REF Nuclease for Site-Specific REF-Mediated DNA Cleavage

• US 20150023943A1
• Filed: 07/21/2014
• Published: 01/22/2015
• 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 Matthew, Gruber, Angela Jo, Olsen, Tayla Maria
• Granted Patent: US 9,611,470 B2
• Time in Patent Office: Days
• US Class Current: 424/94.6
• 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 purified Ref polypeptide comprising an amino acid sequence at least 95% identical to any of SEQ ID NOs:

4 and 5, wherein

(i) the Ref polypeptide does not comprise the entire amino acid sequence of a naturally occurring full length Ref protein, SEQ ID NO;

1, or SEQ ID NO;

2;

in that the Ref polypeptide does not include at least the first ten amino acids of the N-terminal segment of the entire amino acid sequence, and

(ii) the 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 purified Ref polypeptide of claim 1, wherein the amino acid sequence comprises:

(a) one or more of 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;

or

(b) an amino acid sequence at least 95% identical to one or more of the group consisting of SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10; and

SEQ ID NO;

11.

3. The purified Ref polypeptide 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 purified Ref polypeptide of claim 1, wherein the purified Ref polypeptide is a fusion polypeptide.

5. A kit comprising:

(i) the purified Ref polypeptide of claim 1; and

(ii) a purified RecA polypeptide comprising an amino acid sequence at least 95% identical to any of SEQ ID NOs;

12-16.

6. The kit of claim 5, wherein the purified RecA comprises an amino acid at least 95% identical to SEQ ID NO:

13.

7. The kit of claim 5, further comprising a targeting oligonucleotide.

8. The kit of claim 7, wherein the targeting oligonucleotide is from 60-150 nucleotides in length.

9. The kit of claim 6, further comprising a reaction buffer suitable for an in vitro nuclease site-specific targeting reaction in the presence of the purified Ref polypeptide of (i), the purified RecA polypeptide (ii), a targeting oligonucleotide, and a double stranded DNA comprising a target sequence to be cleaved.

10. A recombinant nucleic acid encoding the Ref polypeptide of claim 1.

11. The recombinant nucleic acid of claim 9, further comprising a heterologous nucleic acid sequence on its 5′

or 3′

end.

12. The recombinant nucleic acid of claim 10, wherein the heterologous nucleic acid is a 5′

UTR, a 3′

UTR, a Kozak consensus sequence, a Shine-Delgarno sequence, or a promoter.

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

14. A recombinant cell comprising the recombinant nucleic acid of claim 10.

15. A method for cleaving a DNA duplex within a target-defined nucleotide sequence, the method comprising:

(i) providing a complex of a RecA protein, a double stranded DNA, and a targeting oligonucleotide comprising a sequence homologous to a target sequence found within the double stranded DNA; and

(ii) contacting the complex from (i) with the purified Ref polypeptide of claim 1.

16. The method of claim 15, wherein the wherein the amino acid sequence of the Ref polypeptide comprises:

(a) one or more of 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;

or

(b) an amino acid sequence at least 95% identical to one or more of the group consisting of SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10; and

SEQ ID NO;

11.

17. The method of claim 14, 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.

18. The method of claim 14, wherein the targeting oligonucleotide is from 60-150 nucleotides in length.

19. The method of claim 14, wherein the method is performed as part of a genome editing process.

20. The method of claim 19, wherein the method is performed in vivo.