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

• US 9,115,409 B2
• Filed: 03/11/2013
• Issued: 08/25/2015
• Est. Priority Date: 09/27/2007
• Status: Active Grant

• Current Assignee: Sangamo Biosciences, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Doyon, Yannick, Holmes, Michael C., Li, Tianjian
• Primary Examiner(s): DUNSTON, JENNIFER ANN
• Application Number:
  US13/794,199
• Publication Number:
  US 20130171657A1
• Time in Patent Office: 897 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 multiple target sites for at least one nuclease comprising a TAL-effector domain, the target sites flanked by sequences encoding a disabled reporter gene, wherein the endogenous genome comprises the target sites 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, further comprising a sequence encoding the at least one nuclease.

9. 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) comprising a TAL-effector domain into host cells 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 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.

10. 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 comprising a TAL-effector domain 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.

11. The method of claim 10, further comprising isolating the cells expressing the reporter gene.

12. The method of claim 10, wherein the genomic modification is a gene disruption.

13. The method of claim 10, wherein the method further comprises introducing an exogenous sequence into the host cells such that it is incorporated into the genome.

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

15. The method of claim 10, wherein the reporter gene encodes a fluorescent protein and the selecting comprises fluorescence activated cell sorting.