intractable headache syndromes, neuropathic facial pain, shingles, chronic non-malignant pain, causal pain, chronic inflammatory demyelinating polyneuropathy, bladder pain, myoclonus, post-concussion syndrome, residual limb pain, obstructive sleep apnea, traumatic brain injury (TBI), elevated intraocular pressure, opioid or opiate withdrawal symptoms, and / or loss of appetite.

[0165] In some cases, the cells, organisms, or plants described herein, or extracts or products thereof, may also contain other pharma- ceutically or nutraceutical relevant compounds and extracts, including flavonoids, monoamine oxidase inhibitors, and phytosterols (e.g., apigenin, quercetin, cannflavin A, beta-sitosterol, etc.).

[0166] In some cases, the extract or product may be subjected to methods including extraction that preserves psilociben, dimethyltryptamine, or psilocene. The extracts of the present disclosure are designed to create products for human or animal consumption via inhalation (via combustion, vaporization, and nebulization), oral absorption in the mouth, oral administration, and topical application delivery methods. The present disclosure teaches an optimized method of extracting the compounds of interest by extracting the dried post-harvest plant or fungus when it reaches 5, 10, or 15% moisture weight. The stems are usually still "cool" and "rubbery" from the occurrence of evaporation. This time frame (or if frozen at this point in the process) allows the extractor to minimize loss of active agents due to evaporation. Cool / slow drying is directly correlated with the preservation of essential oils. Thus, there is a direct correlation with EO loss in flowers that dry too quickly or in conditions that are too hot, or simply dry (less than 10% H2O). Chemical extraction of the cells, organisms, or plants described herein, or their extracts or products, can be accomplished with polar and non-polar solvents at various stages, at different pressures and temperatures, to selectively or comprehensively extract flavors, aromas, or other compounds of pharmacological value that are used individually or in combination in the formulation of the product. The extracts can be shaped and formed into single or multi-dose packages, such as dabs, pellets, and loads. Solvents used in the selective extraction of our varieties can include combinations or series of water, carbon dioxide, 1,1,1,2-tetrafluoroethane, butane, propane, ethanol, isopropyl alcohol, hexane, and limonene. Extracts of the present disclosure can also be combined with pure compounds of interest to the extract, such as cannabinoids or terpenes, to further enhance or modify the aroma, flavor, or pharmacology of the resulting formulation. In some embodiments, the extract is supplemented with terpenes or cannabinoids to compensate for the loss of these compounds during the extraction process.

[0167] In some aspects, the genetically modified organisms, derivatives, or extracts of the present disclosure may be used for vaporization, production of e-juice or tinctures for electronic cigarettes, or production of other ingestible products such as edibles, balms, or topical applications. In some aspects, the modified compositions provided herein may be used as supplements, such as dietary supplements. In some embodiments, the cells, organisms, or plants described herein, or their extracts or products, may be used to create edibles. Edible recipes may begin with the extraction of cannabinoids and terpenes, which are then used as ingredients in various edible recipes. Extraction methods for edibles include extraction into cooking oils, milk, cream, balms, flour, and butter. It is believed that lipid-rich extraction media / edibles facilitate absorption into the bloodstream. Lipids may be utilized as excipients in combination with various compositions provided herein. In other aspects, the compositions provided herein may include oral forms, transdermal forms, oil formulations, edibles or food matrices, aqueous dispersions, emulsions, solutions, suspensions, elixirs, gels, syrups, aerosols, mists, powders, tablets, electuaries, gels, lotions, pastes, formulation sticks, balms, creams, or ointments.

[0168] Also provided herein are kits that include the compositions provided herein. The kits may include packaging, instructions, and the various compositions provided herein. In some aspects, the kits may also include additional compositions used to generate the various plants and plant parts provided herein, such as pots, soil, fertilizer, water, and culture tools.

[0169] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. And, those skilled in the art will recognize numerous variations, changes, and substitutions without departing from the present disclosure. It is understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the present disclosure. It is intended that the following claims define the scope of the disclosure, and that methods and structures within the scope of these claims and their equivalents are covered thereby. EXAMPLES

[0170] Example 1: Method for overexpressing the Psi gene in Psliocybe Cubensis Step 1. Construct a psilocybin pathway expression vector.

[0171] A panel of expression vectors with various promoters of different strengths is constructed. Some promoters are specific to mushrooms, others are derived from high-expressing plant systems, etc. (Figure 5A). Then, Agrobacterium is generated from these expression vectors.

[0172] Step 2. Prepare mushroom material for transformation.

[0173] Protoplasts, conidia, ruffle tissue, and mycelium were isolated for transformation as shown in Examples 3-7. The choice of the appropriate protocol depends on the mushroom to be transformed. Here, protoplasts and ruffle tissue were isolated for extraction as exemplified in Examples 3-5 and FIG. 5B. Protoplasts were extracted from mycelium as shown in Example 4. The method of transformation of ruffle tissue using Agrobacterium co-culture is shown in Example 6.

[0174] Step 3. Transformation.

[0175] The cultured protoplasts from step 2 were transfected with the plasmid DNA from step 1 using various protocols. See Examples 3-5. Additionally, the fold tissue from step 2 was transformed with the Agrobacterium from step 1 using various protocols. See Examples 6-7. Transformants that have integrated the plasmid DNA or Agrobacterium are selected, as shown in Figure 5C.

[0176] Step 4. Play.

[0177] Regenerate adult mushrooms from the transformants of step 3, as shown in Figure 5D.

[0178] Step 5. Psilocybin analysis.

[0179] The psilocybin content of the genetically modified mushrooms is analyzed by gas chromatography / mass spectrometry, as shown in Figure 5E. Psilocybin accounts for 0.63% of the dry weight of unmodified P. Cubensis. The aim of the genetic manipulation is to increase the amount of psilocybin to more than 6%.

[0180] Example 2: Vector construct for overexpressing the Psi gene The coding sequences of the four major psilocybin synthesis genes (psiD / psiH / psiK / psiM) were synthesized and cloned into an overexpression vector system (pGWB5) under the control of the 35S promoter, which is a widely used plant overexpression promoter. See Table 4. For example, the PsiD gene was cloned into a vector expressing the PsiD gene under the control of the 35S promoter (Table 5: SEQ ID NO: 18, 17,647 bp; Figure 3A), the PsiH gene was cloned into a vector expressing the PsiH gene under the control of the 35S promoter (Table 5: SEQ ID NO: 17, 18,494 bp; Figure 3B), the PsiK gene was cloned into a vector expressing the PsiK gene under the control of the 35S promoter (Table 5: SEQ ID NO: 16, 17,420 bp; Figure 3C), and the PsiM gene was cloned into a vector expressing the PsiM gene under the control of the 35S promoter (Table 5: SEQ ID NO: 15, 17,267 bp; Figure 3D).

[0181] In addition, an all-in-one expression vector of the four Psi genes in tandem in a polycistronic vector will also be generated and tested.

[0182] Other vectors with different promoters (GPD, EF1a, and actin) were created. The four main psilocybin synthesis genes (psiD / psiH / psiK / psiM) are cloned into these vectors. For example, the GPD and CcDED1 promoters are two fungal-specific overexpression promoters. See Table 4. The Psi gene is cloned into a vector expressing the Psi gene under the control of the GcDED1 promoter (vector backbone Table 5: SEQ ID NO: 19, 9,462 bp; Figure 4A) or into a vector expressing the Psi gene under the control of the GPD promoter (Table 5: SEQ ID NO: 20, 8,067 bp; Figure 4B).

[0183] Example 3: Vector-mediated transfection of protoplasts: Protocol A material

[0184] Pleurotus nebrodensis strains were grown in PDSA medium (20% potato, 2% dextrose, 0.3% KH2PO4, 0.15% MgSO4, 0.0005% vitamin B1, 2% agar) at 25°C and kept at 4°C.

[0185] Vegetative culture of the mycelium was carried out in PDSB medium (PDSA medium without agar) at 25° C. for 1 week.

[0186] Protoplast extraction.

[0187] One gr of mycelium grown on PDSB medium for 7 days was harvested by infiltration through a nylon mesh.

[0188] Washed twice with 0.6M MgSO4.

[0189] The cells were resuspended in 3 ml of lysis buffer containing 1.5% lywallzyme (Guangdong Institute of Micro-biology) and 0.6 M MgSO4, and then incubated at 32°C for 2.5 h with gentle shaking to release the protoplasts.

[0190] Protoplasts were purified by filtration through a glass syringe through a 1 mm layer of loosely absorbent cotton and harvested by centrifugation at 2000 g for 20 min at 4°C.

[0191] The mixture was washed twice with 3 ml of MM buffer containing 0.5 M mannitol and 50 mM maleic acid buffer (pH 5.5).

[0192] Resuspend in 2-3 ml of MMC buffer (0.5 M mannitol, 50 mM maleic acid buffer at pH 5.5, 5 mM CaCl2) and 10 8 ~10 9 Protoplast ml -1 The concentration was set to .

[0193] Protoplast transformation

[0194] 3 ug of desired plasmid, 12.5 ul of PTC buffer (25% PEG4000, 10 mM Tris-HCl at pH 7.5, 25 mM CaCl 2 ) was added to 50 ul of chilled protoplast suspension and mixed well.

[0195] The mixture was kept on ice for 20 min.

[0196] 0.5 ml of PTC buffer was added to the mixture, mixed gently, and then incubated at room temperature for 5 minutes.

[0197] The protoplast mixture was then ready to be plated onto media for regeneration and screening.

[0198] Protoplast regeneration

[0199] The protoplast mixture was diluted with 1 ml of STC buffer (18.2% sorbitol, 10 mM Tris-HCl at pH 7.5, 25 mM CaCl2) and plated on regeneration medium (PDSA and 1.0 M sorbitol) for 24 h at 25 °C.

[0200] After 24 hours of regeneration incubation at 25°C, 20 ml of screening medium (PDSA with 0.8 M sorbitol, 80 ug / ml hygromycin B, 0.8% agar) was added to each plate and incubated in the dark at 25°C for 2 weeks.

[0201] Putative transformants that appeared on the screening medium were further subcultured five times on PDSA medium containing 80ug / ml hygromycin B to screen for stable transformants. Some of the regenerating protoplasts stopped growing at a diameter of 1-2mm. Only those exceeding a size of 1-2mm in diameter were transferred to further selection steps.

[0202] The average transformation efficiency is approximately 3 transformants per microgram of plasmid pAN7-1 DNA.

[0203] DNA extraction and analysis

[0204] Genomic DNA was isolated from mycelia of putative stable transformants and non-transformed controls of P. nebrodensis by the fungal DNA extraction (FDE) method. One gram of mycelia was ground to powder in liquid nitrogen and digested in 10 ml of TESN buffer (50 mM Tris-HCl pH 7.5, 100 mM EDTA pH 8.0, 0.5% SDS, 300 mM NaOAc pH 5.2) at 68°C for 1 h. After adding 3.5 ml of 3 M NaOAc (pH 5.2) and incubating on ice for 20 min, the digestion mixture was centrifuged at 8000 g for 20 min at 4°C. DNA in the supernatant was extracted by phenol / chloroform extraction method.

[0205] Example 4: Vector-mediated transfection of protoplasts: Protocol B Protoplast extraction and collection:

[0206] Step 1: A small block of monokaryotic mycelium was inoculated into CYM medium (1% maltose, 2% glucose, 0.2% yeast extract, 0.2% tryptone, 0.05% MgSO47H2O, 0.46% KH2PO4) and grown at 25°C for 5 days with shaking at 230 rpm.

[0207] Step 2: The mycelium is harvested by centrifugation, washed twice with 0.7 M NaCl, and incubated at 25 °C for 2.0–2.5 h in an enzyme solution (1 M MgSO 4 and treated with 50 mg / ml of lytic enzyme from Trichoderma harzianum (Sigma-Aldrich) in 0.6 M phosphate buffer, pH 6.0.

[0208] Step 3: After incubation, protoplasts were separated from mycelial debris by filtration through sterile Miracloth and collected by centrifugation at 3,000 × g for 10 min.

[0209] Step 4: The protoplasts were washed twice with 1 M sorbitol, so that the protoplast density was adjusted to 108 / ml.

[0210] PEG-mediated transformation:

[0211] Step 1: 50 microliters of protoplasts (108 / ml) were mixed with 10 µg of each plasmid DNA and 12.5 µl of PEG solution (40% PEG 4000, 10 mM Tris-HCl, pH 8.0, 25 mM CaCl 2 ; sterilized by filtration).

[0212] Step 2: The protoplasts were incubated on ice for 20 minutes.

[0213] Step 3: 500 microliters of PEG solution was added, mixed gently and incubated at room temperature for 5 minutes.

[0214] Step 4: Add 1 milliliter of ice-cold STC buffer (1 M sorbitol, 10 mM Tris-HCl, pH 8.0, 25 mM CaCl 2 ) was added and the mixture was then spread onto a plate containing 20 ml of PDAS Regenerated Agar Medium (PDA and 0.6 M sucrose, pH 6.5).

[0215] Step 5: The plates were incubated at 25°C for 48 hours, then 5 ml of PDAS medium containing 600 μg / ml hygromycin B (Duchefa, The Netherlands), 600 μg / ml phleomycin (Invitrogen), or 60 μg / ml carboxin (Duchefa, The Netherlands) was added as an overlay, and the plates were further incubated at 25°C until transformants appeared (5-7 days).

[0216] Protoplast regeneration:

[0217] Step 1: Transformants were individually subcultured onto fresh PDA plates containing 50 μg / ml hygromycin, 50 μg / ml phleomycin, or 5 μg / ml carboxin.

[0218] Step 2: Mature fruiting bodies of Psilocybe cubensis were obtained after cultivation with each selective agent on MMP medium (1% malt extract, 0.5% mycological peptone, 1.5% agar) at 25°C for 20–22 days.

[0219] Example 5: Agrobacterium-mediated transformation of protoplasts. Material: pleated tissue

[0220] The veil was cut from the fruiting body of P. eryngii and the exposed gill tissue was aseptically excised and separated into 1.0 × 0.5 cm pieces.

[0221] Preparation of Agrobacterium

[0222] GV3101 carrying the desired plasmid vector was grown in 50 ml of LB medium supplemented with kanamycin (50 μg / ml) at 28° C. for 2 days to an optical density at 600 nm of 1.6. Bacteria were harvested by centrifugation at 4,000 g for 30 min and then washed once with 50 ml of wash solution containing 100 mM MgCl2 and 100 μM acetosyringone. After another centrifugation at 4,000 g for 30 min, the bacterial pellet was resuspended in wash solution to an optical density at 600 nm of 1.0.

[0223] Transformation (this dark culture method is very effective in growing the mycelium and eliminating Agrobacterium).

[0224] These pieces (from ##) were vacuum infiltrated twice for 10 min in Agrobacterium suspension cultures.

[0225] The aspirated tissue was washed with triple-distilled water and dried under sterile conditions for 10 minutes on sterile Whatman filter paper.

[0226] The tissue was then transferred to a sterile Petri dish without medium and incubated in the dark at 25°C for 7-14 days.

[0227] For selection, dark-grown viable tissues were transferred to PDA (potato dextrose agar) medium (20% potato extract, 2% dextrose, and 1.5% agar) containing 50 μg / ml hygromycin and 100 μg / ml cefotaxime and incubated in the dark at 25°C for 2–3 weeks.

[0228] Putative transformants are then subcultured onto PDA medium in the dark for 1 week at 25° C. Finally, mycelium is grown on liquid medium containing PDB (PDA without agar) for 2 weeks in a shaking incubator at 25° C. and 130 g.

[0229] The mycelium is then separated by filtration through Whatman filter paper and used for further processing.

[0230] DNA extraction: Collect mycelium from putative transgenic and non-transformed mushrooms and grind in liquid nitrogen using a pre-chilled mortar and pestle. Isolate DNA from mycelium after cetyltrimethylammonium bromide (CTAB).

[0231] Example 6: Agrobacterium-mediated transformation of mycelia. Psilocybe cubensis mycelium was routinely maintained on potato dextrose agar (PDA) at 25°C. Mature fruiting bodies of Psilocybe cubensis were obtained after cultivation on MMP medium (1% malt extract, 0.5% mycological peptone, 1.5% agar) at 25°C for 20-22 days.

[0232] A. tumefaciens strain AGL1 containing the desired expression vector was grown for 24 hours in LB medium supplemented with the appropriate antibiotic.

[0233] The bacterial culture was then diluted in Agrobacterium induction medium (AIM) (induction medium (IM) [MM containing 0.5% (w / v) glycerol, 0.2 mM acetosyringone (AS), 40 mM 2-( N -morpholino)ethanesulfonic acid (MES), pH 5.3]) in the presence of 200 μM acetosyringone until the optical density at 660 nm was 0.15 and grown for an additional 5–6 h.

[0234] Five-day-old Psilocybe cubensis mycelium from general purpose growth medium was homogenized using an Ultra-Turrax homogenizer, and the mycelial fragments were transferred to fresh general purpose growth medium and grown for 24 hours to obtain a homogenous mycelial slurry.

[0235] 100 μl of mycelium suspension was mixed with 100 μl of bacterial culture, then spread onto a cellophane disk, overlaid on an AIM agar plate, and incubated at 25° C. for 48 h.

[0236] After co-cultivation, the cellophane disks were transferred to PDA medium containing 200 μg / ml timentin to kill remaining Agrobacterium cells and 100 μg / ml hygromycin to select for fungal transformants.

[0237] These were incubated at 25° C. until hygromycin-resistant colonies appeared, after which individual colonies were transferred to PDA medium containing 50 μg / ml hygromycin.

[0238] Mature fruiting bodies of Psilocybe cubensis were obtained after incubation with each selective agent on MMP medium (1% malt extract, 0.5% mycological peptone, 1.5% agar) at 25°C for 20–22 days.

[0239] Example 7: Agrobacterium-mediated transformation of fruit bodies. P. cubensis was routinely maintained on potato dextrose agar (PDA) at 25°C. Mature fruiting bodies of P. cubensis were obtained after cultivation on MMP medium (1% malt extract, 0.5% mycological peptone, 1.5% agar) at 25°C for 20-22 days.

[0240] A. tumefaciens strain AGL-1 containing the desired expression vector was grown for 24 hours in LB medium supplemented with the appropriate antibiotic.

[0241] The bacterial culture was then diluted in Agrobacterium induction medium (AIM) in the presence of 200 μM acetosyringone to an optical density at 660 nm of 0.15 and grown for an additional 5–6 h.

[0242] Using a scalpel, mature fruiting bodies (mature but before the gills were exposed) were excised from the MMP plates and cut into small pieces.

[0243] Small pieces of fruiting body gill tissue were mixed with induced A. tumefaciens cultures and vacuum infiltrated until no more air bubbles were produced.

[0244] The infiltrated flap pieces were transferred to cellulose discs overlaid on AIM agar plates. Co-cultivation and selection of transformants was performed as described in Example 6.

[0245] After co-cultivation, the cellophane disks were transferred to PDA medium containing 200 μg / ml timentin to kill remaining Agrobacterium cells and 100 μg / ml hygromycin to select for fungal transformants.

[0246] These were incubated at 25° C. until hygromycin-resistant colonies appeared, after which individual colonies were transferred to PDA medium containing 50 μg / ml hygromycin.

[0247] Mature fruiting bodies of P. cubensis were obtained after cultivation with the respective selective agents on MMP medium (1% malt extract, 0.5% mycological peptone, 1.5% agar) at 25°C for 20–22 days.

[0248] Example 8: Transformation, transfection, and regeneration Psilocybe cubensis was propagated and grown on different substrates to produce both mature mushroom fruiting bodies and mycelium, as shown in Figure 6. Psilocybe cubensis was grown in PDA agar (Figures 6A and 6B) and in barley-perlite compost (Figure 6C) at room temperature for 7 days.

[0249] The pGWB5 vector described in Example 2 is used to transform Basidiomycetes using the transformation or transfection protocols described in Examples 3 to 7. Transformations include different Psi genes individually and in combination (using multiple different vectors, or a vector containing multiple Psi genes).

[0250] For example, tissue was extracted from the mushroom gills and transformed with the Psi gene by Agrobacterium-mediated transformation as described in Examples 3-7.

[0251] Protoplasts were generated from the mycelia and transformed with the Psi gene by PEG-mediated transfection.The mycelia were transformed by Agrobacterium-mediated transformation.

[0252] After regeneration of multiple transformed fungi, polynucleotide analysis is performed to confirm gene integration and determine RNA expression level.Furthermore, the mRNA and protein levels of the disrupted gene are determined.The content of one or more bioactive metabolites, such as terpenes or cannabinoids, in plant tissue is also determined.For example, the content of one or more of psilocybin and / or psilocin is determined using procedures known to those skilled in the art.

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Claims

[Claim 1] The invention described in this specification.

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