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

• US 9,121,072 B2
• Filed: 09/15/2011
• Issued: 09/01/2015
• Est. Priority Date: 09/27/2007
• Status: Active Grant

• Current Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Holmes, Michael C., Doyon, Yannick, Li, Tianjian
• Primary Examiner(s): DUNSTON, JENNIFER ANN
• Application Number:
  US13/234,007
• Publication Number:
  US 20120237926A1
• Time in Patent Office: 1,447 Days
• Field of Search: None
• US Class Current: 1/1
• 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. A host cell comprising an endogenous genome and an episomal reporter construct, the episomal reporter construct comprising a sequence consisting essentially of target sequences for multiple pairs of zinc finger nucleases, the target sequences flanked by sequences encoding a disabled reporter gene, wherein the target sequences are the same as sequences in the endogenous genome and further wherein the sequences encoding the disabled reporter comprise identical repeated sequences flanking the target sequences.

2. The host cell of claim 1, wherein the reporter construct further comprises a polyadenylation signal.

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

4. The host cell of claim 3, wherein the promoter is selected from the group of consisting of a constitutive promoter, a regulatable promoter or inducible promoter.

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

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

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

8. The host cell of claim 1, wherein the reporter construct further comprises a regulatable promoter operably linked to the reporter gene.

9. The host cell of claim 1, further comprising a sequence encoding a nuclease, wherein the nuclease comprises a zinc finger protein.

10. The host cell of claim 1, further comprising a donor polynucleotide that is integrated into the endogenous genome.

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

introducing one or more expression constructs that expresses the zinc finger nuclease(s) into a host cell according to claim 1, wherein the reporter construct comprises a target sequence recognized by the nuclease(s);

incubating the cells under conditions such that the zinc finger nuclease(s) are 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.

12. 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 one or more zinc finger nucleases targeted to recognize and cleave a target site in the genome into host cells according to claim 1, 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.

13. The method of claim 12, further comprising isolating the cells expressing the reporter gene.

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

15. The method of claim 12, wherein the method further comprises introducing an exogenous sequence into the host cells such that it is incorporated into the genome.

16. The method of claim 15, wherein the genomic modification is a gene addition.

17. The method of claim 12, wherein the reporter gene encodes a fluorescent protein and the selecting comprises fluorescence activated cell sorting (FACS) analysis.