Ref nuclease for site specific Ref-mediated DNA cleavage

• US 9,752,133 B2
• Filed: 03/10/2017
• Issued: 09/05/2017
• Est. Priority Date: 07/20/2013
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Cox, Michael M., Gruber, Angela J., Olsen, Tayla M.
• Primary Examiner(s): FRONDA, CHRISTIAN L
• Application Number:
  US15/455,255
• Publication Number:
  US 20170183640A1
• Time in Patent Office: 179 Days
• Field of Search: None
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 15/01 Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/00 Structure or type of the nucleic acid
• CPC Class Codes: C12N 2795/10122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/11 Exodeoxyribonucleases producing 5'-phosphomonoesters (3.1.11)

Abstract

Purified Ref polypeptides with increased nuclease site-specific targeting activity, recombinant nucleic acids and cells for expression of such Ref polypeptides, and methods for using the Ref polypeptides in combination with RecA protein and variants thereof to effect targeted nuclease cleavage of a DNA duplex are disclosed.

Claims

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.