US 2024/0287535 A1 · DELAY OR PREVENTION OF BROWNING IN BANANA FRUIT

Abstract

The present invention relates to compositions and methods for the delay or prevention of browning in banana fruit by genetically editing one or more genes encoding a Polyphenol Oxidase (PPO).

Claims35

Dependent claims

A method of reducing the level or activity of at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase encoded by a PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene in a banana plant or banana plant cell.

A method of producing a banana plant characterised by a phenotype of delayed and/or reduced browning of fruit flesh and/or peel as compared to a wild-type banana plant, the method comprising:

(a) providing to a banana plant cell a CRISPR-associated endonuclease or a modified CRISPR-associated endonuclease, and one or more guide RNAs, wherein said CRISPR-associated endonuclease or modified CRISPR-associated endonuclease and said one or more guide RNAs form a complex that enables the CRISPR-associated endonuclease or modified CRISPR-associated endonuclease to introduce a single-strand break or a double-strand break in at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene:
(A) comprising a coding sequence selected from the group consisting of:
(i) SEQ ID NO:
5 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
5;
(ii) SEQ ID NO:
6 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
6;
(iii) SEQ ID NO:
12 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
12;
(iv) SEQ ID NO:
13 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
13;
and
(v) SEQ ID NO:
8 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
8;
(B) encoding a polyphenol oxidase selected from the group consisting of:
(i) SEQ ID NO:
40 or a polypeptide with at least 75% sequence identity to SEQ ID NO:
40;
(ii) SEQ ID NO:
41 or a polypeptide with at least 75% sequence identity to SEQ ID NO:
41;
(iii) SEQ ID NO:
47 or a polypeptide with at least 75% sequence identity to SEQ ID NO:
47;
(iv) SEQ ID NO:
48 or a polypeptide with at least 75% sequence identity to SEQ ID NO:
48;
and
(v) SEQ ID NO:
43 or a polypeptide with at least 75% sequence identity to SEQ ID NO:
43;
or
(C) comprising a polynucleotide sequence selected from the group consisting of:
(i) SEQ ID NO:
151 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
151;
(ii) SEQ ID NO:
152 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
152;
(iii) SEQ ID NO:
158 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
158;
(iv) SEQ ID NO:
159 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
159;
and
(v) SEQ ID NO:
154 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
154;
(b) identifying at least one banana plant cell that comprises a modification of said at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene, wherein the modification is selected from the group consisting of:
(i) at least one nucleotide insertion;
(ii) at least one nucleotide deletion;
(iii) at least one nucleotide substitution;
or
(iv) any combination of (b)(i) to (b)(iii);
and
(c) regenerating a banana plant from said banana plant cell, wherein said banana plant is characterised by a phenotype of delayed and/or reduced browning of fruit flesh and/or peel as compared to a wild-type banana plant.

58 more elements

A banana plant or plant part comprising in its genome at least one modified endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene, wherein said modification results in a reduction in, or loss of function of, the at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase encoded by said modified endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene, wherein said modification is located in at least one endogenous PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene:

(A) comprising a coding sequence selected from the group consisting of:
(a) SEQ ID NO:
5 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
5;
(b) SEQ ID NO:
6 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
6;
(c) SEQ ID NO:
12 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
12;
(d) SEQ ID NO:
13 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
13;
and
(e) SEQ ID NO:
8 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
8;
(B) encoding a polyphenol oxidase selected from the group consisting of:
(a) SEQ ID NO:
40 or a polypeptide with at least 75% sequence identity to SEQ ID NO:
40;
(b) SEQ ID NO:
41 or a polypeptide with at least 75% sequence identity to SEQ ID NO:
41;
(c) SEQ ID NO:
47 or a polypeptide with at least 75% sequence identity to SEQ ID NO:
47;
(d) SEQ ID NO:
48 or a polypeptide with at least 75% sequence identity to SEQ ID NO:
48;
and
(e) SEQ ID NO:
43 or a polypeptide with at least 75% sequence identity to SEQ ID NO:
43;
or
(C) comprising a polynucleotide sequence selected from the group consisting of:
(a) SEQ ID NO:
151 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
151;
(b) SEQ ID NO:
152 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
152;
(c) SEQ ID NO:
158 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
158;
(d) SEQ ID NO:
159 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
159;
and
(e) SEQ ID NO:
154 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
154.

49 more elements

A recombinant DNA construct comprising a promoter operably linked to a nucleotide sequence expressing at least one banana PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase guide RNA, wherein said guide RNA is capable of forming a complex with a CRISPR-associated endonuclease or modified CRISPR-associated endonuclease, and wherein said complex is capable of binding to and creating a double-strand or single-strand break in the at least one endogenous banana PPO1, PPO2, PPO8, PPO9, or PPO4 polyphenol oxidase gene:

(A) comprising a coding sequence selected from the group consisting of:
(a) SEQ ID NO:
5 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
5;
(b) SEQ ID NO:
6 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
6;
(c) SEQ ID NO:
12 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
12;
(d) SEQ ID NO:
13 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
13;
and
(e) SEQ ID NO:
8 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
8;
(B) encoding a polyphenol oxidase selected from the group consisting of:
(a) SEQ ID NO:
40 or a polypeptide with at least 75% sequence identity to SEQ ID NO:
40;
(b) SEQ ID NO:
41 or a polypeptide with at least 75% sequence identity to SEQ ID NO:
41;
(c) SEQ ID NO:
47 or a polypeptide with at least 75% sequence identity to SEQ ID NO:
47;
(d) SEQ ID NO:
48 or a polypeptide with at least 75% sequence identity to SEQ ID NO:
48;
and
(e) SEQ ID NO:
43 or a polypeptide with at least 75% sequence identity to SEQ ID NO:
43;
or
(C) comprising a polynucleotide sequence selected from the group consisting of:
(a) SEQ ID NO:
151 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
151;
(b) SEQ ID NO:
152 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
152;
(c) SEQ ID NO:
158 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
158;
(d) SEQ ID NO:
159 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
159;
and
(e) SEQ ID NO:
154 or a polynucleotide with at least 75% sequence identity to SEQ ID NO:
154;
in the banana genome.

50 more elements

Dependencies are inferred from the claim text.

Facts

Application

18/571,140Filed 30 Jun 2022

Priority

GB 2109585.62 Jul 2021

Published

29 Aug 2024

Term ends

Not estimatedThe source records don’t support an estimate.

Classification16 codes

A01H 5/08
Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy: fruits
C12N 15/8262
With agronomic traits, e.g. crop yield: involving plant development
C12N 9/226
Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes: class 2 CAS enzyme complex, e.g. single CAS protein
C12N 15/11
Recombinant DNA-technology: dNA or RNA fragments; Modified forms thereof ; Non-coding nucleic acids having a biological activity

Abstract

The present invention relates to compositions and methods for the delay or prevention of browning in banana fruit by genetically editing one or more genes encoding a Polyphenol Oxidase (PPO).

Delay or prevention of browning in banana fruit

Life of this patent

Abstract

Claims35

Delay or prevention of browning in banana fruit

Life of this patent

Abstract

Claims35