Nuclease fusion protein and uses thereof
The present invention is concerned with nuclease fusion proteins and various uses thereof. Specifically, it relates to a polynucleotide encoding a polypeptide comprising (i) a first module comprising at least a first DNA binding domain derived from a homing endonuclease, (ii) a linker, and (iii) a second module comprising at least a second DNA binding domain and a cleavage domain derived from a restriction endonuclease, wherein the polypeptide functionally interacts only with DNA comprising a DNA recognition site for the first DNA binding domain and a DNA recognition site for the second DNA binding domain, and wherein the cleavage domain cleaves DNA within a specific DNA cleavage site upon binding of the polypeptide. Further contemplated are a vector and a non-human transgenic organism comprising the polynucleotide as well as a polypeptide encoded by the polynucleotide of the invention. Finally, the present invention relates to a method for introducing a nucleic acid of interest into a genome of a non-human organism wherein the polypeptide of the invention is applied.
1. A polynucleotide encoding a polypeptide comprising:
(i) a first module comprising at least a first DNA binding domain from I-SceI, wherein the first module comprises (a) the amino acid sequence of SEQ ID NO:1 or (b) the amino acid sequence of SEQ ID NO:1 having one or more of the following changes: (1) substitution D44S, (2) substitution D44S in combination with D145A, (3) substitution D44N in combination with D145A, (4) a deletion of 5 to 9 amino acids from the C-terminal end;
(ii) a linker; and
(iii) a second module comprising at least a second DNA binding domain and a cleavage domain from PvuII, wherein the second module comprises (a) the amino acid sequence of SEQ ID NO:2 or (b) the amino acid sequence of SEQ ID NO:2 having one of the following changes: (1) substitution T46G, (2) substitution H83A, (3) substitution Y94F, (4) substitution T46G in combination with H83A, (5) substitution T46G in combination with Y94F, or (6) substitution T46G in combination with H83A and Y94F;
wherein said polypeptide functionally interacts only with DNA comprising a DNA recognition site for the first DNA binding domain and a DNA recognition site for the second DNA binding domain, and
wherein said cleavage domain cleaves DNA within a specific DNA cleavage site upon binding of the polypeptide.
2. The polynucleotide of claim 1 , wherein said specific DNA cleavage site and said DNA recognition site of the second DNA binding domain of the restriction endonuclease are identical.
3. The polynucleotide of claim 1 , wherein said second DNA binding domain and the cleavage domain comprised in the second module are derived from a type IIP restriction endonuclease.
4. The polynucleotide of claim 1 , wherein said second DNA binding domain and the cleavage domain comprised in the second module are derived from a restriction endonuclease which exhibits reduced DNA binding and/or reduced catalytic activity when compared to the wild type restriction endonuclease.
5. The polynucleotide of claim 1 , wherein said first module exhibits reduced or no catalytic activity.
6. The polynucleotide of claim 1 , wherein said linker consists essentially of 6 to 10 amino acids.
7. The polynucleotide of claim 6 , wherein said linker has an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.
8. A vector comprising the polynucleotide of claim 1 .
9. A microorganism, a plant, plant part or isolated cell thereof; or an isolated animal tissue or isolated cell thereof comprising the polynucleotide of claim 1 .
10. The polynucleotide of claim 1 , wherein the polynucleotide encodes a polypeptide having the amino acid sequence of SEQ ID NO: 9.
11. The polynucleotide of claim 1 , wherein the polynucleotide encodes a polypeptide having the amino acid sequence of SEQ ID NO: 10.