Targeted genomic modification with partially single-stranded donor molecules

• US 20110287545A1
• Filed: 07/08/2011
• Published: 11/24/2011
• Est. Priority Date: 02/09/2010
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Urnov, Fyodor, Cost, Gregory J., Guschin, Dmitry M.
• Granted Patent: US 9,005,973 B2
• Time in Patent Office: Days
• US Class Current: 435/440
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

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.

Claims

1. A method of integrating a linear nucleic acid molecule comprising a double-stranded sequence of interest having first and second ends and a first single-stranded nucleotide at the first end of the double-stranded sequence into the genome of a cell, the method comprising:

creating a double-stranded break using a nuclease comprising a TAL-effector domain in the genome of the cell such that the linear nucleic acid molecule is integrated at the site of the double-stranded break.

2. The method of claim 1, wherein the linear nucleic kid molecule further comprises a second single-stranded sequence at the second end of the double-stranded sequence.

3. The method of claim 1, wherein the first single-stranded sequence is between 1 and 10 nucleotides in length.

4. 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.

5. The method of claim 1, wherein one or more nucleic acids are methylated or comprise non-natural nucleotide analogs.

6. The method of claim 1, wherein the sequence of interest comprises a polynucleotide encoding a functional polypeptide.

7. The method of claim 6, 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.

8. The method of claim 1, wherein the sequence of interest Comprises a sequence encoding a functional RNA.

9. The method of claim 1, wherein the sequence of interest comprises an integration site.