Optimal Soybean Loci

• US 20150128307A1
• 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, CAO, Zehui, SRIRAM, Shreedharan, WEBB, Steven R., CAMPER, Debra L.
• Granted Patent: US 10,233,465 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 15/902 using homologous recombination

Abstract

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

Claims

1. A recombinant sequence, said recombinant sequence comprising:

a nongenic soybean genomic sequence of at least 1 Kb, said nongenic sequence being hypomethylated, targetable, located proximal to a genic region within a soybean genome, and exemplifying evidence of recombination; and

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

2. The recombinant sequence of claim 1, wherein said nongenic sequence comprises the following characterstics:

a. the level of methylation of said nongenic sequence is 1% or less

b. said nongenic sequence shares less than 40% sequence identity with any other sequence contained in the soybean genome;

c. said nongenic sequence is located within a 40 Kb region of a known or predicted expressive soybean coding sequence; and

d. said nongenic sequence exhibits a recombination frequency within the soybean genome of greater than 0.01574 cM/Mb.

3. The recombinant sequence of claim 1, wherein said nongenic sequence comprises 1% or less nucleotide methylation.

4. The recombinant sequence of claim 3, wherein said nongenic sequence is 1 Kb to 5.73 Kb in length and contains no methylated cytosine residues.

5. The recombinant sequence of claim 4, wherein said nongenic sequence does not align with greater than 40% sequence identity to any other sequence within the soybean genome.

6. The recombinant sequence of claim 4, wherein said nongenic sequence exemplifies evidence of recombination at a recombination frequency of greater than 0.01574 cM/Mb.

7. The recombinant sequence of claim 5, wherein a 40 Kb region of native soybean genome comprising said nongenic sequence also comprises at least one known or predicted soybean coding sequence, or a sequence comprising a 2 Kb upstream and/or 1 Kb downstream sequence of a known soybean gene.

8. The recombinant sequence of claim 1, wherein one end of said nongenic sequence is within 40 Kb of an expressed endogenous gene.

9. 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.

10. The recombinant sequence of claim 1, wherein said recombinant sequence comprises the following characteristics:

a. said nongenic sequence contains less than 1% DNA methylation

b. said nongenic sequence exhibits a 0.01574 to 83.52 cM/Mb recombination frequency within the soybean genome;

c. said nongenic sequence exhibits a 0 to 0.494 level of nucleosome occupancy of the soybean genome;

d. said nongenic sequence shares less than 40% sequence identity with any other sequence contained in the soybean genome;

e. said nongenic sequence has a relative location value from 0 to 0.99682 ratio of genomic distance from a soybean chromosomal centromere;

f. said nongenic sequence has a guanine/cytosine percent content range of 14.36 to 45.9%;

g. said nongenic sequence is located proximally to a genic sequence; and

,

h. said nongenic sequence is located in a 1 Mb region of soybean genomic sequence that comprises one or more additional nongenic sequences.

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

12. 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.

13. A method of making a transgenic plant cell comprising a DNA of interest targeted to one nongenic soybean genomic sequence, the method comprising:

a. selecting an optimal nongenic soybean genomic locus;

b. introducing a site specific nuclease into a plant cell, wherein the site specific nuclease cleaves said nongenic soybean genomic sequence;

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

d. targeting the DNA of interest into said nongenic locus, wherein the cleavage of said nongenic sequence facilitates integration of the polynucleotide sequence into said nongenic locus; and

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

14. The method of making a transgenic plant cell of claim 13, 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.

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

16. The method of making a transgenic plant cell of claim 13, wherein said DNA of interest is integrated within said nongenic locus via a non-homologous end joining integration method.

17. The method of making a transgenic plant cell of claim 13, wherein said selected nongenic locus is at least 1 Kb in length and comprises the following characteristics:

a. said nongenic locus has less than 1% DNA methylation within the sequence;

b. said nongenic locus exhibits a 0.001574 to 83.52 cM/Mb rate of recombination within the soybean genome;

c. said nongenic locus exhibits a 0 to 0.494 level of nucleosome occupancy of the soybean genome;

d. said nongenic locus shares less than 40% sequence identity with any other 1 Kb sequence contained in the soybean genome;

e. said nongenic locus has a relative location value from 0 to 0.99682 ratio of genomic distance from a soybean chromosomal centromere;

f. said nongenic locus has a guanine/cytosine percent content range of 14.36 to 45.9%;

g. said nongenic locus is located proximally to an genic sequence; and

,

h. a 1 Mb region of soybean genomic sequence comprising said nongenic locus comprises at least one second nongenic sequence.

18. The method of making a transgenic plant cell of claim 13, wherein said DNA of interest and/or said nongenic locus are modified during said integration of the polynucleotide sequence into said nongenic locus of step (d).

19. A recombinant targetable nongenic soybean genomic sequence comprising:

a nongenic sequence of at least 1 Kb selected from the group consisting of cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or a sequence sharing 99% sequence identity with a sequence selected from the group consisting of cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, and their respective complements thereof, wherein said clusters are generated from a PCA statistical algorithm comprising PCA values defined in Table 6; and

a DNA of interest inserted into said nongenic sequence.

20. The targetable nongenic soybean genomic sequence of claim 19, wherein said DNA of interest comprises a gene expression cassette comprising a transgene.

21. The targetable nongenic soybean genomic sequence of claim 19, wherein said insertion of said DNA of interest into said nongenic sequence results in a modification of said DNA of interest and said nongenic sequences.