Methods and compositions for nuclease-mediated targeted integration of transgenes
Disclosed herein are methods and compositions for homology-independent targeted insertion of donor molecules into the genome of a cell.
1. A method of integrating a transgene into an endogenous locus of a cell, the method comprising;
introducing one or more nucleases into the cell, the cell comprising a circular double-stranded plasmid donor, the double-stranded plasmid donor comprising: a transgene; and first and second sequences flanking the transgene, the first and second sequences flanking the transgene comprising paired target sites for one or more nucleases, the target sites flanking a spacer sequence, wherein at least one spacer sequence differs from the spacer sequence present in the genome of the cell, wherein the double-stranded plasmid does not comprise homology arms and further wherein the nucleases cleave the double-stranded plasmid in the sequences flanking the transgene and cleave the endogenous locus such that the transgene is integrated into the endogenous locus via homology-independent mechanisms.
2. The method of claim 1 , wherein the transgene is integrated in a forward orientation.
3. The method of claim 1 , wherein the transgene is integrated in a reverse orientation.
4. The method of claim 1 , wherein the same nucleases cleave the endogenous locus and the double-stranded plasmid.
5. The method of claim 1 , wherein different nucleases cleave the endogenous locus and the double-stranded plasmid.
6. The method of claim 1 , wherein the spacer sequences comprise at least 5 nucleotides.
7. The method of claim 1 , wherein the nucleases generate a deletion in the endogenous locus and the transgene is integrated into the deletion.
8. The method of claim 1 , wherein the cell is a eukaryotic cell.
9. The method of claim 8 , wherein the cell is a plant or mammalian cell.
10. The method of claim 9 , wherein the plant cell is a dicotyledonous or a monocotyledonous plant cell.