Methods and compositions for identifying and enriching for cells comprising site specific genomic modifications
The present invention relates to methods and compositions for modifying a target site in the genome of a plant cell. Such modifications include integration of a transgene and mutations. The present invention also relates to methods and compositions for identifying and enriching for cells which comprise a modified target site.
1. A method for modifying a target site in the genome of a plant cell, comprising introducing into the plant cell:
a) a first nucleic acid molecule comprising a genomic target site, wherein the nucleic acid molecule comprises at least 100 contiguous nucleotides with at least 90% identity to at least 100 contiguous nucleotides with a target site in the genome of the cell, and further comprising a heterologous nucleotide sequence; and
b) a second nucleic acid molecule comprising a nucleotide sequence encoding a nuclease for site-directed cleavage at a genomic nuclease cleavage site in the genome of the cell adjacent to the nucleotide sequence in the genome of the cell that corresponds to the at least 100 contiguous nucleotides of (a),
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 in the genome of the cell and modify the target site in the genome of the plant cell, wherein the nuclease is a modified Cas9 nuclease comprising SEQ ID NO: 30.
2. The method of claim 1 , wherein the plant cell is a maize cell.
3. The method of claim 2 , wherein the maize cell is a transgenic maize cell.
4. The method of claim 3 , wherein the transgenic maize cell is an event MIR604 transgenic maize cell.
5. The method of claim 1 , wherein the heterologous nucleotide sequence comprises a transgene.
6. The method of claim 1 , wherein said modifying a target site comprises inserting the heterologous nucleotide sequence at the genomic nuclease cleavage site.
7. A method of producing a maize plant, plant part, or progeny thereof comprising a modification at a target site in the genome of the plant cell, comprising introducing into the plant cell:
a) a first nucleic acid comprising at least 100 contiguous nucleotides with at least 90% identity to at least 100 contiguous nucleotides with a target site in the genome of the cell, and further comprising a heterologous nucleotide sequence;
b) a second nucleic acid molecule comprising a nucleotide sequence encoding a nuclease for site-directed cleavage at a genomic nuclease cleavage site in the genome of the cell adjacent to the nucleotide sequence in the genome of the cell that corresponds to the at least 100 contiguous nucleotides of (a),
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 in the genome of the cell and modify the target site in the genome of the plant cell; and
c) regenerating a plant, plant part, or progeny thereof from the plant cell of (a), wherein the nuclease is a modified Cas9 nuclease comprises SEQ ID NO: 30.
8. A maize plant, plant part, or progeny thereof produced by the method of claim 7 .
9. The method of claim 7 , wherein said modification at a target site comprises inserting the heterologous nucleotide sequence at the genomic nuclease cleavage site.