Precision gene targeting to a particular locus in maize
The present invention claims methods for the stable integration of exogenous DNA into a specific locus, E32, in the maize genome through the use of zinc finger nucleases. Maize plants and plant parts that were transformed by the methods of the invention are claimed. The invention is useful for creating desirable traits such as herbicide resistance, herbicide tolerance, insect resistance, insect tolerance, disease resistance, disease tolerance, stress tolerance, and stress resistance in maize The E32 locus represents a superior site for inserting foreign genes because native agronomic phenotypes are not disturbed.
1. A method for integrating one or more exogenous nucleic acid sequences into the genome of a maize cell having an E32 locus, said method comprising the steps of:
making a double-stranded cleavage in the E32 locus using a site specific zinc finger nuclease, wherein the site specific zinc finger nuclease comprises three or more zinc finger motifs selected from the group consisting of SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52; and
ligating a polynucleotide comprising one or more exogenous nucleic acid sequences into the cleavage using a ligase or a recombinase, wherein the polynucleotide is integrated into the cleavage.
2. The method of claim 1 , further comprising the step of expressing a product of the one or more exogenous sequences.
3. The method of claim 1 , wherein the one or more exogenous nucleic acid sequences comprise a coding sequence, a regulatory sequence, or a target site for a DNA-binding domain.
4. The method of claim 2 , wherein the one or more exogenous nucleic acid sequences comprise a coding sequence, a regulatory sequence, or a target site for a DNA-binding domain.
5. The method of claim 3 , wherein the coding sequence encodes for a product that confers: herbicide resistance; herbicide tolerance; insect resistance; insect tolerance; disease resistance; disease tolerance; stress tolerance; stress resistance; a change in oxidative stress; increased yields of oil; a change in food content and makeup; a change in physical appearance; male sterility; drydown; standability; prolificacy; a change in starch quantity or quality; a change in oil quality; a change in protein quality or quantity; a change in amino acid composition or combinations thereof.
6. The method of claim 4 , wherein the coding sequence encodes for a product that confers: herbicide resistance; herbicide tolerance; insect resistance; insect tolerance; disease resistance; disease tolerance; stress tolerance; stress resistance; a change in oxidative stress; increased yields of oil; a change in food content and makeup; a change in physical appearance; male sterility; drydown; standability; prolificacy; a change in starch quantity or quality; a change in oil quality; a change in protein quality or quantity; a change in amino acid composition or combinations thereof.
7. The method of claim 2 wherein the exogenous sequence is chose from the group consisting of the phosphinothricin-N-acetyl-transferase (PAT) gene and the aryloxyalkanoate dioxygenase (AAD-1).
8. The method of claim 1 , wherein the polynucleotide further comprises nucleotide sequences that are homologous to sequences in the E32 locus.
9. The method according to claim 8 , wherein the homologous nucleotide sequences flank the exogenous sequence.
10. The method of claim 1 , wherein the polynucleotide further comprises a promoter.
11. The method of claim 1 , wherein one or more of the integrated exogenous nucleic acid sequences are transmitted to progeny in subsequent generations.
12. A maize plant or maize plant part comprising a maize cell having an E32 locus, wherein the genome of the maize cell comprises one or more nucleic acid exogenous sequences, wherein the one or more nucleic acid exogenous sequences are integrated into the genome of the maize cell within the E32 locus according to the method of claim 1 .
13. A maize seed comprising a maize cell having an E32 locus, wherein the genome of the maize cell comprises one or more nucleic acid exogenous sequences, wherein the one or more nucleic acid exogenous sequences are integrated into the genome of the maize cell within the E32 locus according to the method of claim 1 .