Optimal maize loci
View Patent ↗As disclosed herein, optimal native genomic loci have been identified in monocot plants, such as maize plants, that represent best sites for targeted insertion of exogenous sequences.
1. A maize plant, maize plant part, or maize plant cell comprising a recombinant nucleic acid molecule, said recombinant nucleic acid molecule comprising a nongenic maize genomic nucleic acid of at least 1 Kb determined to comprise the following characteristics:
a. the level of methylation of said nongenic nucleic acid is 1% or less;
b. said nongenic nucleic acid shares less than 40% sequence identity with any other nucleic acid contained in the Zea mays genome;
c. said nongenic nucleic acid is located within a 40 Kb region of a known or predicted expressive maize coding nucleic acid; and
d. said nongenic nucleic acid exhibits a recombination frequency within the maize genome of greater than 0.00041 cM/Mb wherein said nongenic nucleic acid comprises a 1Kb nucleic acid having at least 95% sequence identity with a nucleic acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 100, SEQ ID NO: 203, SEQ ID NO: 295, SEQ ID NO: 384, SEQ ID NO: 687, SEQ ID NO: 781, SEQ ID NO: 843, SEQ ID NO: 967, SEQ ID NO: 1190, SEQ ID NO: 1252, SEQ ID NO: 1571, SEQ ID NO: 1663, SEQ ID NO: 1906, SEQ ID NO: 2027, SEQ ID NO: 2171, SEQ ID NO: 2256, SEQ ID NO: 2428, SEQ ID NO: 2632, and SEQ ID NO: 2649; and
a DNA of interest, wherein the DNA of interest comprises a non-native exogenous nucleic acid, and said DNA of interest is inserted into said nongenic nucleic acid.
2. The maize plant, maize plant part, or maize plant cell of claim 1 , wherein said nongenic nucleic acid sequence is 1 Kb to 8.3 Kb in length and contains no methylated cytosine residues.
3. The maize plant, maize plant part, or maize plant cell of claim 1 , wherein a 40 Kb region of native maize genome comprising said nongenic nucleic acid also comprises at least one known or predicted maize coding nucleic acid, or a nucleic acid comprising a 2 Kb upstream and/or 1 Kb downstream nucleic acid of a known maize gene.
4. The maize plant, maize plant part, or maize plant cell 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, or selectable marker gene.
5. The maize plant, maize plant part, or maize plant cell of claim 1 , wherein said DNA of interest comprises a gene expression cassette comprising an insecticidal resistance gene or herbicide tolerance gene.
6. A method of making a transgenic maize plant cell comprising a DNA of interest targeted to one nongenic maize genomic nucleic acid, the method comprising:
a. selecting a nongenic maize genomic nucleic acid of at least 1 Kb, wherein
said nongenic nucleic acid comprises the following characteristics:
i). the level of methylation of said nongenic nucleic acid is 1% or less;
ii). said nongenic nucleic acid shares less than 40% sequence identity with any other nucleic acid contained in the Zea mays genome;
iii). said nongenic nucleic acid is located within a 40 Kb region of a known or predicted expressive maize coding nucleic acid; and
iv). said nongenic nucleic acid exhibits a recombination frequency within the maize genome of greater than 0.00041 cM/Mb; further wherein said nongenic nucleic acid comprises a 1 Kb nucleic acid having at least 95% sequence identity with a nucleic acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 100, SEQ ID NO: 203, SEQ ID NO: 295, SEQ ID NO: 384, SEQ ID NO: 687, SEQ ID NO: 781, SEQ ID NO: 843, SEQ ID NO: 967, SEQ ID NO: 1190, SEQ ID NO: 1252, SEQ ID NO: 1571, SEQ ID NO: 1663, SEQ ID NO: 1906, SEQ ID NO: 2027, SEQ ID NO: 2171, SEQ ID NO: 2256, SEQ ID NO: 2428, SEQ ID NO: 2632, and SEQ ID NO: 2649;
b. introducing a site specific nuclease into a plant cell, wherein the site specific nuclease cleaves said nongenic nucleic acid;
c. introducing the DNA of interest into the plant cell;
d. targeting the insertion of the DNA of interest into said nongenic nucleic acid, wherein the cleavage of said nongenic nucleic acid facilitates integration of the DNA of interest into said nongenic nucleic acid; and
e. selecting transgenic plant cells comprising the DNA of interest inserted into said nongenic nucleic acid.
7. The method of making a transgenic plant cell of claim 6 , wherein said DNA of interest comprises a gene expression cassette comprising a transgene.
8. The method of making a transgenic plant cell of claim 6 , wherein said site specific nuclease is selected from the group consisting of a zinc finger nuclease, a CRISPR nuclease, a TALEN, a homing endonuclease and a meganuclease.
9. The method of making a transgenic plant cell of claim 6 , wherein said DNA of interest is integrated within said nongenic nucleic acid via a homology directed repair integration method.
10. The method of making a transgenic plant cell of claim 6 , wherein said DNA of interest is integrated within said nongenic nucleic acid via a non-homologous end joining integration method.