Methods and compositions for increasing nuclease activity

• US 20110129898A1
• Filed: 05/18/2010
• Published: 06/02/2011
• Est. Priority Date: 05/18/2009
• Status: Active Grant

• Current Assignee: Sangamo Biosciences, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Doyon, Yannick
• Granted Patent: US 8,772,008 B2
• Time in Patent Office: Days
• US Class Current: 435/196
• CPC Class Codes: C12N 2523/00 Culture process characterised by temperature
• CPC Class Codes: C12N 5/00 Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; plant reproduction by tissue culture techniques A01H4/00
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed herein are methods and compositions for increasing nuclease activity to increase nuclease-mediated genomic modifications.

Claims

1. A method for increasing the nuclease activity of at least one exogenous nuclease in a cell, the method comprising:

subjecting the cells to cold shock conditions following introduction of the exogenous nuclease into the cell, and

returning the cells to an appropriate temperature for growth.

2. The method of claim 1, wherein the cold shock temperature is between 27°

and 33°

C.

3. The method of claim 1, wherein the cells are subjected to cold shock for between 1 and 4 days.

4. The method of claim 1, wherein the nuclease is introduced into the cell using a lentiviral vector.

5. The method of claim 4, wherein the lentiviral vector is an Integration Defective Lentiviral vector (IDLV) construct.

6. The method of claim 1, wherein the cell is a eukaryotic cell.

7. The method of claim 1, wherein the nuclease is a zinc finger nuclease.

8. The method of claim 1, wherein the nuclease is a naturally occurring of non-naturally occurring homing endonuclease.

9. The method of claim 1, wherein the nuclease is a TAL-effector domain nuclease fusion protein.

10. A kit for increasing the activity of a nuclease in a cell, the kit comprising

one or more nucleases,

instructions for introducing the nucleases into cells and cold shocking the cells to increase nuclease activity.

11. The kit of claim 10, further comprising the cells into which the nucleases are introduced.

12. The kit of claim 10, further comprising an exogenous sequence that is introduced into the cell and integrated into the genome of at least a portion of the cells following nuclease-induced cleavage of the genome.