OPTIMAL SOYBEAN LOCI

• US 20180142250A1
• Filed: 01/11/2018
• Published: 05/24/2018
• 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, CAO, Zehui, SRIRAM, Shreedharan, WEBB, Steven R., CAMPER, Debra L., AINLEY, W. Michael
• Granted Patent: US 10,106,804 B2
• Time in Patent Office: Days
• 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 soybean cell produced by the following steps:

a. providing a soybean cell wherein said cell comprises a target nongenic soybean genomic nucleic acid molecule selected from the group consisting of

 

SEQ ID NO;

639, identified as soy_OGL_1423,

 

SEQ ID NO;

137, identified as soy_OGL_1434,

 

SEQ ID NO;

76, identified as soy_OGL_4625,

 

SEQ ID NO;

440, identified as soy_OGL_6362,

 

SEQ ID NO;

43, identified as soy_OGL_308,

 

SEQ ID NO;

566, identified as soy_OGL_307,

 

SEQ ID NO;

4236, identified as soy_OGL_310,

 

SEQ ID NO;

47, identified as soy_OGL_684,

 

SEQ ID NO;

2101, identified as soy_OGL_682, and

 

SEQ ID NO;

48, identified as soy_OGL_685

b. selecting a site specific nuclease that specifically binds and cleaves said target nongenic soybean genomic nucleic acid molecule;

c. introducing said site specific nuclease activity into said soybean cell;

d. introducing a DNA of interest into the soybean cell;

e. inserting the DNA of interest into said target nongenic soybean genomic nucleic acid molecule; and

,

f. selecting transgenic soybean cells comprising the DNA of interest targeted to said nongenic nucleic acid molecule.

2. A soybean plant regenerated from the soybean cell of claim 1.

3. A seed or other soybean plant part of the plant of claim 2.

4. The soybean cell of claim 1 wherein the site specific nuclease is selected from the group consisting of Zinc Finger Nucleases (ZFNs), Meganucleases, Transcription Activator-Like Effector Nucelases (TALENS) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease and the DNA of interest is inserted within 2 Kb, 1.75 Kb, 1.5 Kb, 1.25 Kb, 1.0 Kb, 0.75 Kb, 0.5 Kb, or 0.25 Kb of a target site of said site specific nuclease.

5. The soybean cell of claim 1 wherein the site specific nuclease is a Zinc Finger Nuclease and said DNA of interest is inserted within 2 Kb, 1.75 Kb, 1.5 Kb, 1.25 Kb, 1.0 Kb, 0.75 Kb, 0.5 Kb, or 0.25 Kb of a zinc finger target site.

6. The soybean cell of claim 5, wherein said DNA of interest is inserted between a pair of zinc finger target sites.

7. The soybean cell of claim 1 wherein the DNA of interest comprises an open reading frame.

8. The soybean cell of claim 1 wherein the DNA of interest comprises a gene expression cassette.

9. The soybean cell of claim 8 wherein the gene expressions cassette comprises an insecticidal resistance gene, herbicide tolerance gene, nitrogen use efficiency gene, water use efficiency gene, nutritional quality gene, DNA binding gene, or selectable marker gene.

10. A soybean plant, soybean plant part, or soybean plant cell comprising a recombinant nucleic acid molecule, said recombinant nucleic acid molecule comprising

a nongenic nucleic acid molecule of at least 1 Kb, wherein

a. the level of methylation of said nongenic nucleic acid molecule is 1% or less;

b. the nongenic nucleic acid molecule shares less than 40% sequence identity with any other nucleic acid molecules contained in the soybean genome;

c. the nongenic nucleic acid molecule is located within a 40 Kb region of a known or predicted expressive soybean coding nucleic acid molecule; and

d. the nongenic nucleic acid molecule exhibits a recombination frequency within the soybean genome of greater than 0.01574 cM/Mb, wherein said nongenic nucleic acid molecule has at least 95% sequence identity with a nongenic nucleic acid molecule selected from the group consisting of

 

SEQ ID NO;

639, identified as soy_OGL_1423,

 

SEQ ID NO;

137, identified as soy_OGL_1434,

 

SEQ ID NO;

76, identified as soy_OGL_4625,

 

SEQ ID NO;

440, identified as soy_OGL_6362,

 

SEQ ID NO;

43, identified as soy_OGL_308,

 

SEQ ID NO;

566, identified as soy_OGL_307,

 

SEQ ID NO;

4236, identified as soy_OGL_310,

 

SEQ ID NO;

47, identified as soy_OGL_684,

 

SEQ ID NO;

2101, identified as soy_OGL_682, and

 

SEQ ID NO;

48, identified as soy_OGL_685; and

a DNA of interest, wherein the DNA of interest comprises an open reading frame and said DNA of interest is inserted into said nongenic nucleic acid molecule to produce said recombinant nucleic acid molecule.

11. The soybean plant, soybean plant part, or soybean plant cell of claim 10 wherein said nongenic nucleic acid molecule comprises a nongenic nucleic acid molecule selected from the group consisting of

SEQ ID NO:

639, identified as soy_OGL_1423,

SEQ ID NO;

137, identified as soy_OGL_1434,

SEQ ID NO;

76, identified as soy_OGL_4625,

SEQ ID NO;

440, identified as soy_OGL_6362,

SEQ ID NO;

43, identified as soy_OGL_308,

SEQ ID NO;

566, identified as soy_OGL_307,

SEQ ID NO;

4236, identified as soy_OGL_310,

SEQ ID NO;

47, identified as soy_OGL_684,

SEQ ID NO;

2101, identified as soy_OGL_682, and

SEQ ID NO;

48, identified as soy_OGL_685

12. The soybean plant, soybean plant part, or soybean plant cell of claim 10 wherein the nongenic soybean genomic nucleic acid molecule has at least 95% sequence identity with a nucleic acid molecule selected from the group consisting of

SEQ ID NO:

639, identified as soy_OGL_1423,

SEQ ID NO;

137, identified as soy_OGL_1434,

SEQ ID NO;

76, identified as soy_OGL_4625,

SEQ ID NO;

440, identified as soy_OGL_6362,

SEQ ID NO;

43, identified as soy_OGL_308,

SEQ ID NO;

566, identified as soy_OGL_307, and

SEQ ID NO;

4236, identified as soy_OGL_310.

13. The soybean plant, soybean plant part, or soybean plant cell of claim 10 wherein the nongenic soybean genomic nucleic acid molecule has at least 95% sequence identity with a nucleic acid molecules selected from the group consisting of

SEQ ID NO:

43, identified as soy_OGL_308,

SEQ ID NO;

566, identified as soy_OGL_307, and

SEQ ID NO;

4236, identified as soy_OGL_310.

14. The soybean plant cell of claim 10 wherein said open reading frame is part of a gene expression cassette.

15. The soybean plant cell of claim 14 wherein the gene expressions cassette comprises an insecticidal resistance gene, herbicide tolerance gene, nitrogen use efficiency gene, water use efficiency gene, nutritional quality gene, DNA binding gene, or selectable marker gene.

16. The soybean plant, soybean plant part, or soybean plant cell of claim 10, wherein two or more of said nongenic nucleic acid molecules each comprise an inserted DNA of interest to produce two or more recombinant nucleic acid molecules wherein each DNA of interest comprises an open reading frame.

17. The soybean plant, soybean plant part, or soybean plant cell of claim 16 wherein each of said open reading frames independently comprise an insecticidal resistance gene, herbicide tolerance gene, nitrogen use efficiency gene, water use efficiency gene, nutritional quality gene, DNA binding gene, or selectable marker gene.

18. A soybean plant, soybean plant part, or soybean plant cell comprising a nucleic acid molecule encoding a site specific nuclease that specifically binds and cleaves a target nongenic soybean genomic nucleic acid molecule selected from the group consisting of

SEQ ID NO:

639, identified as soy_OGL_1423,

SEQ ID NO;

137, identified as soy_OGL_1434,

SEQ ID NO;

76, identified as soy_OGL_4625,

SEQ ID NO;

440, identified as soy_OGL_6362,

SEQ ID NO;

43, identified as soy_OGL_308,

SEQ ID NO;

566, identified as soy_OGL_307,

SEQ ID NO;

4236, identified as soy_OGL_310,

SEQ ID NO;

47, identified as soy_OGL_684,

SEQ ID NO;

2101, identified as soy_OGL_682, and

SEQ ID NO;

48, identified as soy_OGL_685.

19. The soybean plant, soybean plant part, or soybean plant cell of claim 18 wherein the site specific nuclease is selected from the group consisting of Zinc Finger Nucleases (ZFNs), Meganucleases, Transcription Activator-Like Effector Nucelases (TALENS) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease.

20. The soybean plant, soybean plant part, or soybean plant cell of claim 18 wherein the nucleic acid molecule encoding a site specific nuclease is operably linked to an inducible promoter.

21. The soybean plant, soybean plant part, or soybean plant cell of claim 18 wherein the nucleic acid molecule encoding a site specific nuclease is operably linked to a constitutive promoter.