# US 2014/0154397 A1 · Tal-mediated transfer dna insertion

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- Number: US 2014/0154397 A1
- Document ID: US20140154397A1
- Status: Published application
- Kind: A1
- Application number: 14/084,406
- Filed: 2013-11-19
- Published: 2014-06-05
- Earliest claimed: 2012-11-20
- Assignees: J.R. Simplot Company
- Applicants: J.R. Simplot Company
- Inventors: Caius M. Rommens; Hui Duan; J. Troy Weeks
- Web page: https://patentgrep.com/patent/US20140154397A1

## CPC

- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- A23L 19/18: Roasted or fried products, e.g. snacks or chips
- C12N 15/8209: {Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers}
- C12N 15/8245: {involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis}
- C12N 15/825: {involving pigment biosynthesis}
- C12N 15/8251: {Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis}
- C12N 15/8274: {for herbicide resistance}
- C12N 15/8278: {Sulfonylurea}
- C12N 15/8282: {for fungal resistance}
- A23V 2002/00: Food compositions, function of food ingredients or processes for food or foodstuffs
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B)
- A23V: INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION

## Abstract

The invention relates to methods for stably integrating a desired polynucleotide into a plant genome, comprising transforming plant material with a first vector comprising nucleotide sequences encoding TAL proteins designed to recognize a target sequence; transforming the plant material with a second vector comprising (i) a marker gene that is not operably linked to a promoter (“promoter-free marker cassette”) and which comprises a sequence homologous to the target sequence; and (ii) a desired polynucleotide; and identifying transformed plant material in which the desired polynucleotide is stably integrated.

## Claims

### Claim 1

A method for stably integrating a desired polynucleotide into a plant genome, comprising
(A) transforming plant material with a first vector comprising nucleotide sequences encoding TAL proteins designed to recognize a target sequence;
(B) transforming the plant material with a second vector comprising (i) a marker gene that is not operably linked to a promoter (“promoter-free marker cassette”) and which comprises a sequence homologous to the target sequence; and (ii) a desired polynucleotide; and
(C) identifying transformed plant material in which the desired polynucleotide is stably integrated.

### Claim 2 (depends on 1)

The method of claim 1, wherein the transformed plant material is exposed to conditions that reflect the presence or absence of the marker gene in the transformed plant.

### Claim 3 (depends on 2)

The method of claim 2, wherein the marker gene is a herbicide resistance gene and the transformed plant material is exposed to herbicide.

### Claim 4 (depends on 3)

The method of claim 3, wherein the herbicide resistance gene is the ALS gene.

### Claim 5 (depends on 1)

The method of claim 1, wherein the promoter-free marker cassette is stably integrated into the plant genome.

### Claim 6

A method for the targeted insertion of exogenous DNA into a plant comprising the steps of (i) transforming isolated plant cells with
(A) a first binary vector comprising a promoter-less cassette comprising (a) a right border sequence linked to (b) a partial sequence of the Ubi7 intron 5′-untranslated region; (c) an Ubi7 monomer-encoding sequence fused to a mutated acetolactate synthase (ALS) gene; (d) a desired nucleotide sequence; and (e) a terminator sequence, wherein the desired nucleotide sequence is not operably linked to a promoter; and
(B) a second binary vector comprising (a) a right border; (b) a forward expression cassette and a reverse expression cassette, each comprising a modified TAL effector operably linked to a strong constitutive promoter, and a terminator sequence; and (c) a sequence encoding isopentenyl transferase (ipt), wherein the modified TAL effector is designed to bind the desired nucleotide sequence within an intron of potato'subiquitin-7 (Ubi7) gene;
and (ii) culturing the isolated plant cells under conditions that promote growth of plants that express the desired nucleotide sequence; wherein no vector backbone DNA is permanently inserted into the plant genome.

### Claim 7 (depends on 6)

The method of claim 6, wherein the modified TAL effector comprises (a) a truncated C-terminal activation domain comprising a Fok1 endonuclease catalytic domain; (b) a codon-optimized target sequence binding domain comprising 16.5 repeat variable diresidues corresponding to the Ubi7 5′-untranslated intron sequence; and (c) an N-terminal region comprising a SV40 nuclear localization sequence.

### Claim 8 (depends on 7)

The method of claim 7, wherein the desired nucleotide sequence is a silencing cassette targeting one or more genes selected from the group consisting of asparagine synthase 1 (Asn1), polyphenol oxidase (Ppo), and vacuolar invertase (Inv) genes.

### Claim 9 (depends on 8)

The method of claim 8, wherein the first binary vector further comprises a late blight resistance gene Vnt1 operably linked to its native promoter and terminator sequences.

### Claim 10

A transformed plant comprising in its genome an endogenous Ubi7 promoter operably linked to a desired exogenous nucleotide sequence operably linked to an exogenous terminator sequence.

### Claim 11 (depends on 10)

The transformed plant of claim 10, wherein the expression of one or more genes selected from the group consisting of asparagine synthase 1 (Asn1), polyphenol oxidase (Ppo), and vacuolar invertase (Inv) genes is down-regulated.

### Claim 12 (depends on 11)

The transformed plant of claim 11, wherein the plant further expresses a late blight resistance gene Vnt1.

### Claim 13 (depends on 12)

The transformed plant of claim 12, wherein the plant is a tuber-bearing plant.

### Claim 14 (depends on 13)

The transformed plant of claim 13, wherein the tuber-bearing plant is a potato plant.

### Claim 15 (depends on 14)

The transformed plant of claim 14, wherein the plant has a phenotype characterized by one or more of late blight resistance, black spot bruise tolerance, reduced cold-induced sweetening and reduced asparagine levels in its tubers.

### Claim 16 (depends on 15)

A heat-processed product of the transformed plant of claim 15.

### Claim 17 (depends on 16)

The heat-processed product of claim 16, wherein the product is a French fry, chip, crisp, potato, dehydrated potato or baked potato.

### Claim 18 (depends on 17)

The heat-processed product of claim 17, wherein the heat-processed product has a lower level of acrylamide than a heat-processed product of a non-transformed plant of the same species.

### Claim 19

A modified TAL effector designed to bind to a desired sequence comprising (a) a truncated C-terminal activation domain comprising a catalytic domain; (b) a codon-optimized target sequence binding domain; and (c) an N-terminal region comprising a nuclear localization sequence.

### Claim 20 (depends on 19)

The modified TAL effector of claim 19, wherein the modified TAL effector is designed to bind the desired sequence within an intron of potato'subiquitin-7 (Ubi7) gene.

### Claim 21 (depends on 20)

The modified TAL effector of claim 20, wherein (a) the catalytic domain in the C-terminal activation domain comprises a Fok1 endonuclease; (b) the target sequence binding domain comprises 16.5 repeat variable diresidues corresponding to the Ubi7 5′-untranslated intron sequence; and (c) the nuclear localization sequence in the N-terminal region is a SV40 nuclear localization sequence.

### Claim 22 (depends on 16)

A binary vector comprising (a) a right border; (b) a forward expression cassette and a reverse expression cassette, each comprising a modified TAL effector according to claim 16 operably linked to a strong constitutive promoter and a terminator sequence; and (c) a sequence encoding isopentenyl transferase (ipt).

### Claim 23

A DNA construct comprising a promoter-less cassette comprising (a) a right border sequence linked to (b) a partial Ubi7 5′-untranslated intron sequence; (c) an Ubi7 monomer-encoding sequence fused to a mutated acetolactate synthase (ALS) gene; (d) a desired nucleotide sequence; (e) a terminator sequence; and (f) a left border, wherein the desired nucleotide sequence is not operably linked to a promoter.

### Claim 24 (depends on 23)

The DNA construct of claim 23, wherein the desired nucleotide sequence is a silencing cassette targeting one or more genes selected from the group consisting of asparagine synthase 1 (Asn1), polyphenol oxidase (Ppo), and vacuolar invertase (Inv) genes.

### Claim 25 (depends on 24)

The DNA construct of claim 24, wherein the DNA construct further comprises a late blight resistance gene Vnt1 operably linked to its native promoter and terminator sequences.

### Claim 26

A kit for targeted insertion of exogenous DNA into a plant comprising:
(A) a first binary vector comprising a promoter-less cassette comprising (a) a right border sequence linked to (b) a partial sequence of the Ubi7 intron 5′-untranslated region; (c) an Ubi7 monomer-encoding sequence fused to a mutated acetolactate synthase (ALS) gene; (d) a desired nucleotide sequence; and (e) a terminator sequence, wherein the desired nucleotide sequence is not operably linked to a promoter; and
(B) a second binary vector comprising (a) a right border; (b) a forward expression cassette and a reverse expression cassette, each comprising a modified TAL effector operably linked to a strong constitutive promoter, and a terminator sequence; and (c) a sequence encoding isopentenyl transferase (ipt), wherein the modified TAL effector is designed to bind the desired nucleotide sequence within an intron of potato'subiquitin-7 (Ubi7) gene.

### Claim 27 (depends on 26)

The kit of claim 26, wherein the modified TAL effector comprises (a) a truncated C-terminal activation domain comprising a Fok1 endonuclease catalytic domain; (b) a codon-optimized target sequence binding domain comprising 16.5 repeat variable diresidues corresponding to the Ubi7 5′-untranslated intron sequence; and (c) an N-terminal region comprising a SV40 nuclear localization sequence.

### Claim 28 (depends on 27)

The kit of claim 27, wherein the desired nucleotide sequence is a silencing cassette targeting one or more genes selected from the group consisting of asparagine synthase 1 (Asn1), polyphenol oxidase (Ppo), and vacuolar invertase (Inv) genes.

### Claim 29 (depends on 28)

The kit of claim 28, wherein the first binary vector further comprises a late blight resistance gene Vnt1 operably linked to its native promoter and terminator sequences.