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 targeted integration of an exogenous sequence into a specified region of interest in the genome of a cell.
1. An isolated mammalian, plant, insect, xenopus or nematode cell comprising a linear donor nucleic acid molecule comprising homology arms of between 50 and 100 base pairs and a sequence of interest, wherein the homology arms flank the sequence of interest, wherein the one or more base pairs of the homology arms are joined with a phosphorothioate phosphodiester bond.
2. The cell of claim 1 , wherein the phosphorothioate phosphodiester bonds are positioned at the first and, optionally, second bonds of the 5′ and 3′ ends of the donor nucleic acid.
3. The cell of claim 1 , further comprising, between the homology arms, a sequence encoding a 2A peptide.
4. The cell of claim 1 , further comprising, between the homology arms, a sequence comprising an SA site.
5. The cell of claim 1 , further comprising, between the homology arms, a sequence comprising an IRES sequence.
6. The cell of claim 1 , wherein the sequence of interest does not encode a polypeptide.
7. The cell of claim 1 , further comprising a promoter sequence operably linked to the sequence of interest.
8. The cell of claim 1 , wherein the sequence of interest encodes a polypeptide.
9. The cell of claim 8 , 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.
10. The cell of claim 1 , wherein the sequence contains a non-coding nucleic acid.
11. The cell of claim 10 , wherein the non-coding nucleic acid is selected from the group consisting of a miRNA, and SH-RNA, or siRNA.