# US 2014/0208457 A1 · Nuclease Fusion Protein and Uses Thereof

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- Number: US 2014/0208457 A1
- Document ID: US20140208457A1
- Status: Published application
- Kind: A1
- Application number: 14/124,968
- Filed: 2012-06-08
- Published: 2014-07-24
- Earliest claimed: 2011-06-10
- Assignees: BASF Plant Science GmbH
- Inventors: Ines Fonfara; Wolfgang Wende; Alfred Pingoud
- Web page: https://patentgrep.com/patent/US20140208457A1

## CPC

- C12N 15/902: {using homologous recombination}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C07K 2319/00: Fusion polypeptide
- C07K 2319/80: containing a DNA binding domain, e.g. Lacl or Tet-repressor
- 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)

## Abstract

The present invention is concerned with nuclease fusion proteins and various uses thereof. Specifically, it relates to a polynucleotide encoding a polypeptide comprising (i) a first module comprising at least a first DNA binding domain derived from a homing endonuclease, (ii) a linker, and (iii) a second module comprising at least a second DNA binding domain and a cleavage domain derived from a restriction endonuclease, wherein said polypeptide functionally interacts only with DNA comprising a DNA recognition site for the first DNA binding domain and a DNA recognition site for the second DNA binding domain, and wherein said cleavage domain cleaves DNA within a specific DNA cleavage site upon binding of the polypeptide. Further contemplated are a vector and a non-human transgenic organism comprising said polynucleotide as well as a polypeptide encoded by the polynucleotide of the invention. Finally, the present invention relates to a method for introducing a nucleic acid of interest into a genome of a non-human organism wherein the polypeptide of the invention is applied.

## Claims

### Claim at position 1

1-18. (canceled)

### Claim 19

A polynucleotide encoding a polypeptide comprising:
(i) a first module comprising at least a first DNA binding domain derived from a homing endonuclease;
(ii) a linker; and
(iii) a second module comprising at least a second DNA binding domain and a cleavage domain derived from a restriction endonuclease;
wherein said polypeptide functionally interacts only with DNA comprising a DNA recognition site for the first DNA binding domain and a DNA recognition site for the second DNA binding domain, and
wherein said cleavage domain cleaves DNA within a specific DNA cleavage site upon binding of the polypeptide.

### Claim 20 (depends on 19)

The polynucleotide of claim 19, wherein said specific DNA cleavage site and said DNA recognition site of the second DNA binding domain of the restriction endonuclease are identical.

### Claim 21 (depends on 19)

The polynucleotide of claim 19, wherein said second DNA binding domain and the cleavage domain comprised in the second module are derived from a type IIP restriction endonuclease.

### Claim 22 (depends on 19)

The polynucleotide of claim 19, wherein said second DNA binding domain and the cleavage domain comprised in the second module are derived from a restriction endonuclease which exhibits reduced DNA binding and/or reduced catalytic activity when compared to the wild type restriction endonuclease.

### Claim 23 (depends on 19)

The polynucleotide of claim 19, wherein said second DNA binding domain and the cleavage domain comprised in the second module are derived from PvuII.

### Claim 24 (depends on 23)

The polynucleotide of claim 23, wherein said second DNA binding domain and the cleavage domain comprised in the second module comprise at least one of following modifications: substitution T46G, substitution H83A, substitution Y94F, substitution T46G in combination with H83A, substitution T46G in combination with Y94F or substitution T46G in combination with H83A and Y94F.

### Claim 25 (depends on 19)

The polynucleotide of claim 19, wherein said first module exhibits reduced or no catalytic activity.

### Claim 26 (depends on 19)

The polynucleotide of claim 19, wherein said first DNA binding domain comprised in the first module is derived from a LAGLIDADG-family homing endonuclease.

### Claim 27 (depends on 26)

The polynucleotide of claim 26, wherein said first DNA binding domain comprised in the first module is derived from I-SceI.

### Claim 28 (depends on 27)

The polynucleotide of claim 27, wherein said first DNA binding domain comprised in the first module comprise at least one of following modifications: substitution D44S alone or in combination with D145A, or substitution D44N in combination with D145A.

### Claim 29 (depends on 19)

The polynucleotide of claim 19, wherein said linker consists essentially of 6 to 10 amino acids.

### Claim 30 (depends on 29)

The polynucleotide of claim 29, wherein said linker has an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

### Claim 31 (depends on 19)

A vector comprising the polynucleotide of claim 19.

### Claim 32 (depends on 19)

A non-human organism comprising the polynucleotide of claim 19.

### Claim 33 (depends on 19)

A polypeptide encoded by the polynucleotide of claim 19.

### Claim 34

A nucleic acid molecule comprising in 5′ to 3′ direction:
i. a DNA recognition site of I-SceI,
ii. a first nucleic acid linker,
iii. a DNA recognition site of PvuII,
iv. a second nucleic acid linker, and
v. the reverse complement sequence of a DNA recognition site of I-SceI.

### Claim 35 (depends on 34)

A vector comprising the polynucleotide of claim 34.

### Claim 36 (depends on 34)

A non-human transgenic organism comprising the polynucleotide of claim 34.

### Claim 37 (depends on 19)

A non-human transgenic organism comprising the polynucleotide of claim 19 and further comprising a nucleic acid molecule comprising in 5′ to 3′ direction:
i. a DNA recognition site of I-SceI,
ii. a first nucleic acid linker,
iii. a DNA recognition site of PvuII,
iv. a second nucleic acid linker, and
v. the reverse complement sequence of a DNA recognition site of I-SceI.

### Claim 38 (depends on 33)

A method for introducing a nucleic acid of interest into a genome of a non-human organism comprising:
a) introducing into a non-human organism a nucleic acid of interest to be introduced into the genome of the said organism
b) expressing the polypeptide of claim 33 in said organism; and
c) cultivating said organism under conditions allowing said polypeptide to cleave the genome and allowing the nucleic acid of interest to become introduced into the genome.

### Claim 39 (depends on 38)

The method of claim 38, wherein said non-human transgenic organism is selected from the group consisting of: a microorganism, a plant, plant part or isolated cell thereof, or an animal tissue or isolated cell thereof.

### Claim 40 (depends on 38)

The non-human transgenic organism of claim 38, wherein said non-human transgenic organism is selected from the group consisting of: a microorganism, a plant, plant part or isolated cell thereof, or an animal tissue or isolated cell thereof.

### Claim 41 (depends on 36)

The non-human transgenic organism of claim 36, wherein said non-human transgenic organism is selected from the group consisting of: a microorganism, a plant, plant part or isolated cell thereof, or an animal tissue or isolated cell thereof.