Targeted genomic modification with partially single-stranded donor molecules

• US 9,005,973 B2
• Filed: 07/08/2011
• Issued: 04/14/2015
• Est. Priority Date: 02/09/2010
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Cost, Gregory J., Guschin, Dmitry M., Urnov, Fyodor
• Primary Examiner(s): Dunston, Jennifer
• Assistant Examiner(s): Ault, Addison D
• Application Number:
  US13/135,558
• Publication Number:
  US 20110287545A1
• Time in Patent Office: 1,376 Days
• Field of Search: None
• 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 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.