Ref nuclease for site specific Ref-mediated DNA cleavage

1. A recombinant nucleic acid encoding an engineered truncated Ref polypeptide, the engineered truncated Ref polypeptide comprising an amino acid sequence comprising a deletion of ten (10) to sixty-six (66) amino acids of the N-terminus of a naturally occurring full length Ref protein, SEQ ID NO:1, or SEQ ID NO:2, and wherein the engineered truncated purified Ref polypeptide exhibits an increased nuclease site-specific targeting activity relative to nuclease site-specific targeting activity of a naturally occurring full length wildtype Ref protein or full length Ref protein comprising the amino acid of any of SEQ ID NOs:1-2.

2. The recombinant nucleic acid of claim 1 , wherein the engineered truncated Ref polypeptide comprising the amino acid sequence comprising:

(a) a deletion of the first ten (10) amino acids of the N-terminus of the full length wildtype Ref protein, wherein the amino acid sequence comprises SEQ ID NO: 10 or 11

(b) a deletion of the first twenty one (21) amino acids of the N-terminus of the full length wildtype Ref protein, wherein the amino acid sequence comprises SEQ ID NO: 8 or 9;

(c) a deletion of the first forty-seven (47) amino acids of the N-terminus of the full length wildtype Ref protein, wherein the amino acid sequence comprises SEQ ID NO:6 or SEQ ID NO:7,

(d) a deletion of the first fifty-nine (59) amino acids of the N-terminus of the full length wildtype Ref protein, wherein the amino acid sequence comprises residues 60-186 of SEQ ID NO:1 or residues 60-186 of SEQ ID NO:2; or

(e) a deletion of the first sixty-six (66) amino acids of the N-terminus of the full length wildtype Ref protein, wherein the amino acid comprises SEQ ID NO:4 or 5.

3. The recombinant nucleic acid of claim 2 , wherein the Ref polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10; SEQ ID NO:11, residues 60-186 of SEQ ID NO:1, and residues 60-186 of SEQ ID NO:2.

4. The recombinant nucleic acid of claim 1 , further comprising a heterologous nucleic acid sequence on its 5′ or 3′ end.

5. The recombinant nucleic acid of claim 4 , wherein the heterologous nucleic acid is a 5′ UTR, a 3′ UTR, a Kozak consensus sequence, a Shine-Delgarno sequence, or a promoter.

6. The recombinant nucleic acid of claim 1 , wherein the recombinant nucleic acid is a linear double-stranded DNA cassette for in vitro transcription.

7. The recombinant nucleic acid of claim 6 , where the cassette comprises a promoter for an RNA polymerase followed by a 5′ UTR, an open reading frame encoding the Ref polypeptide, and a 3′ UTR.

8. A vector comprising the recombinant nucleic acid of claim 1 .

9. A vector comprising the recombinant nucleic acid of claim 2 .

10. A vector comprising the recombinant nucleic acid of claim 3 .

11. A vector comprising the recombinant nucleic acid of claim 4 .

12. A recombinant cell comprising the recombinant nucleic acid of claim 1 .

13. The recombinant cell of claim 12 , wherein the recombinant cell is a prokaryotic or eukaryotic cell.

14. The recombinant cell of claim 13 , wherein the cell is a mammalian host cell.

15. The recombinant cell of claim 13 , wherein the cell further comprises a RecA polypeptide, and at least one targeting oligonucleotide of at least 30 to about 200 nucleotides, wherein the nucleotide sequence of the targeting oligonucleotide is at least 95% identical over its entire length to an endogenous genomic sequence in the recombinant mammalian host cell.

16. A recombinant cell comprising the recombinant nucleic acid of claim 2 .

17. A recombinant cell comprising the recombinant nucleic acid of claim 3 .

18. A recombinant cell comprising the recombinant nucleic acid of claim 4 .

19. A recombinant cell comprising the vector of claim 8 .