Methods for inducing targeted stimulation of meiotic recombination and kits for performing said methods
View Patent ↗The Invention pertains to methods, kits, molecules and cells to increase the rate or recombination and/or target recombination in dividing cells. In a particular aspect, the invention concerns methods and kits to induce targeted meiotic recombination.
1. A method for increasing recombination between homologous chromosomes in a dividing cell, comprising the steps of: (i) introducing into said cell, a nucleic acid sequence encoding a fusion protein comprising a DNA binding domain operably linked to a Spo11 protein, wherein said DNA binding domain recognizes a DNA sequence in said homologous chromosomes and (ii) having the cell divide; so that the recombination between said homologous chromosomes in said cell is increased.
2. The method of claim 1 , further comprising, before step (ii), a step of introducing into said cell's genome, a DNA sequence recognized by said DNA binding domain operably linked to the Spo11 protein.
3. The method of claim 1 , wherein the cell division performed in step (ii) is meiosis.
4. The method of claim 3 , wherein in step (i), the sequence encoding the fusion protein is under the control of a meiosis-specific promoter.
5. The method of claim 4 , wherein said meiosis-specific promoter is the IME1 promoter or the REC8 promoter.
6. The method of claim 1 , wherein said cell is an eukaryotic cell.
7. The method of claim 6 , wherein said cell is a plant cell.
8. The method of claim 1 , wherein said cell comprises artificial chromosomes carrying one or more sequence(s) recognized by said DNA binding domain operably linked to the Spo11 protein.
9. The method of claim 1 , wherein the nucleic acid sequence introduced into the cell in step (i) is stably integrated into the cell genome.
10. A method for performing a targeted recombination between two or more polymorphisms carried by same chromosome in a cell, comprising the steps of: (i) introducing into said cell, a nucleic acid sequence encoding a fusion protein comprising a DNA binding domain operably linked to a Spo11 protein, wherein said DNA binding domain recognizes a sequence situated between said two or more polymorphisms and wherein the sequence encoding thefusion protein is under the control of a meiosis-specific promoter, and (ii) having the cell divide; so that the recombination between said two or more polymorphisms in said cell occurs.
11. The method of claim 10 , further comprising before step (ii) a step of introducing into said cell's genome, between said two or more polymorphisms, a DNA sequence recognized by said DNA binding domain operably linked to a Spo11 protein.
12. The method of claim 10 , wherein the cell division performed in step (ii) is meiosis.
13. The method of claim 10 , wherein said mejosis-specific promoter is the IME1 promoter or the REC8 promoter.
14. The method of claim 10 , wherein said cell is a eukaryotic cell.
15. The method of claim 14 , wherein said cell is a plant cell.
16. The method of claim 10 , wherein said cell comprises artificial chromosomes carrying one or more sequence(s) recognized by said DNA binding domain operably linked to the Spo11 protein.
17. A method for introducing a gene conversion in a cell, comprising the steps of: (i) introducing into said cell, a nucleic acid sequence encoding a fusion protein comprising a DNA binding domain operably linked to a Spo11 protein, wherein said DNA binding domain recognizes a sequence situated between polymorphisms, and having the cell divide; so that gene conversion occurs.
18. A method for inducing meiotic recombination in artificial chromosomes in a cell, comprising the steps of: (i) introducing into said cell, a nucleic acid encoding a fusion protein comprising a DNA binding domain operably linked to a Spo11 protein, wherein said DNA binding domain recognizes one or more sequence(s) carried by said artificial chromosome, and (ii) having the cell divide; so that the meiotic recombination occurs between homologous artificial chromosomes, and (ii) having the cell divide; so that the meiotic recombination occurs between homologous artificial chromosomes.
19. A method for increasing recombination between homologous chromosomes in a dividing cell, comprising the steps of:
(i) introducing into said cell a nucleic acid sequence encoding a fusion
(ii) protein comprising a Gal4BD-Spo11 fusion protein, and having the cell divide; so that the recombination between homologous chromosomes in said cell is increased.
20. A method for performing a targeted recombination between two or more polymorphisms carried by a same chromosome in a cell, comprising the steps of: (i) introducing into said cell, a nucleic acid encoding a fusion protein comprising a Gal4BD-Spo11 fusion protein, wherein said DNA binding domain recognizes sequence situated between said two or more polymorphisms and wherein the sequence encoding the fusion protein is under the control of a meiosis-specific promoter, and (ii) having the cell divide; so that the recombination between said two or more polymorphisms in said cell occurs.
21. A method for introducing a gene conversion in a cell, comprising the steps of: (i) introducing into said cell, a nucleic acid encoding a fusion protein comprising a Gal4BD-Spo11 fusion protein, wherein said DNA binding domain recognizes a sequence situated between polymorphisms, and (ii) having the cell divide; so that gene conversion occurs.
22. A method for inducing meiotic recombination in artificial chromosomes in a cell, comprising the steps of: (i) introducing into said cell, a nucleic acid encoding a fusion protein comprising a Gal4BD-Spo11 fusion protein, wherein said DNA binding domain recognizes one or more sequence(s) carried by said artificial chromosome, and (ii) having the cell divide; so that the melotic recombination occurs between homologous artificial chromosomes.