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 mammalian cell comprising:
a genome comprising a double-stranded break with single-stranded overhangs;
a non-naturally occurring nuclease comprising a DNA-binding molecule that binds to a target site of at least 9 nucleotides in length and endonuclease cleavage domain, which nuclease is capable of generating the double-stranded break with single-stranded overhangs in the genome at the target site of interest; and
an exogenous linear donor nucleic acid molecule comprising a double-stranded exogenous sequence of interest having first and second ends and first and second single-stranded sequences of 4 to 10 nucleotides at the first and second ends of the double-stranded sequence,
wherein the first and second single-stranded sequences are 100% complementary to the single-stranded overhangs in the genome such that the exogenous linear donor sequence is capable of being directly integrated at the target site of interest by annealing of the complementary first and second-single stranded sequences and non-homologous end joining (NHEJ)-mediated targeted integration.
2. The cell of claim 1 , wherein the exogenous linear donor further comprises one or more phosphorothioate or phosphodiester bonds between one or more nucleic acids.
3. The cell of claim 1 , wherein one or more nucleic acids of the exogenous linear donor are methylated or comprise non-natural nucleotide analogs.
4. The cell of claim 1 , wherein the sequence of interest comprises a polynucleotide encoding a functional polypeptide.
5. The cell 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, and a reporter.
6. The cell of claim 1 , wherein the sequence of interest comprises a sequence encoding a functional RNA.
7. The cell of claim 1 , wherein the sequence of interest comprises an integration site.
8. A method of integrating a linear nucleic acid molecule in to the genome of a cell, the method comprising:
culturing the cell according to claim 1 under conditions such that the linear nucleic acid molecule is integrated at the site of the double-stranded break.
9. The method of claim 8 , wherein the linear nucleic acid molecule further comprises one or more phosphorothioate or phosphodiester bonds between one or more nucleic acids.
10. The method of claim 8 , wherein one or more nucleic acids are methylated or comprise non-natural nucleotide analogs.
11. The method of claim 8 , wherein the sequence of interest comprises a polynucleotide encoding a functional polypeptide.
12. The method of claim 11 , 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.
13. The method of claim 8 , wherein the sequence of interest comprises a sequence encoding a functional RNA.
14. The method of claim 8 , wherein the sequence of interest comprises an integration site.