Chimeric DNA-binding proteins
Chimeric proteins containing composite DNA-binding regions are disclosed together with DNA constructs encoding them, compositions containing them and applications in which they are useful.
1. A nucleic acid encoding a chimeric protein comprising a cleavage domain and a composite nucleic acid-binding region, wherein the composite nucleic acid-binding region comprises at least two component polypeptide portions which are individually capable of binding specific nucleotide sequences, and wherein the at least two component polypeptide portions:
(i) do not occur in the same protein in nature;
(ii) do not occur in the same protein in the order in which they are present in the chimeric protein; or
(iii) do not occur in nature with the same spacing that is present in the chimeric protein.
2. The nucleic acid of claim 1 , wherein at least one of the component polypeptide portions is a zinc finger module.
3. The nucleic acid of claim 1 , wherein the at least two component polypeptide portions are zinc finger modules.
4. The nucleic acid of claim 1 , wherein at least one of the component polypeptide portions is a homeodomain.
5. The nucleic acid of claim 2 , wherein at least one of the component polypeptide portions is a homeodomain.
6. The-nucleic acid of claim 1 , wherein the chimeric protein is capable of binding a nucleotide sequence with a K d value less than or equal to, or about, 10 −8 .
7. The nucleic acid of claim 6 , wherein the chimeric protein is capable of binding a DNA sequence with a K d value less than or equal to, or about, 10 −8 .
8. The nucleic acid of claim 6 , wherein the chimeric protein is capable of binding an RNA sequence with a K d value less than or equal to, or about, 10 −8 .
9. The nucleic acid of claim 1 , wherein the cleavage domain is a FokI cleavage domain.
10. A eukaryotic expression construct comprising the nucleic acid of claim 1 , operably linked to at least one expression control element permitting gene expression in eukaryotic cells.
11. The construct of claim 10 , wherein at least one expression control element is inducible.
12. The construct of claim 10 , wherein at least one expression control element is tissue-specific.
13. The construct of claim 10 , wherein at least one expression control element is cell-type-specific.
14. A method for genetically engineering cells to express a chimeric protein comprising a cleavage domain and a composite nucleic acid-binding region, wherein the composite nucleic acid-binding region comprises at least two component polypeptide portions which are individually capable of binding specific nucleotide sequences, and wherein the at least two component polypeptide portions:
(i) do not occur in the same protein in nature;
(ii) do not occur in the same protein in the order in which they are present in the chimeric protein; or
(iii) do not occur in nature with the same spacing that is present in the chimeric protein;
the method comprising:
(a) providing an expression construct of claim 10 for directing the expression in a eukaryotic cell of the chimeric protein; and
(b) introducing the expression construct into cells in a manner permitting expression of the introduced construct in at least a portion of the cells.
15. The method of claim 14 , wherein the cells are in culture.
16. The method of claim 14 , wherein the cells are present within an organism.
17. The method of claim 16 , wherein the organism is a mammal.
18. A genetically engineered cell containing and capable of expressing a nucleic acid of claim 1 .