Targeted genomic modification with partially single-stranded donor molecules

• US 9,255,259 B2
• Filed: 02/09/2011
• Issued: 02/09/2016
• Est. Priority Date: 02/09/2010
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Cost, Gregory J., Guschin, Dmitry M., Urnov, Fyodor
• Primary Examiner(s): Ault, Addison D
• Application Number:
  US12/931,760
• Publication Number:
  US 20110207221A1
• Time in Patent Office: 1,826 Days
• Field of Search: None
• US Class Current: 1/1
• 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 mammalian or plant cell comprising:

a genome comprising a target site of interest;

a non-naturally occurring zinc finger nuclease that is capable of generating a 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 at the target site of interest 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 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:

providing the cell according to claim 1; and

creating a double-stranded break in the genome of the cell using a zinc finger nuclease 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 nuclease comprises a naturally occurring or non-naturally occurring cleavage domain from a Type IIS restriction endonuclease.