Some content of this application is unavailable at the moment.
If this situation persist, please contact us atFeedback&Contact
1. (WO2014173955) IMPROVED GENE TARGETING AND NUCLEIC ACID CARRIER MOLECULE, IN PARTICULAR FOR USE IN PLANTS
Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

Claims

A nucleic acid carrier molecule, comprising the general formula


,

wherein M is a first polypeptide specifically binding to a donor nucleic acid sequence to be transferred into an organelle of a cell,

W is a second polypeptide specifically binding to a target nucleic acid sequence, wherein said target nucleic acid sequence is located in an organelle of a cell,

L is missing or is linking group allowing M and W flexibility and semi-independence, and

S1 and S2 independently of each other are missing or are a signal peptide sequence, wherein said donor nucleic acid sequence is brought into close proximity with said target nucleic acid sequence when both nucleic acid sequences are bound to said carrier molecule.

The nucleic acid carrier molecule according to claim 1 , wherein M is selected from the group of a TAL effector (TALe) polypeptide, a zinc finger polypeptide, a relaxase, such as, for example, VirD2-like polypeptide, a VirE2-like polypeptide, an RNA binding polypeptide, CRISPR associated protein 9 (Cas9) family proteins and their derivatives, programmable Argonautes, such as TtAgo, and a transcription factor polypeptide, wherein said polypeptide specifically binds to said donor nucleic acid, and preferably is a fusion protein, for example comprising an enzyme, such as an endonucle-ase.

The nucleic acid carrier molecule according to claim 1 or 2, wherein W is selected from the group of a TAL effector (TALe) polypeptide, a zinc finger polypeptide, a VirD2-like polypeptide, a VirE2-like polypeptide, an RNA binding polypeptide, TtAgo, and a transcription factor polypeptide, wherein said polypeptide specifically binds to said target nucleic acid sequence that is located in said organelle, and wherein W preferably is a fusion protein, for example comprising an enzyme, such as an endonuclease.

4. The nucleic acid carrier molecule according to any of claims 1 to 3, wherein L is selected from a polypeptide linker or an organic linker group which is covalently connecting M and W.

5. The nucleic acid carrier molecule according to any of claims 1 to 4, wherein S1 and S2 are selected from the group of translocation signal polypeptides, such as type IV translocation signal (D2TS) peptides, organelle targeting peptides, and type III translocation signals.

6. The nucleic acid carrier molecule according to any of claims 1 to 5, wherein said target nucleic acid sequence is selected from viral sequences, mutated sequences, trans- poson sequences or any other foreign sequence in a genome and sequences that are or- ganelle-specific.

7. The nucleic acid carrier molecule according to any of claims 1 to 6, wherein said cell is an animal or plant cell, such as, for example, a protoplast.

8. The nucleic acid carrier molecule according to any of claims 1 to 7, wherein said organelle is selected from the group of a nucleus, a chloroplast and a mitochondrium.

9. A recombinant nucleic acid, encoding for a nucleic acid carrier molecule according to any of claims 1 to 8, which is preferably part of an expression vector or an expression cassette.

10. An in vitro method for recombinant ly transforming a nucleic acid into an organelle in a cell, comprising the steps of

a) providing a cell to be transformed comprising organelles,

b) providing the nucleic acid carrier molecule according to any of claims 1 to 8 in said organelles,

c) providing a donor nucleic acid sequence in said organelles, and

d) selecting cells comprising organelles wherein said donor nucleic acid sequence has been recombinantly transformed into the DNA of said organelles.

11. The method according to claim 10, wherein said cell is a bacterial cell, a fungal cell, an animal cell or plant cell, such as, for example, a protoplast, or a stem cell, wherein human embryonic stem cells are excluded, and wherein said organelle preferably is selected from the group of a nucleus, a chloroplast and a mitochondrium.

12. The method according to any of claims 10 or 11, wherein said method comprises the use of a bacterium, such as, for example, Agrobacterium tumeƒaciens, Sinorhizobium meliloti, Wolbachia Sp., Bartonella henselae, Helicobacter pylori, Pseudomonas aeruginosa, Pseudomonas syringae, Bacillus megaterium, and E. coli.

13. The method according to any of claims 10 to 12, wherein said transformation comprises gene targeting of a specific gene, such as a viral gene sequence, a mutated gene sequence, and a gene sequence that is organelle-specific.

14. The method according to any of claims 10 to 13, wherein said transformation comprises bringing said first target nucleic acid sequence into close proximity with said second target nucleic acid sequence when both target nucleic acid sequences are bound to said carrier molecule.

15. Use of the nucleic acid carrier molecule according to any of claims 1 to 8, or the expression vector or the expression cassette according to claim 9 for recombinantly transforming a nucleic acid in an organelle in a cell in vitro or group of cells in vivo, wherein said cell preferably is an animal or plant cell, such as, for example, a protoplast, and wherein said organelle preferably is selected from the group of a nucleus, a chloroplast, and a mitochondrium.