# US 11,999,958 B2 · Methods of gene editing and transforming Cannabis

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- Number: US 11,999,958 B2
- Document ID: US11999958B2
- Status: Granted
- Kind: B2
- Application number: 16/930,936
- Filed: 2020-07-16
- Published: 2021-03-11
- Granted: 2024-06-04
- Earliest claimed: 2019-07-17
- Assignees: Wisconsin Alumni Research Foundation
- Applicants: Wisconsin Alumni Research Foundation
- Inventors: Michael W. Petersen; Edward James Williams; Robert Harnish; Heidi Flewelling Kaeppler; Brian Martinell; Ray Collier; Frank McFarland; Shawn Michael Kaeppler
- Web page: https://patentgrep.com/patent/US11999958B2

## CPC

- C12N 15/8201: {Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation}
- C12N 15/8205: {Agrobacterium mediated transformation}
- A01H 4/005: {Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques}
- A01H 6/28: Cannabaceae, e.g. cannabis
- C07K 14/415: from plants
- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C12N 15/8243: {involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Disclosed herein are methods for the production of Cannabis meristem explants from dry seeds. Also described are methods of transforming and gene editing using the Cannabis meristem explants disclosed herein.

## Claims

### Claim 1

A method of transforming a Cannabis plant, the method comprising:
growing a sanitized and imbibed Cannabis seed on a non-selective culture medium suitable for supporting the growth and survival of the Cannabis seed;
harvesting an internode, leaf, or petiole tissue to form a Cannabis explant;
inoculating the Cannabis explant with a heterologous nucleic acid;
co-culturing the Cannabis explant in a co-culture medium for between about 1 day and about 6 days,
culturing the Cannabis explant on a selection medium comprising meta-topolin to select for transformed Cannabis explants.

### Claim 2 (depends on 1)

The method of claim 1, wherein the Cannabis explant is inoculated using a method selected from the group consisting of particle bombardment, high velocity microprojection, microinjection, electroporation, direct DNA uptake, cell-penetrating peptides, silica carbide fibers, nanoparticles, and bacterially-mediated transformation.

### Claim 3 (depends on 1)

The method of claim 1, wherein Agrobacterium spp. is used to inoculate the Cannabis explant.

### Claim 4 (depends on 1)

The method of claim 1, additionally comprising the step of force treating the Cannabis explant prior to or following inoculation.

### Claim 5 (depends on 4)

The method of claim 4, wherein the force treatment is selected from the group consisting of sonication, vortexing, centrifugation, heat-shock, increased pressure, vacuum infiltration, and addition of chemicals.

### Claim 6 (depends on 1)

The method of claim 1, wherein the heterologous nucleic acid modulates the expression or activity of an endogenous Cannabis gene selected from the group consisting of tetrahydrocannabinolic acid synthase (THCA synthase), cannabidiolic acid synthase (CBDA synthase), O-methyltransferase (CsOMT21), lipid transfer protein 2 (LTP2), prenyltransferase 3 (CsPT3), and prenyltransferase 1 (CsPT1).

### Claim 7 (depends on 1)

The method of claim 1, wherein the Cannabis seed is a Cannabis sativa seed.

### Claim 8 (depends on 1)

The method of claim 1, wherein the heterologous nucleic acid encodes a polypeptide at least 90% identical to SEQ ID NO:28.

### Claim 9 (depends on 1)

The method of claim 1, wherein the heterologous nucleic acid encodes a guide RNA that targets Cannabis sativa THCA synthase gene, Cannabis sativa CBDA synthase gene, or Cannabis sativa EPSP synthase gene.

### Claim 10 (depends on 1)

The method of claim 1, wherein the tissue harvested is from an internode, leaf, or petiole tissue, and wherein the selection medium further comprises gibberellic acid (GA3).

### Claim 11 (depends on 1)

The method of claim 1, wherein culturing is performed under a light/dark photoperiod.