Site-specific serine recombinases and methods of their use
The present invention provides a method for obtaining site-specific recombination in a eukaryotic cell, the method comprising providing a eukaryotic cell that comprises a first recombination attachment site and a second recombination attachment site; contacting the first and second recombination attachment sites with a prokaryotic recombinase polypeptide, resulting in recombination between the recombination attachment sites, wherein the recombinase polypeptide can mediate recombination between the first and second recombination attachment sites, the first recombination attachment site is a phage genomic recombination attachment site (attP) or a bacterial genomic recombination attachment site (attB), the second recombination site is attB or attP, and the recombinase is selected from the group consisting of a Listeria monocytogenes phage recombinase, a Streptococcus pyogenes phage recombinase, a Bacillus subtilis phage recombinase, a Mycobacterium tuberculosis phage recombinase and a Mycobacterium smegmatis phage recombinase, provided that when the first recombination attachment site is attB, the second recombination attachment site is attP and when the first recombination attachment site is attP, the second recombination attachment site is attB. The invention also describes compositions, vectors, and methods of use thereof, for the generation of transgenic cells, tissues, plants, and animals. The compositions, vectors and methods of the present invention are also useful in gene therapy applications.
1. A method for obtaining site-specific recombination in a eukaryotic cell, the method comprising: providing a eukaryotic cell that comprises a first recombination site and a second recombination site; a stop sequence positioned between said first recombination site and said second recombination site; contacting the first and second recombination sites with a prokaryotic recombinase polypeptide, resulting in recombination between the recombination sites and deletion of said stop sequence, wherein the recombinase polypeptide can mediate recombination between the first and second recombination sites, the first recombination site is a phage genomic recombination attachment site (attP) or a bacterial genomic recombination attachment site (attB), the second recombination site is attB or attP, and the recombinase is a Mycobacterium smegmatis Bxb1 phage recombinase, provided that when the first recombination attachment site is attB, the second recombination attachment site is attP, and when the first recombination attachment site is attP, the second recombination attachment site is attB.
2. A method for obtaining site-specific recombination in a eukaryotic cell, the method comprising: providing a eukaryotic cell that comprises a first recombination site and a second recombination site; contacting the first and second recombination sites with a prokaryotic recombinase polypeptide, resulting in recombination between the recombination sites, wherein the recombinase polypeptide can mediate recombination between the first and second recombination sites, the first recombination site is attP or attB, the second recombination site is a pseudo attachment site, and the recombinase is a Mycobacterium smegmatis Bxb1 phage recombinase.
3. The method of claim 1 , wherein the recombinase-encoding polynucleotide is operably linked to a promoter which mediates expression of the polynucleotide in the eukaryotic cell.
4. The method of claim 1 , wherein the recombinase polypeptide is introduced into the eukaryotic cell by expression of a polynucleotide that encodes the recombinase polypeptide.
5. The method of claim 1 , wherein the recombinase polypeptide is introduced into the eukaryotic cell as a polypeptide.
6. The method of claim 1 , wherein the recombinase polypeptide is introduced into the eukaryotic cell by messenger RNA that encodes the recombinase polypeptide.
7. The method of claim 1 , wherein the site-specific recombination results in integration, deletion, inversion, translocation or exchange of DNA.
8. A method for obtaining a eukaryotic cell having a stably integrated polynucleotide sequence, the method comprising: introducing a polynucleotide into a eukaryotic cell that comprises a first recombination attB or attP site, wherein the polynucleotide comprises a plurality of nucleic acid sequences of interest and a second recombination attP or attB site, and contacting the first and the second recombination sites with a prokaryotic recombinase polypeptide, wherein the recombinase polypeptide can mediate site-specific recombination between the first and second recombination sites, and the recombinase is a Mycobacterium smegmatis Bxb1 phage recombinase, provided that when the first recombination site is attB, the second recombination site is attP and when the first recombination site is attP, the second recombination site is attB.
9. A method for obtaining a eukaryotic cell having a stably integrated polynucleotide sequence, the method comprising: introducing a polynucleotide into a eukaryotic cell that comprises a first recombination pseudo attachment site, wherein the polynucleotide comprises a nucleic acid sequence and a second recombination attP or attB site, and contacting the first and the second recombination sites with a prokaryotic recombinase polypeptide, wherein the recombinase polypeptide can mediate site-specific recombination between the first and second recombination sites, and the recombinase is a Mycobacterium smegmatis Bxb1 phage recombinase.
10. The method of claim 8 , wherein the recombinase-encoding polynucleotide is operably linked to a promoter which mediates expression of the polynucleotide in the eukaryotic cell.
11. The method of claim 8 , wherein the recombinase polypeptide is introduced into the eukaryotic cell by expression of a polynucleotide that encodes the recombinase polypeptide.
12. The method of claim 8 , wherein the recombinase polypeptide is introduced into the eukaryotic cell as a polypeptide.
13. The method of claim 8 , wherein the recombinase polypeptide is introduced into the eukaryotic cell by expression of RNA that encodes the recombinase polypeptide.
14. A method for obtaining site-specific recombination in a eukaryotic cell, the method comprising: providing a eukaryotic cell that comprises a first recombination site and a second recombination site with a polynucleotide sequence flanked by a third recombination site and a fourth recombination site; contacting the recombination sites with a prokaryotic recombinase polypeptide, resulting in recombination between the recombination sites, wherein the recombinase polypeptide can mediate recombination between the first and third recombination sites and the second and fourth recombination sites, the first and second recombination sites are attP or attB, the third and fourth recombination sites are attB or attP, and the recombinase is a Mycobacterium smegmatis Bxb1 phage recombinase, provided that when the first and second recombination attachment sites are attB, the third and fourth recombination attachment sites are attP, and when the first and second recombination attachment sites are attP, the third and fourth recombination attachment sites are attB.
15. The method of claim 14 , wherein the recombinase polypeptide is introduced into the eukaryotic cell by expression of a polynucleotide that encodes the recombinase polypeptide.
16. The method of claim 14 , wherein the recombinase polypeptide is introduced into the eukaryotic cell as a polypeptide.
17. The method of claim 14 , wherein the recombinase polypeptide is introduced into the eukaryotic cell by messenger RNA that encodes the recombinase polypeptide.
18. A method for the site-specific integration of a polynucleotide of interest into the genome of a transgenic subject, wherein said genome comprises a first recombination attB or attP site or pseudo attB or pseudo attP site, the method comprising: introducing a nucleic acid that comprises the polynucleotide of interest and a second recombination attP or attB site; contacting the first and the second recombination sites with a prokaryotic recombinase polypeptide, wherein the recombinase polypeptide can mediate site-specific recombination between the first and second recombination sites, and the recombinase is a Mycobacterium smegmatis Bxb1 phage recombinase, provided that when the first recombination site is attB or pseudo attB, the second recombination site is attP and when the first recombination site is attP or pseudo attP, the second recombination site is attB.
19. The method of claim 2 , wherein the recombinase-encoding polynucleotide is operably linked to a promoter which mediates expression of the polynucleotide in the eukaryotic cell.
20. The method of claim 2 , wherein the recombinase polypeptide is introduced into the eukaryotic cell by expression of a polynucleotide that encodes the recombinase polypeptide.
21. The method of claim 2 , wherein the recombinase polypeptide is introduced into the eukaryotic cell as a polypeptide.
22. The method of claim 2 , wherein the recombinase polypeptide is introduced into the eukaryotic cell by messenger RNA that encodes the recombinase polypeptide.
23. The method of claim 2 , wherein the site-specific recombination results in integration, deletion, inversion, translocation or exchange of DNA.
24. The method of claim 9 , wherein the recombinase-encoding polynucleotide is operably linked to a promoter which mediates expression of the polynucleotide in the eukaryotic cell.
25. The method of claim 9 , wherein the recombinase polypeptide is introduced into the eukaryotic cell by expression of a polynucleotide that encodes the recombinase polypeptide.
26. The method of claim 9 , wherein the recombinase polypeptide is introduced into the eukaryotic cell as a polypeptide.
27. The method of claim 9 , wherein the recombinase polypeptide is introduced into the eukaryotic cell by expression of RNA that encodes the recombinase polypeptide.
28. The method of claim 1 , wherein the recombinase polypeptide comprises the amino acid sequence of SEQ ID NO: 6.