Optimal Soybean Loci

• US 20150128308A1
• Filed: 11/03/2014
• Published: 05/07/2015
• Est. Priority Date: 11/04/2013
• Status: Active Grant

• Current Assignee: Corteva Agriscience LLC (Corteva, Inc.)
• Original Assignee: Dow Agrosciences LLC (Corteva, Inc.)
• Inventors: SASTRY-DENT, Lakshmi, WEBB, Steven R., AINLEY, W. Michael, CAO, Zehui, SRIRAM, Shreedharan, CAMPER, Debra L.
• Granted Patent: US 9,909,131 B2
• Time in Patent Office: Days
• US Class Current: 800/312
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8243 involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
• CPC Class Codes: C12N 15/8273 for drought, cold, salt resistance
• CPC Class Codes: C12N 15/8274 for herbicide resistance
• CPC Class Codes: C12N 15/8286 for insect resistance
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

As disclosed herein, optimal native genomic loci of soybean plants have been identified that represent best sites for targeted insertion of exogenous sequences.

Claims

1. A recombinant sequence, said recombinant sequence comprising:

a nucleic acid sequence of at least 1 Kb and having at least 90% sequence identity with a nongenic sequence selected from the group consisting of

soy_OGL_—

1423 (SEQ ID NO;

639),

soy_OGL_—

1434 (SEQ ID NO;

137),

soy_OGL_—

4625 (SEQ ID NO;

76),

soy_OGL_—

6362 (SEQ ID NO;

440),

soy_OGL_—

308 (SEQ ID NO;

43),

soy_OGL_—

307 (SEQ ID NO;

566),

soy_OGL_—

310 (SEQ ID NO;

4236),

soy_OGL_—

684 (SEQ ID NO;

47),

soy_OGL_—

682 (SEQ ID NO;

2101),

soy_OGL_—

685 (SEQ ID NO;

48), and

a DNA of interest, wherein the DNA of interest is inserted into said nongenic sequence to produce said recombinant sequence.

2. The recombinant sequence of claim 1 wherein said DNA of interest is inserted proximal to a zinc finger target site.

3. The recombinant sequence of claim 1, wherein said DNA of interest is inserted between a pair of zinc finger target sites.

4. The recombinant sequence of claim 1, wherein said DNA of interest comprises a gene expression cassette comprising an insecticidal resistance gene, herbicide tolerance gene, nitrogen use efficiency gene, water use efficiency gene, nutritional quality gene, DNA binding gene, and selectable marker gene.

5. The recombinant sequence of claim 1, wherein said DNA of interest comprises two or more gene expression cassettes.

6. The recombinant sequence of claim 1, wherein two or more of said nongenic sequences each comprise an inserted DNA of interest to produce two or more recombinant sequences wherein the two or more recombinant sequences are located on a same chromosome.

7. The recombinant sequence of claim 1, wherein said DNA of interest and/or said nongenic sequence are modified during insertion of said DNA of interest into said nongenic sequence.

8. The recombinant sequence of claim 1 wherein the nongenic soybean genomic locus has at least 90% sequence identity with a sequence selected from the group consisting of

soy_OGL_—

1423 (SEQ ID NO;

639),

soy_OGL_—

1434 (SEQ ID NO;

137),

soy_OGL_—

4625 (SEQ ID NO;

76),

soy_OGL_—

6362 (SEQ ID NO;

440),

soy_OGL_—

308 (SEQ ID NO;

43),

soy_OGL_—

307 (SEQ ID NO;

566), and,

soy_OGL_—

310 (SEQ ID NO;

4236).

9. The recombinant sequence of claim 1 wherein the nongenic soybean genomic locus has at least 90% sequence identity with a sequence sleeted from the group consisting of

soy_OGL_—

308 (SEQ ID NO;

43),

soy_OGL_—

307 (SEQ ID NO;

566), and

soy_OGL_—

310 (SEQ ID NO;

4236).

10. A soybean plant, soybean plant part, or soybean plant cell comprising a recombinant sequence of claim 1.

11. A method of making a transgenic plant cell comprising a DNA of interest, the method comprising:

a. selecting a target nongenic soybean genomic locus having at least 90% sequence identity from the group consisting of soy_OGL_—

1423 (SEQ ID NO;

639),

soy_OGL_—

1434 (SEQ ID NO;

137),

soy_OGL_—

4625 (SEQ ID NO;

76),

soy_OGL_—

6362 (SEQ ID NO;

440),

soy_OGL_—

308 (SEQ ID NO;

43),

soy_OGL_—

307 (SEQ ID NO;

566),

soy_OGL_—

310 (SEQ ID NO;

4236),

soy_OGL_—

684 (SEQ ID NO;

47),

soy_OGL_—

682 (SEQ ID NO;

2101), and,

soy_OGL_—

685 (SEQ ID NO;

48);

b. selecting a site specific nuclease that specifically binds and cleaves said target nongenic soybean genomic locus

c. introducing said site specific nuclease into a soybean plant cell;

d. introducing the DNA of interest into the plant cell;

e. inserting the DNA of interest into said target nongenic soybean genomic loci; and

,

f. selecting transgenic plant cells comprising the DNA of interest targeted to said nongenic locus.

12. The method of making a transgenic plant cell of claim 11, wherein said site specific nuclease is selected from the group consisting of a zinc finger nuclease, a CRISPR nuclease, a TALEN, a homing endonuclease or a meganuclease.

13. The method of making a transgenic plant cell of claim 11, wherein said DNA of interest is integrated within said nongenic locus via a homology directed repair integration method.

14. The method of making a transgenic plant cell of claim 11, wherein said DNA of interest is integrated within said nongenic locus via a non-homologous end joining integration method.

15. The method of making a transgenic plant cell of claim 11, wherein two or more of said DNA of interest are inserted into two or more of said target nongenic soybean genomic loci, optionally on a same chromosome.

16. The method of making a transgenic plant cell of claim 11, wherein said DNA of interest and/or said target nongenic soybean genomic loci are modified during insertion of said DNA of interest into said target nongenic soybean genomic loci.

17. The method of making a transgenic plant cell of claim 11, wherein the nongenic soybean genomic locus has at least 90% sequence identity with a sequence sleeted from the group consisting of

soy_OGL_—

1423 (SEQ ID NO;

639),

soy_OGL_—

1434 (SEQ ID NO;

137),

soy_OGL_—

4625 (SEQ ID NO;

76),

soy_OGL_—

6362 (SEQ ID NO;

440),

soy_OGL_—

308 (SEQ ID NO;

43),

soy_OGL_—

307 (SEQ ID NO;

566), and,

soy_OGL_—

310 (SEQ ID NO;

4236).

18. The method of making a transgenic plant cell of claim 11, wherein the nongenic soybean genomic locus has at least 90% sequence identity with a sequence selected from the group consisting of

soy_OGL_—

308 (SEQ ID NO;

43),

soy_OGL_—

307 (SEQ ID NO;

566), and

soy_OGL_—

310 (SEQ ID NO;

4236).

19. A plant comprising a recombinant sequence, said recombinant sequence comprising:

a nucleic acid sequence having at least 90% sequence identity with a nongenic sequence, and

a DNA of interest, wherein the DNA of interest is inserted into said nongenic sequence.

20. The plant of claim 19, wherein said nongenic sequence is selected from the group consisting of

soy_OGL_—

1423 (SEQ ID NO;

639),

soy_OGL_—

1434 (SEQ ID NO;

137),

soy_OGL_—

4625 (SEQ ID NO;

76),

soy_OGL_—

6362 (SEQ ID NO;

440),

soy_OGL_—

308 (SEQ ID NO;

43),

soy_OGL_—

307 (SEQ ID NO;

566),

soy_OGL_—

310 (SEQ ID NO;

4236),

soy_OGL_—

684 (SEQ ID NO;

47),

soy_OGL_—

682 (SEQ ID NO;

2101), and

soy_OGL_—

685 (SEQ ID NO;

48).

21. The plant of claim 19, wherein said nongenic sequence is selected from the group consisting of

soy_OGL_—

308 (SEQ ID NO;

43),

soy_OGL_—

307 (SEQ ID NO;

566), and

soy_OGL_—

310 (SEQ ID NO;

4236).