# US 2021/0340554 A1 · Method for producing genome-modified plants from plant protoplasts at high efficiency

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- Number: US 2021/0340554 A1
- Document ID: US20210340554A1
- Status: Published application
- Kind: A1
- Application number: 17/366,320
- Filed: 2021-07-02
- Published: 2021-11-04
- Earliest claimed: 2015-10-06
- Assignees: Institute for Basic Science; Seoul National University R&Db Foundation; Aict
- Applicants: Institute for Basic Science; Seoul National University R&Db Foundation; Aict
- Inventors: Jin Soo KIM; Jungeun KIM; Sunghwa CHOE; Je Wook WOO; Soon Il KWON; Hyeran KIM
- Web page: https://patentgrep.com/patent/US20210340554A1

## CPC

- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/8206: {by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated}
- C12N 15/8207: {by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers}
- C12N 15/8298: {Brassinosteroids}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to a method of increasing the production efficiency of gene-edited plants, regenerated from plant protoplasts, by use of a Cas protein-guide RNA ribonucleoprotein (RNP). According to the present invention, the method of increasing the production efficiency of gene-edited plants makes it possible to efficiently produce target gene-mutated plants and to minimize the introduction of foreign DNA into plants. Thus, the present invention can be very advantageously used in a wide variety of fields, including agriculture, food and biotechnology.

## Claims

### Claim 1

A method for increasing the production efficiency of a genome-edited plant from a plant protoplast, comprising the steps of:
(i) editing a genome of the plant protoplast by a direct introduction of a Cas protein-guide RNA ribonucleoprotein (RNP) in which a Cas protein and a guide RNA in the form of naked RNA are pre-assembled into an isolated plant protoplast without using a vector; and
(ii) producing a genome-edited plant by regenerating the plant protoplast.

### Claim 2 (depends on 1)

The method of claim 1, wherein the guide RNA is specific for a DNA encoding a target gene.

### Claim 3 (depends on 2)

The method of claim 2, wherein the target gene is a Brassinosteroid Insensitive 2 (BIN2) gene or a Glucosinolate-oxoglutarate-dependent dioxygenase homolog (GSL-ALK) gene.

### Claim 4 (depends on 1)

The method of claim 1, wherein the editing of a genome is performed by knocking-out or knocking-in.

### Claim 5 (depends on 1)

The method of claim 1, wherein the guide RNA is in the form of a dual RNA comprising a crRNA and a tracrRNA, or a single-chain guide RNA (sgRNA).

### Claim 6 (depends on 5)

The method of claim 5, wherein the single-chain guide RNA comprises a part of crRNA and a part of tracrRNA.

### Claim 7 (depends on 1)

The method of claim 1, wherein the Cas protein is a Cas9 protein or a variant of Cas9 Protein in which the catalytic aspartate residue is substituted with another amino acid.

### Claim 8 (depends on 1)

The method of claim 1, wherein the Cas protein recognizes NGG trinucleotide.

### Claim 9 (depends on 1)

The method of claim 1, wherein the Cas protein is linked to a protein transduction domain.

### Claim 10 (depends on 7)

The method of claim 7, wherein the amino acid is alanine.

### Claim 11 (depends on 1)

The method of claim 1, wherein the Cas9 protein is derived from the genus Streptococcus.

### Claim 12 (depends on 11)

The method of claim 11, wherein the genus Streptococcus is Streptococcus pyogenes.

### Claim 13 (depends on 1)

The method of claim 1, wherein the plant protoplast is derived from Lactuca sativa or Brassica oleracea.

### Claim 14 (depends on 1)

The method of claim 1, wherein the introduction is performed by the method selected from the group consisting of microinjection, electroporation, DEAE-dextran treatment, lipofection, nanoparticle-mediated transfection, protein transduction domain-mediated transduction, and PEG-mediated transfection.

### Claim 15 (depends on 1)

A plant regenerated from the genome-edited plant protoplast produced by the method of claim 1.