# US 2023/0056748 A1 · Method for suppressing methylation of target dna in plant

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- Number: US 2023/0056748 A1
- Document ID: US20230056748A1
- Status: Published application
- Kind: A1
- Application number: 17/789,075
- Filed: 2020-12-25
- Published: 2023-02-23
- Earliest claimed: 2019-12-26
- Assignees: National Institute of Advanced Industrial Science and Technology
- Applicants: National Institute of Advanced Industrial Science and Technology
- Inventors: Go ATSUMI; Takeshi MATSUMURA; Chikara MASUTA; Tsuyoshi INUKAI; Wataru MATSUNAGA; Reika ISODA
- Web page: https://patentgrep.com/patent/US20230056748A1

## CPC

- 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)}
- C12N 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]}
- A01H 1/00: Processes for modifying genotypes {; Plants characterised by associated natural traits} (A01H4/00 takes precedence)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES

## Abstract

According to the present invention, methylation of a target DNA in a plant is suppressed quickly and easily without using recombinant technology, and a plant having desired traits is thereby created. In an RNA-directed DNA methylation mechanism, binding between scaffold RNA produced by transcription of the target DNA and an siRNA-AGO4 complex is inhibited.

## Claims

### Claim 1

A method for inhibiting methylation of a target DNA in a plant cell, comprising a step of inhibiting binding of a scaffold RNA produced by transcription of the target DNA with an siRNA-AGO4 complex, in an RNA-directed DNA methylation mechanism.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the step of inhibiting binding of the scaffold RNA with the siRNA-AGO4 complex is accomplished by introducing into the plant cell a short dummy RNA comprising a sequence complementary to the siRNA incorporated in the siRNA-AGO4 complex.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the step of inhibiting binding of the scaffold RNA with the siRNA-AGO4 complex is accomplished by introducing into the plant cell a short dummy RNA comprising a sequence complementary to at least a portion of the scaffold RNA.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the target DNA is a promoter that controls a gene expressing a desired trait for the plant cell.

### Claim 5 (depends on 4)

The method according to claim 4, wherein the gene that expresses the desired trait is a gene coding for the amino acid sequence of an enzyme that controls synthesis or accumulation of a plant-derived functional component.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the short dummy RNA is introduced into the plant cell by a plant virus vector method, an agroinfiltration method, a magnICON® system or a particle gun method.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the plant cell is a non-isolated cell of a plant body.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the plant cell is a cultured cell.

### Claim 9 (depends on 1)

A method for creating a plant with a desired trait, comprising a step of inhibiting methylation of a DNA involved in expression of the desired trait for the plant by using the method according to claim 1.

### Claim 10 (depends on 1)

A method for producing a plant-derived functional component, comprising steps of inhibiting methylation of DNA involved in synthesis or accumulation of a plant-derived functional component by using the method according to claim 1, thereby causing accumulation of the functional component in a plant cell, and recovering the functional component from the plant cell.

### Claim 11

An expression system for accumulation of a plant-derived functional component, comprising:
(A) a plant body or a plant cell that produces a scaffold RNA produced by transcription of a DNA involved in synthesis or accumulation of the plant-derived functional component, and siRNA-AGO4 complex, in an RNA-directed DNA methylation mechanism, and
(B) a short dummy RNA comprising a sequence complementary to the siRNA incorporated in the siRNA-AGO4 complex, or a sequence complementary to at least a portion of the scaffold RNA.