Targeted genomic modification with partially single-stranded donor molecules
Disclosed herein are donor molecules comprising single-stranded complementary regions 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. A method of non-homologous end joining (NHEJ)-mediated targeted integration of a linear nucleic acid molecule into the genome of a mammalian cell, the method comprising:
introducing a linear nucleic acid molecule into the mammalian cell, the linear nucleic acid molecule comprising a double-stranded sequence of interest having first and second ends and first and second single-stranded nucleotides between 1 and 10 nucleotides in length at the first and second ends of the double-stranded sequence; and
creating a double-stranded break using a nuclease in an endogenous locus of the genome of the mammalian cell such that the linear nucleic acid molecule is integrated at the site of the double-stranded break and further wherein the nuclease comprises a TALE domain and the first and second single-stranded nucleotides are 100% complementary to the overhangs of the double-stranded break in the mammalian genome created by the nuclease; and
annealing the first and second single-stranded nucleotides of the donor to the overhangs of the double-stranded break in the mammalian genome, thereby directly integrating the donor at the site of double-stranded break by NHEJ-mediated targeted integration.
2. The method of claim 1 , wherein the linear nucleic acid molecule further comprises one or more phosphorothioate phosphodiester bonds between one or more nucleic acids.
3. The method of claim 1 , wherein one or more nucleic acids are methylated or comprise non-natural nucleotide analogs.
4. The method of claim 1 , wherein the sequence of interest comprises a polynucleotide encoding a functional polypeptide.
5. The method of claim 4 , wherein the polypeptide is selected from the group consisting of an antibody, an antigen, an enzyme, a growth factor, a receptor, a hormone, a lymphokine, a cytokine, a reporter and combinations therefore.
6. The method of claim 1 , wherein the sequence of interest comprises a sequence encoding a functional RNA.
7. The method of claim 1 , wherein the sequence of interest comprises an integration site.