# US 2021/0340548 A1 · Photoactivatable tet expression control system

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- Number: US 2021/0340548 A1
- Document ID: US20210340548A1
- Status: Published application
- Kind: A1
- Application number: 17/271,037
- Filed: 2019-08-30
- Published: 2021-11-04
- Earliest claimed: 2018-08-31
- Assignees: Japan Science and Technology Agency
- Applicants: Japan Science and Technology Agency
- Inventors: Itaru IMAYOSHI; Mayumi YAMADA; Yusuke SUZUKI; Shinji NAGASAKI
- Web page: https://patentgrep.com/patent/US20210340548A1

## CPC

- C12N 15/62: DNA sequences coding for fusion proteins
- C12N 15/66: General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
- C07K 14/4702: {Regulators; Modulating activity}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/635: {Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline}
- 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 provides a photoactivatable Tet-OFF/ON system that can precisely control temporal and spatial gene expression. The present invention is a PA-Tet-OFF/ON system that includes a target gene expression cassette including a TRE having a TetO sequence, a promoter which is controlled by the TRE, and a target gene whose expression is controlled by the promoter; a first fusion protein expression cassette containing a gene which encodes a first fusion protein containing TetR or rTetR and a first protein; and a second fusion protein expression cassette containing a gene which encodes a second fusion protein containing p65AD and a second protein, in which the first protein and the second protein bind to each other to form a heterodimer only in a state of being irradiated with light at a specific wavelength.

## Claims

### Claim 1

A photoactivatable tetracycline gene expression control system, comprising:
a target gene expression cassette including a tetracycline response element having a TetO sequence, a promoter which is positioned downstream of the tetracycline response element and controlled by the tetracycline response element, and a target gene which is positioned downstream of the promoter and of which expression is controlled by the promoter;
a first fusion protein expression cassette including a gene which encodes a first fusion protein containing a Tet repressor protein or a reverse Tet repressor protein and a first protein; and
a second fusion protein expression cassette including a gene which encodes a second fusion protein containing a transactivation domain of a transactivation element p65 and a second protein,
wherein the first protein and the second protein bind to each other and form a heterodimer only in a state of being irradiated with light at a specific wavelength.

### Claim 2 (depends on 1)

The photoactivatable tetracycline gene expression control system according to claim 1,
wherein the Tet repressor protein or the reverse Tet repressor protein has a threonine residue as an amino acid residue corresponding to the 194th isoleucine of a wild-type Tet repressor protein of Escherichia coli.

### Claim 3 (depends on 1)

The photoactivatable tetracycline gene expression control system according to claim 1,
wherein in the first fusion protein, the Tet repressor protein or the reverse Tet repressor protein is linked to the first protein through a peptide linker consisting of an amino acid sequence represented by SPKKK.

### Claim 4 (depends on 1)

The photoactivatable tetracycline gene expression control system according to claim 1,
wherein the first protein is CIB1 or a variant thereof and the second protein is Cry2 or a variant thereof, or
the first protein is Cry2 or a variant thereof and the second protein is CIB1 or a variant thereof.

### Claim 5 (depends on 4)

The photoactivatable tetracycline gene expression control system according to claim 4,
wherein the first protein is CIB1 or a variant thereof, and the second protein is Cry2 or a variant thereof.

### Claim 6 (depends on 5)

The photoactivatable tetracycline gene expression control system according to claim 5,
wherein in the first fusion protein, CIB1 or a variant thereof is linked to a C-terminal side of the Tet repressor protein or the reverse Tet repressor protein.

### Claim 7 (depends on 5)

The photoactivatable tetracycline gene expression control system according to claim 5,
wherein the CIB1 or a variant thereof contained in the first fusion protein is a C-terminal deletion variant of CIB1 that consists of a partial protein corresponding to a region consisting of the 1st to 170th amino acids of wild-type CIB1 of Arabidopsis thaliana or a variant that is obtained by deleting a nuclear localization signal from the C-terminal deletion variant of CIB1.

### Claim 8 (depends on 7)

The photoactivatable tetracycline gene expression control system according to claim 7,
wherein CIB1 or a variant thereof contained in the first fusion protein is a variant obtained by deleting a nuclear localization signal from a C-terminal deletion variant of CIB1 consisting of a partial protein corresponding to a region consisting of the 1st to 170th amino acids of wild-type CIB1 of Arabidopsis thaliana, and
the second fusion protein contains a nuclear localization signal on the N-terminal or the C-terminal.

### Claim 9 (depends on 5)

The photoactivatable tetracycline gene expression control system according to claim 5,
wherein Cry2 or a variant thereof contained in the second fusion protein is a C-terminal deletion variant having an N-terminal photolyase homology region or a variant obtained by substituting an amino acid residue in the C-terminal deletion variant with phenylalanine, and the amino acid residue corresponds to the 348th leucine of wild-type Cry2 of Arabidopsis thaliana.

### Claim 10 (depends on 1)

The photoactivatable tetracycline gene expression control system according to claim 1,
wherein the first protein is Bphp1 or a variant thereof and the second protein is Q-PAS1 or a variant thereof, or
the first protein is Q-PAS1 or a variant thereof and the second protein is Bphp1 or a variant thereof.

### Claim 11 (depends on 10)

The photoactivatable tetracycline gene expression control system according to claim 10,
wherein the first protein is Bphp1 or a variant thereof, and the second protein is Q-PAS1 or a variant thereof.

### Claim 12 (depends on 11)

The photoactivatable tetracycline gene expression control system according to claim 11,
wherein the second fusion protein contains a nuclear localization signal on the N-terminal, and Q-PAS1 or a variant thereof is linked to the C-terminal side of the transactivation domain of the transactivation element p65.

### Claim 13 (depends on 11)

The photoactivatable tetracycline gene expression control system according to claim 11,
wherein in the first fusion protein, Bphp1 or a variant thereof is linked to the N-terminal side of the Tet repressor protein or the reverse Tet repressor protein, and
in the second fusion protein, Q-PAS1 or a variant thereof is linked to the C-terminal side of the transactivation domain of the transactivation element p65.

### Claim 14 (depends on 1)

The photoactivatable tetracycline gene expression control system according to claim 1, further comprising, in addition to the target gene expression cassette:
an expression cassette for a protein in which the first fusion protein and the second fusion protein are linked to each other through a T2A self-cleaving peptide; or
an expression cassette for bicistronically expressing the first fusion protein and the second fusion protein.

### Claim 15 (depends on 1)

The photoactivatable tetracycline gene expression control system according to claim 1,
wherein the target gene is a gene that encodes a protein modified with ubiquitin.

### Claim 16

(canceled)

### Claim 17 (depends on 16)

A method for controlling target gene expression, comprising:
controlling expression of the target gene in the cell according to claim 16 by adjusting conditions so that the cell is irradiated or not irradiated with blue light or near-infrared light and treated or not treated with a tetracycline-based compound.

### Claim 18

A kit for a photoactivatable tetracycline gene expression control system, comprising:
a target gene expression vector including a tetracycline response element having a TetO sequence, a promoter which is positioned downstream of the tetracycline response element and controlled by the tetracycline response element, and a multicloning site which is positioned downstream of the promoter and into which a target gene will be inserted;
a first expression vector including a first fusion protein expression cassette containing a gene that encodes a first fusion protein in which a Tet repressor protein or a reverse Tet repressor protein is linked to CIB 1 or a variant thereof; and
a second expression vector including a second fusion protein expression cassette containing a gene that encodes a second fusion protein in which a transactivation domain of a transactivation element p65 is linked to Cry2 or a variant thereof.

### Claim 19

A kit for a photoactivatable tetracycline gene expression control system, comprising:
a target gene expression vector including a tetracycline response element having a TetO sequence, a promoter which is positioned downstream of the tetracycline response element and controlled by the tetracycline response element, and a multicloning site which is positioned downstream of the promoter and into which a target gene will be inserted;
a first expression vector including a first fusion protein expression cassette containing a gene that encodes a first fusion protein in which a Tet repressor protein or a reverse Tet repressor protein is linked to Bphp1 or a variant thereof; and
a second expression vector including a second fusion protein expression cassette containing a gene that encodes a second fusion protein in which a transactivation domain of a transactivation element p65 is linked to Q-PAS1 or a variant thereof.

### Claim 20 (depends on 18)

The kit for a photoactivatable tetracycline gene expression control system according to claim 18, comprising, instead of the first expression vector and the second expression vector:
an expression vector including an expression cassette for a protein in which the first fusion protein and the second fusion protein are linked to each other through a T2A self-cleaving peptide, or an expression cassette for bicistronically expressing the first fusion protein and the second fusion protein.

### Claim 21 (depends on 18)

The kit for a photoactivatable tetracycline gene expression control system according to claim 18,
wherein the Tet repressor protein or the reverse Tet repressor protein has a threonine residue as an amino acid residue corresponding to the 194th isoleucine of a wild-type Tet repressor protein of Escherichia coli.

### Claim at position 22

22-27. (canceled)