Rapid screening of biologically active nucleases and isolation of nuclease-modified cells

• US 20110014616A1
• Filed: 06/29/2010
• Published: 01/20/2011
• Est. Priority Date: 06/30/2009
• Status: Abandoned Application

• Current Assignee: Sangamo Biosciences, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: LI, TIANJIAN, HOLMES, MICHAEL C.
• Application Number:
  US12/803,552
• Publication Number:
  US 20110014616A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

Disclosed herein are methods and compositions for rapidly identifying active nucleases and cells having nuclease-mediated genomic modifications.

Claims

1. An episomal reporter construct comprising at least one target sequence for a nuclease flanked by sequences encoding a reporter gene.

2. The reporter construct of claim 1, further comprising a polyadenylation signal.

3. The reporter construct of claim 1, wherein the reporter construct further comprises a promoter sequence operably linked to the reporter gene.

4. The reporter construct of claim 2, wherein the promoter is selected from the group of consisting of a constitutive promoter, a regulatable promoter or inducible promoter.

5. The reporter construct of claim 1, wherein the reporter gene encodes a light-generating protein, an enzyme, a cell surface receptor, or a selectable marker.

6. A host cell comprising a reporter construct according to claim 1.

7. The host cell of claim 6, wherein the cell is a eukaryotic cell.

8. The host cell of claim 7, wherein the cell is a mammalian cell.

9. The host cell of claim 6, wherein the reporter construct is transiently expressed in the host cell.

10. The host cell of claim 6, further comprising a sequence encoding a nuclease.

11. The host cell of claim 10, wherein the nuclease comprises a zinc finger protein, a meganuclease or a TAL-effector domain nuclease fusion protein.

12. A method of identifying one or more nucleases that induce cleavage at a specific target site, the method comprising the steps of:

introducing one or more expression constructs that expresses the nuclease(s) into a host cell according to claim 6, wherein the reporter construct comprises a target sequence recognized by the nuclease;

incubating the cells under conditions such that the nuclease is expressed; and

measuring the levels of reporter gene expression in the cells, wherein increased levels of reporter gene expression are correlated with increased nuclease-induced cleavage of the target sequence.

13. A method of enriching a population of cells for cells having a nuclease-mediated genomic modification, the method comprising the steps of:

introducing one or more expression constructs encoding nucleases targeted to recognize and cleave a target site in the genome into host cells according to claim 6, wherein the reporter construct in the host cells comprises the target sequence recognized by the nuclease (s);

incubating the cells under conditions such that the nuclease(s) are expressed;

measuring the levels of reporter gene expression in the cells; and

selecting cells that express the reporter gene, thereby enriching the population of cells for cells with nuclease-mediated genomic modifications and an active nuclease.

14. The method of claim 13, further comprising isolating the cells expressing the reporter gene.

15. The method of claim 13, wherein the genomic modification is a gene disruption.

16. The method of claim 13, wherein the genomic modification is a gene addition.

17. The method of claim 16, wherein the method further comprises introducing an exogenous sequence into the host cells such that it is incorporated into the genome.

18. The method of claim 13, wherein the reporter gene expresses light and the selecting comprises FACS analysis.