GENE-EDITING IN CANNABIS PLANT
The present technology is in the field of molecular biology and plant biology, particularly as it pertains to gene editing in plants of the genus Cannabis . More specifically, there are provided systems and methods for targeted DNA modifications and gene editing in Cannabis plant, including systems and methods for making such modifications using a CRISPR/Cas-system nuclease complex, kits comprising the CRISPR/Cas-system nuclease complex, and constructs encoding the same.
1 . A method for genetically editing a target gene in the genome of cells from a Cannabis plant, the method comprising:
(a) introducing one or more exogenous nucleic acid having gene-editing activity into the cells;
(b) optionally, cultivating the cells under conditions allowing gene-editing of the target gene in the genome of said cells; and
(c) optionally, selecting cells which have been gene-edited by the gene-editing activity of the one or more exogenous nucleic acid molecule.
2 . The method of claim 1 , wherein the one or more exogenous nucleic acid molecule is a DNA.
3 . The method of claim 2 , wherein the DNA is an expression vector.
4 . The method of claim 1 , wherein the one or more exogenous nucleic acid molecule is an RNA.
5 . The method of claim 1 , wherein the one or more exogenous nucleic acid molecule encodes a CRISPR endonuclease.
6 . The method of claim 5 , wherein the CRISPR endonuclease is a Cas enzyme, optionally selected from Cas9, Cas12, Cas12a, Cas13a, Cpf1, Csm1, CasX, and CasY.
7 . The method of claim 6 , wherein the Cas enzyme is Cas9, optionally selected from SpCas9, SaCas9, SauriCas9, StCas9, NmCas9, and FnCas9.
8 . The method of claim 7 , wherein the Cas9 is SpCas9-NLS.
9 . The method of claim 6 , wherein the Cas enzyme is Cas12, optionally Cas12a, optionally selected from AsCas12a, FnCas12a, and LbCas12a.
10 . The method of claim 9 , wherein the Cas12 is LbCas12a-NLS.
11 . The method of claim 1 , wherein the one or more exogenous nucleic acid molecule encodes a guide RNA, optionally selected from a crRNA, a tracrRNA, an sgRNA, and combinations thereof.
12 . The method of claim 4 , wherein the one or more exogenous nucleic acid molecule is a guide RNA, optionally selected from a crRNA, a tracrRNA, an sgRNA, and combinations thereof.
13 . The method of claim 11 , wherein the guide RNA comprises or consists of the sequence set forth in any one of SEQ ID NOs: 1-42, or has at least, greater than or about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
14 . The method of claim 13 , wherein the guide RNA has the sequence set forth in any one of SEQ ID NOs: 1-42.
15 . The method of claim 11 , wherein the guide RNA binds the target gene in the genome of the cells, wherein the target gene is CBDAS, CBCAS, R3-MYB1, R3-MYB2, R3-MYB3, R3-MYB4, or eIF4e.
16 . The method of claim 1 , wherein the target gene is CBDAS, CBCAS, R3-MYB1, R3-MYB2, R3-MYB3, R3-MYB4, eIF4e, or a combination thereof.
17 . The method of claim 1 , wherein the one or more exogenous nucleic acid molecule comprises a first exogenous nucleic acid molecule and a second exogenous nucleic acid molecule, the first exogenous nucleic acid molecule encoding the CRISPR endonuclease as defined in claim 6 , and the second exogenous nucleic acid molecule encoding the guide RNA as defined in claim 11 .
18 . The method of claim 1 , wherein said step of introducing one or more exogenous nucleic acid having gene-editing activity into the cells comprises transformation, optionally transformation via a DNA virus, via an RNA virus, via protoplasts, via T-DNA delivery or via particle bombardment.
19 . The method of claim 18 , wherein said transformation comprises transformation with an Agrobacterium carrying said one or more exogenous nucleic acid molecule.
20 . The method of claim 18 , wherein said transformation comprises transformation via biolistic transformation.