Targeted genomic alteration
Disclosed herein are methods and compositions for targeted integration and/or targeted excision of one or more sequences into a cell, for example, for expression of one or more polypeptides of interest.
1. A host cell or cell line comprising an endogenous genome and an exogenous nucleic acid molecule integrated into the endogenous genome, the exogenous nucleic acid comprising a non-coding multiple insertion site, the multiple insertion site comprising three or more different paired target sites for heterodimeric pairs of zinc finger nucleases, wherein the target sites of each paired target site are recognized by different zinc finger nucleases and further wherein (i) the target sites of each paired target site are separated from each other by 0 to 18 nucleotides; (ii) the paired target sites are separated from each other by non-coding sequences; (iii) the paired target sites are not present in the endogenous genome; and (iv) upon cleavage of one of the paired target sites by a pair of zinc finger nucleases and in the presence of a donor sequence in the host cell or cell line, the donor sequence is integrated into the genome in place of the paired target site and further wherein the donor sequence does not comprise the paired target sites cleaved by the pair of zinc finger nucleases.
2. The cell or cell line of claim 1 , where one target site from each paired target site comprises the same sequence.
3. The cell or cell line of claim 1 , wherein the exogenous nucleic acid molecule further comprises one or more coding sequences.
4. The cell or cell line of claim 1 , wherein the cell is a eukaryotic cell.
5. The cell or cell line of claim 4 , wherein the eukaryotic cell is a plant or mammalian cell.
6. A method for integrating one or more exogenous sequences into the genome of a cell, the method comprising
(a) providing one or more pairs of zinc finger nucleases to a cell according to claim 1 , wherein the zinc finger nucleases bind to a paired target site and cleave the multiple insertion site; and
(b) contacting the cell with a donor polynucleotide comprising an exogenous sequence, wherein the exogenous sequence is integrated into the genome of the cell within the multiple insertion site and in place of the paired target site and further wherein the donor polynucleotide does not comprise the paired target sites cleaved by the pair of zinc finger nucleases.
7. The method of claim 6 , further comprising repeating steps (a) and (b) with additional zinc finger nucleases that cleave additional target sites in the integrated nucleic acid molecule in the presence of additional exogenous sequences, thereby inserting the additional exogenous sequences into the genome of the cell.
8. The method of claim 6 , wherein one or more of the exogenous sequences comprise one or more target sites for zinc finger nucleases.
9. The method of claim 6 , wherein the target site is a zinc finger nuclease half target site, wherein, upon integration of the half target site, a target site is created.
10. The method of claim 6 , wherein one or more of the exogenous sequences comprises a coding sequence and the cell expresses the product of the coding sequence.
11. A method of deleting one or more sequences inserted into the genome of a cell, the method comprising,
(a) integrating a plurality of exogenous sequences according to claim 7 ; and
(b) expressing the appropriate nucleases in the cell such that one or more of the exogenous sequences are deleted from the genome.
12. A method of providing a genomically altered cell, the method comprising
(a) integrating one or more exogenous sequences in at least a first cell according to the method of claim 6 ;
(b) allowing the first cell to develop into a sexually mature organism; and
(c) crossing the organism with a second organism comprising at least one genomic alteration to generate a second cell with at least one of the genomic alterations of the first and second organisms.
13. The method of claim 12 , wherein the second cell genomic alterations comprise a plurality of heterologous genes at a single genomic location in the second cell.
14. The method of claim 12 , wherein the first cell further comprises one or more deletions within the integrated nucleic acid molecule.
15. The method of claim 6 , wherein the cell further comprises modifications of its genome outside the integrated nucleic acid molecule.
16. The method of claim 6 , wherein the cell is a plant cell.
17. The cell of claim 5 , wherein the cell is a plant cell and the paired target sites comprise sequences from a mammalian genome.