Linear donor constructs for targeted integration
Disclosed herein are linear donor molecules comprising homology arms of 50-750 base pairs (e.g., 50-100 base pairs) flanking one or more sequences of interest. The donor molecules and/or compositions comprising these molecules can be used in methods for integration of an exogenous sequence into a specified region of interest in the genome of a cell.
1. A linear donor nucleic acid molecule comprising homology arms of between 50 and 100 base pairs, a sequence encoding a 2A peptide and a sequence of interest, wherein the homology arms flank the sequence of interest and the sequence encoding the 2A peptide.
2. The linear donor nucleic acid of claim 1 , further comprising, between the homology arms, a sequence comprising an SA site.
3. The linear donor nucleic acid of claim 1 , further comprising, between the homology arms, a sequence comprising an IRES sequence.
4. The linear donor nucleic acid of claim 1 , wherein the sequence of interest does not encode a polypeptide.
5. The linear donor nucleic acid of claim 1 , further comprising a promoter sequence operably linked to the sequence of interest.
6. The linear donor nucleic acid of claim 1 , wherein the sequence of interest encodes a polypeptide.
7. The linear donor nucleic acid according to claim 6 , wherein the polypeptide is selected from the group consisting of an antibody, an antigen, an enzyme, a growth factor, a cell surface receptor, a nuclear receptor, a hormone, a lymphokine, a cytokine, a reporter gene, a selectable marker, a secreted factor, an epitope tag and functional fragments thereof and combinations thereof.
8. The linear donor nucleic acid of claim 1 , wherein the sequence contains a non-coding nucleic acid.
9. The linear donor nucleic acid according to claim 8 , wherein the non-coding nucleic acid is selected from the group consisting of a miRNA, and SH-RNA, or siRNA.
10. A method for homology-dependent targeted integration of a sequence of interest into a region of interest in the genome of a cell, the method comprising the steps of:
(a) expressing a fusion protein in the cell, the fusion protein comprising a DNA-binding domain and cleavage domain or a cleavage half-domain, wherein the DNA-binding domain has been engineered to bind to a target site in the region of interest;
(b) contacting the cell with a donor polynucleotide of claim 1 ,
wherein binding of the fusion protein to the target site cleaves the genome of the cell in the region of the interest, thereby resulting in homology-dependent targeted integration of the sequence of interest into the genome of the cell.
11. A method for homology-dependent targeted integration of a sequence of interest into a cell, the method comprising:
(a) expressing a first fusion protein in the cell, the first fusion protein comprising a first DNA-binding domain and a first cleavage half-domain, wherein the first DNA-binding domain has been engineered to bind to a first target site in a region of interest in the genome of the cell;
(b) expressing a second fusion protein in the cell, the second fusion protein comprising a second DNA-domain and a second cleavage half domain, wherein the second zinc finger binding domain binds to a second target site in the region of interest in the genome of the cell, wherein the second target site is different from the first target site; and
(c) contacting the cell with a polynucleotide comprising a donor nucleic acid according to claim 1 ;
wherein binding of the first fusion protein to the first target site, and binding of the second fusion protein to the second target site, positions the cleavage half-domains such that the genome of the cell is cleaved in the region of interest, thereby resulting in homology-dependent integration of the donor nucleic said into the genome of the cell.
12. The method of claim 10 , wherein at least one DNA-binding domain is a zinc finger binding domain.
13. The method of claim 10 , wherein at least one DNA-binding domain is a meganuclease DNA-binding domain.
14. The method of claim 10 , wherein the sequence of interest from the integrated donor nucleic acid expresses a polypeptide.
15. The method of claim 10 , wherein the sequence in interest from the integrated donor comprises a non-coding nucleic acid sequence.
16. The method of claim 10 , wherein the cleavage domain is from a meganuclease.
17. The method of claim 11 , wherein the first and second cleavage half-domains are from a Type IIS restriction endonuclease.
18. The method according to claim 17 , wherein the Type IIS restriction endonuclease is selected from the group consisting of Fold and StsI.
19. The method according to claim 10 , wherein the cell is arrested in the G2 phase of the cell cycle.
20. The method according to claim 10 , wherein at least one of the fusion proteins comprises an alteration in the amino acid sequence of the dimerization interface of the cleavage half-domain.
21. The method according to claim 10 , wherein the cell is a mammalian cell.
22. The method according to claim 10 , wherein the cell is a human cell.
23. The method according to claim 10 , wherein the cell is a plant cell.