# US 2020/0399648 A1 · Compositions and methods for modification of fatty acids in soybean

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- Number: US 2020/0399648 A1
- Document ID: US20200399648A1
- Status: Published application
- Kind: A1
- Application number: 16/977,608
- Filed: 2019-03-01
- Published: 2020-12-24
- Earliest claimed: 2018-03-09
- Assignees: Pioneer Hi-Bred International, Inc.
- Applicants: Pioneer Hi-Bred International, Inc.
- Inventors: JOHN D EVERARD; ANTHONY J KINNEY; ZHAN-BIN LIU
- Web page: https://patentgrep.com/patent/US20200399648A1

## CPC

- A01H 6/542: {Glycine max [soybean]}
- C12N 15/8247: {involving modified lipid metabolism, e.g. seed oil composition}
- C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)}
- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C12Q 1/6895: for plants, fungi or algae
- C12Q 2600/13: Plant traits
- C12Q 2600/156: Polymorphic or mutational markers
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Methods and compositions to modify the fatty acid profiles of seeds and the oils produced therefrom include the modification of four or more alleles of fatty acid desaturases in the genome of a plant. Compositions, polynucleotide constructs, transformed and modified host cells, and plants and seeds exhibiting altered fatty characteristics are provided.

## Claims

### Claim 1

A method of altering the fatty acid profile in the seed of a soybean plant, the method comprising introducing four or more nucleotide modifications through four or more targeted DNA breaks at four or more genomic loci of a plant, wherein
(a) the genomic loci comprise a first polynucleotide encoding a FAD2 polypeptide and second polynucleotide encoding a FAD3 polypeptide, and
(b) wherein the oleic acid content is increased to at least 75% by weight of the total fatty acids and the linolenic acid content is decreased to less than 3% by weight of the total fatty acids in the seed compared to the seed of a control plant not comprising the four or more introduced nucleotide modifications.

### Claim 2 (depends on 1)

The method of claim 1, wherein four nucleotide modifications are introduced through targeted DNA breaks.

### Claim 3 (depends on 1)

The method of claim 1, wherein the nucleotide modifications are introduced at the same time.

### Claim 4 (depends on 1)

The method of claim 1, wherein the nucleotide modifications are introduced into polynucleotides encoding FAD2-1A, FAD2-1B, FAD3a and FAD3b.

### Claim 5 (depends on 1)

The method of claim 1, wherein the nucleotide modifications are introduced by targeted DNA breaks using no more than two guide RNAs.

### Claim 6 (depends on 1)

The method of any claim 1, wherein the genetic modifications target more than four distinct genomic loci that are involved in fatty acid metabolism.

### Claim 7

(canceled)

### Claim 8

(canceled)

### Claim 9 (depends on 1)

The method of claim 1, wherein the altered fatty acid profile does not substantially affect yield.

### Claim 10

(canceled)

### Claim 11

(canceled)

### Claim 12 (depends on 1)

The method of claim 1, wherein the modifications target the genomic locus at a target site such that more than one genetic modifications are present within (a) the same coding region; (b) non-coding region; (c) regulatory sequence; or (d) untranslated region of an endogenous polynucleotide encoding a polypeptide that is involved in fatty acid metabolism.

### Claim 13

(canceled)

### Claim 14 (depends on 1)

The method of claim 1, wherein the target site comprises SEQ ID NO: 6 or SEQ ID NO:7.

### Claim 15 (depends on 1)

The method of claim 1, wherein the first polynucleotide encodes a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NOS: 70, 72 or a combination thereof, and the second polynucleotide encodes a polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NOS: 74, 76 or a combination thereof.

### Claim 16 (depends on 1)

The method of claim 1, wherein the first polynucleotide comprises at least one of SEQ ID NOS: 35-58 and wherein the second polynucleotide comprises at least one of SEQ ID NOS:59-64.

### Claim 17 (depends on 1)

The method of claim 1, wherein the first polynucleotide encodes a FAD2-1A polypeptide, the second polynucleotide encodes a FAD3a polypeptide, and wherein the genomic loci comprise a third polynucleotide encoding a FAD2-1B polypeptide and a fourth polynucleotide encoding a FAD3b polypeptide.

### Claim 18 (depends on 17)

The method of claim 17, wherein the first polynucleotide encodes a FAD2-1A polypeptide having at least 90% identity to SEQ ID NO: 70, the second polynucleotide encodes a FAD3a polypeptide having at least 90% identity to SEQ ID NO: 74, and wherein the genomic loci comprise a third polynucleotide encoding a FAD2-1B polypeptide having at least 90% identity to SEQ ID NO: 72 and a fourth polynucleotide encoding a FAD3b polypeptide having at least 90% identity to SEQ ID NO: 76.

### Claim 19 (depends on 18)

The method of claim 18, wherein the first polynucleotide comprises SEQ ID NO: 54, the second polynucleotide comprises SEQ ID NO: 60, the third polynucleotide comprises SEQ ID NO: 57, and the fourth polynucleotide comprises SEQ ID NO: 63.

### Claim 20 (depends on 18)

The method of claim 18, wherein the first polynucleotide comprises SEQ ID NO: 55, the second polynucleotide comprises SEQ ID NO: 61, the third polynucleotide comprises SEQ ID NO: 58, and the fourth polynucleotide comprises SEQ ID NO: 64.

### Claim 21 (depends on 1)

The method of claim 1, wherein the genomic loci comprise an edit in a polynucleotide that encodes a FAD3 polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOS: 74 or 76 such that the edit results in one or more of the following:
(a) reduced expression of a polynucleotide encoding the FAD3 polypeptide;
(b) reduced activity of the FAD3 polypeptide;
(c) generation of one or more alternative spliced transcripts of a polynucleotide encoding the FAD3 polypeptide;
(d) deletion of one or more active sites of the FAD3 polypeptide;
(e) frameshift mutation in one or more exons of a polynucleotide encoding the FAD3 polypeptide;
(f) deletion of a substantial portion of the polynucleotide encoding the FAD3 polypeptide or deletion of the polynucleotide encoding the full-length FAD3 polypeptide;
(g) repression of an enhancer motif present within a regulatory region encoding the FAD3 polypeptide; and
(h) modification of one or more nucleotides or deletion of a regulatory element operably linked to the expression of the polynucleotide encoding the FAD3 polypeptide, wherein the regulatory element is present within a promoter, intron, 3′UTR, terminator or a combination thereof.

### Claim 22 (depends on 1)

The method of claim 1, wherein the genomic loci comprise an edit in a polynucleotide that encodes a FAD2-1 polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOS: 70 or 72 such that the edit results in one or more of the following:
(a) reduced expression of a polynucleotide encoding the FAD2-1 polypeptide;
(b) reduced activity of the FAD2-1 polypeptide;
(c) generation of one or more alternative spliced transcripts of a polynucleotide encoding the FAD2-1 polypeptide;
(d) deletion of one or more domains of the Br2 polypeptide;
(e) frameshift mutation in one or more exons of a polynucleotide encoding the FAD2-1 polypeptide;
(f) deletion of a substantial portion of the polynucleotide encoding the FAD2-1 polypeptide or deletion of the polynucleotide encoding the FAD2-1 polypeptide;
(g) repression of an enhancer motif present within a regulatory region encoding the FAD2-1 polypeptide;
(h) modification of one or more nucleotides or deletion of a regulatory element operably linked to the expression of the polynucleotide encoding the FAD2-1 polypeptide, wherein the regulatory element is present within a promoter, intron, 3′UTR, terminator or a combination thereof.

### Claim 23

(canceled)

### Claim 24 (depends on 1)

The method of claim 1, wherein the targeted DNA break is not effected using a TALEN (transcription activator-like effector nuclease).

### Claim 25

A soybean plant comprising a first, second, third and fourth modified genomic locus, each locus comprising one or more mutations compared to a control plant and wherein the first, second, third and fourth modified genomic loci encode a polypeptide that is at least 95% identical to the amino acid sequence of SEQ ID NOS: 70, 72, 74 and 76, respectively, and wherein the soybean plant has an altered fatty acid profile, produces seeds comprising at least 75% oleic acid by weight of the total fatty acids and less than 3% linolenic acid by weight of the total fatty acids, and contains no heterologous or foreign DNA at the first, second, third and fourth modified genomic loci.

### Claim at position 26

-38. (canceled)

### Claim 39

A method of detecting the presence of a polynucleotide comprising SEQ ID NOs: 36-43, 45-52, 54, 55, 57, 58, 60, 61, 63, 64, 77, 84-91, 93-100, 102-109, 111-118 or 136-145, the polynucleotide indicating the presence of a deletion modification in a FAD2-1 or FAD3 allele, the method comprising the steps of:
(a) contacting a DNA sample obtained from a soybean plant or part thereof with a first and a second DNA primer molecule, the first DNA primer molecule comprising at least 12 contiguous nucleotides of SEQ ID NOs: 36-43, 45-52, 54, 55, 57, 58, 60, 61, 63, 64, 77, 84-91, 93-100, 102-109, 111-118 or 136-145, the contiguous nucleotides comprising the junction sequence nucleotides at positions 18-19 for SEQ ID NOs: 43, 123, 130 and 134; positions 19-20 for SEQ ID NO: 143; positions 20-21 for SEQ ID NOs: 60-61, 63-64, 116, 120-122, 127, 131, 133 and 144; positions 20-22 for SEQ ID NO: 145; positions 21-22 for SEQ ID NOs: 78-79, 124, 125, 128, 129, 132 and 135; positions 22-23 for SEQ ID NOs: 36 and 117; positions 23-24 for SEQ ID NOs: 55, 94 and 100; positions 24-25 for SEQ ID NOs: 106, 137 and 142; positions 25-26 for SEQ ID NOs: 47 and 88; positions 26-27 for SEQ ID NOs: 46, 48, 87, 91, 98, 99, 108, 112, 114 and 141; positions 27-28 for SEQ ID NOs: 37-40, 45, 50-52, 54, 57, 58, 84, 90, 93, 97, 103, 105, 107, 111, 115, 136 and 138; positions 27-29 for SEQ ID NOs: 49 and 139; positions 28-29 for SEQ ID NOs: 41, 42, 85, 86, 89, 95, 96, 102, 104, 109, 113 and 118; positions 29-30 for SEQ ID NO: 140; or the reverse complement thereof, and the second DNA molecule complementary to at least 12 contiguous nucleotides of soybean genomic DNA in proximity to and upstream or downstream of the binding site of the first DNA primer molecule;
(b) providing a nucleic acid amplification reaction condition;
(c) performing the nucleic acid amplification reaction, thereby producing a DNA amplicon molecule indicating the presence of a deletion modification in a FAD2-1 or FAD3 allele; and
(d) detecting the DNA amplicon molecule.

### Claim at position 28

40-43. (canceled)