# US 9,752,133 B2 · Ref nuclease for site specific Ref-mediated DNA cleavage

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- Number: US 9,752,133 B2
- Document ID: US9752133B2
- Status: Granted
- Kind: B2
- Application number: 15/455,255
- Filed: 2017-03-10
- Published: 2017-06-29
- Granted: 2017-09-05
- Earliest claimed: 2013-07-20
- Assignees: Wisconsin Alumni Research Foundation
- Applicants: Wisconsin Alumni Research Foundation
- Inventors: Michael M. Cox; Angela J. Gruber; Tayla M. Olsen
- Web page: https://patentgrep.com/patent/US9752133B2

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C07K 14/005: from viruses
- C12N 15/01: Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
- 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)}
- C12Y 301/11: Exodeoxyribonucleases producing 5'-phosphomonoesters (3.1.11)
- C12N 2310/00: Structure or type of the nucleic acid
- C12N 2795/10122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Y: ENZYMES

## 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

### Claim 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 2)

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.

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 4)

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.

### Claim 6 (depends on 1)

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

### Claim 7 (depends on 6)

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.

### Claim 8 (depends on 1)

A vector comprising the recombinant nucleic acid of claim 1.

### Claim 9 (depends on 2)

A vector comprising the recombinant nucleic acid of claim 2.

### Claim 10 (depends on 3)

A vector comprising the recombinant nucleic acid of claim 3.

### Claim 11 (depends on 4)

A vector comprising the recombinant nucleic acid of claim 4.

### Claim 12 (depends on 1)

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

### Claim 13 (depends on 12)

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

### Claim 14 (depends on 13)

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

### Claim 15 (depends on 13)

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.

### Claim 16 (depends on 2)

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

### Claim 17 (depends on 3)

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

### Claim 18 (depends on 4)

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

### Claim 19 (depends on 8)

A recombinant cell comprising the vector of claim 8.