Rapid in vivo identification of biologically active nucleases

• US 20090111119A1
• Filed: 09/25/2008
• Published: 04/30/2009
• Est. Priority Date: 09/27/2007
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Therapeutics, Inc.
• Inventors: Urnov, Fyodor, Doyon, Yannick
• Granted Patent: US 9,506,120 B2
• 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 1/34 involving hydrolase
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: G01N 2333/922 Ribonucleases (RNAses); Deoxyribonucleases (DNAses)
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

Disclosed herein are methods and compositions for rapidly identifying and ranking nucleases for specific cleavage of a target sequence.

Claims

1. A reporter construct for detecting double-stranded cleavage of a target sequence by one or more nucleases, the reporter construct comprising overlapping and non-functional sequences of a reporter gene separated by a target sequence recognized by the nuclease.

2. The reporter construct of claim 1, wherein the reporter gene is operably linked to a promoter sequence.

3. The reporter construct of claim 2, wherein the promoter is constitutive.

4. The reporter construct of claim 2, wherein the promoter is an inducible promoter.

5. The reporter construct of claim 1, wherein the reporter gene encodes an enzyme.

6. The reporter construct of claim 5, further comprising a sequence encoding a selectable marker.

7. The reporter construct of claim 1, further comprising regions of homology flanking the discontinuous reporter gene sequences.

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

9. The host cell of claim 8, wherein the cell is a eukaryotic cell.

10. The host cell of claim 9, wherein the cell is a yeast cell.

11. The host cell of claim 8, wherein the reporter construct is transiently expressed in the host cell.

12. The host cell of claim 8, wherein the reporter construct is stably integrated into the host cell.

13. A method of identifying a nuclease that induces cleavage at a specific target site, the method comprising the steps of:

introducing one or more expression constructs that expresses the nuclease into a host cell according to claim 8, 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.

14. A method of ranking a panel of nucleases for their activity in inducing cleavage at a specific target site, the method comprising the steps of:

introducing one or more expression constructs encoding nucleases of the panel into separate host cells according to claim 8, wherein the reporter construct in the host cell comprises a target sequence recognized by the nuclease;

incubating the cells under conditions such that the nucleases are expressed;

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

ranking the nucleases according to levels of reporter gene activity induced in the host cell.

15. A methods of predicting the in vivo cleavage activity of a nuclease, the method comprising the steps of:

introducing an expression construct encoding the nuclease into a host cell according to claim 8, 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 higher levels or reporter gene expression are predictive of a nuclease that will be active in vivo.

17. A method of selecting a biologically active nuclease, the method comprising the steps of:

identifying a nuclease cleaves at a selected target site according to claim 13; and

introducing an expression construct encoding the nuclease into a host cell;

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

culturing the cells over a period of time; and

measuring the growth of cells in culture at various time intervals, wherein biologically active nuclease(s) exhibiting cleavage activity and low toxicity are selected.

20. The method of claim 13, the nuclease comprises a homing endonuclease.

21. The method of claim 13, wherein the nuclease comprises an engineered zinc finger nuclease or pair of engineered zinc finger nucleases.

16. A method of determining toxic effects on a host cell caused by a nuclease, the method comprising the steps of:

introducing an expression construct encoding the nuclease into a host cell;

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

culturing the cells over a period of time; and

measuring the growth of cells in culture at various time intervals, wherein diminished growth is correlated with increased toxic effects of the nuclease.

18. The method of claim 16, wherein the growth of the cells is determined by spectrophotometry.

19. The method of claim 16, wherein the host cell comprises a reporter construct comprising overlapping and non-functional sequences of a reporter gene separated by a target sequence recognized by the nuclease.