Targeted insertion sites in the maize genome
The present invention relates to methods and compositions for targeted insertion of polynucleotide molecules into ideal target sites in the genome of a maize plant. The present invention relates to maize recombinant molecules comprising heterologous sequences and also to methods of integrating a DNA of interest into a target maize genomic locus in a maize genome. The present invention also relates to regenerated maize plants or plant parts comprising the recombinant molecules and/or a DNA of interest.
1. A method of integrating DNAs of interest into a target maize genomic locus in a maize genome, wherein the target maize genomic locus comprises a nucleic acid sequence having at least 100 contiguous nucleotides of SEQ ID NO: 67 or the complement thereof, comprising introducing into a maize cell:
a) a first nucleic acid molecule comprising at least 100 contiguous nucleotides of SEQ ID NO: 67 or the complement thereof, and further comprising the DNA of interest; and
b) a second nucleic acid molecule comprising a nucleotide sequence encoding a nuclease for site-directed cleavage at a genomic nuclease cleavage site within the target maize genomic locus, under conditions wherein expression of the second nucleic acid molecule can occur to produce the nuclease and the nuclease can cleave the nucleotide sequence at the genomic nuclease cleavage site, whereby the DNA of interest is integrated into the target maize genomic locus in the maize genome.
2. The method of claim 1 , wherein two or more DNAs of interest are inserted into two or more targeted maize genomic loci.
3. The method of claim 1 , wherein the DNAs of interest is inserted into the target maize genomic locus via homologous recombination.
4. The method of claim 1 , wherein the DNAs of interest is inserted into the target maize genomic locus via non-homologous end-joining.
5. The method of claim 1 , wherein the DNAs of interest and/or the target maize genomic locus are modified during the insertion of said DNA of interest into said target maize genomic locus.
6. A method of producing a maize plant, plant part, or progeny thereof comprising a DNA of interest, comprising regenerating a maize plant from the maize cell produced by the method of claim 1 .
7. A maize plant, plant part, or progeny thereof comprising a DNA of interest, produced by the method of claim 6 .
8. A method of making a maize plant cell comprising a DNA of interest, said method comprising:
a. selecting a target maize genomic locus, wherein the target maize genomic locus comprises a nucleic acid sequence having a sequence selected from the group consisting of SEQ ID NO: 67, and the complement thereof;
b. selecting a site specific nuclease that specifically binds to and cleaves a genomic nuclease cleavage site within said target maize genomic locus;
c. introducing said site specific nuclease and a DNA of interest into the maize plant cell;
d. allowing the DNA of interest to insert into the target maize genomic locus; and
e. selecting maize plant cells that comprise the DNA of interest inserted into the target maize genomic locus.
9. The method of claim 8 , wherein two or more DNAs of interest are inserted into two or more targeted maize genomic loci.
10. The method of claim 8 , wherein the DNA of interest is inserted into the target maize genomic locus via homologous recombination.
11. The method of claim 8 , wherein the DNA of interest is inserted into the target maize genomic locus via non-homologous end-joining.
12. The method of claim 8 , wherein the DNAs of interest and/or the target maize genomic locus are modified during the insertion of said DNA of interest into said target maize genomic locus.
13. The method of claim 8 , wherein the site specific nuclease is a Cas-associated nuclease and wherein a third nucleic acid molecule encoding a guide RNA is introduced into the maize cell.
14. The method of claim 8 , wherein the site specific nuclease is a Cas-associated nuclease and wherein a third nucleic acid molecule comprising a guide RNA is introduced into the maize cell.
15. A method of producing a maize plant or plant part, or progeny thereof, comprising a transgene integrated into a genomic nuclease cleavage site in the maize genome, comprising regenerating a maize plant from the maize cell produced by the method of claim 8 .
16. A maize plant or plant part, or progeny thereof, comprising a transgene integrated into a genomic nuclease cleavage site in the maize genome, produced by the method of claim 15 .
17. A maize recombinant molecule, wherein the recombinant molecule comprises a nucleic acid sequence of at least 100 nucleotides and has at least 100 contiguous nucleotides of SEQ ID NO: 67 or the complement thereof, and wherein the recombinant molecule further comprises a DNA of interest, wherein the DNA of interest is inserted into the nucleic acid sequence to produce said recombinant molecule.
18. The recombinant molecule of claim 17 , wherein the recombinant sequence comprises a nucleic acid sequence of at least 1 Kb and has a sequence of SEQ ID NO: 67 or the complement thereof.
19. A maize plant, plant part, or plant cell comprising the recombinant molecule of claim 17 .