Rapid in vivo identification of biologically active nucleases

• US 9,506,120 B2
• Filed: 09/25/2008
• Issued: 11/29/2016
• Est. Priority Date: 09/27/2007
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Doyon, Yannick, Urnov, Fyodor
• Primary Examiner(s): DUNSTON, JENNIFER ANN
• Application Number:
  US12/284,887
• Publication Number:
  US 20090111119A1
• Time in Patent Office: 2,987 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 1/34 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 yeast host cell comprising a genome and an integrated exogenous sequence, the integrated exogenous sequence comprising a reporter construct for detecting double-stranded cleavage by a pair of zinc finger nucleases, the reporter construct comprising

(i) overlapping and non-functional sequences of a reporter gene separated by an exogenous sequence comprising a counterselectable gene, a sequence encoding antibiotic resistance and a non-coding sequence comprising target sequences recognized by at least two different pairs of zinc finger nucleases, wherein the target sequences comprise endogenous mammalian or plant sequences not present in the yeast cell;

and

(ii) regions of homology to the genome of the yeast host cell flanking the overlapping and non-functional reporter gene sequence,

wherein the reporter gene is capable of being reconstituted when the target sequences are cleaved by the pair of zinc finger nucleases.

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

3. The host cell of claim 2, wherein the promoter is constitutive.

4. The host cell of claim 2, wherein the promoter is an inducible promoter.

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

6. The host cell of claim 5, further comprising a sequence encoding a selectable marker.

7. A method of identifying a pair of 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 pair of zinc finger nucleases into host cells according to claim 1, wherein the reporter construct comprises a target sequence recognized by the pair of zinc finger nucleases;

incubating the cells under conditions such that the pair of zinc finger nucleases is expressed;

selecting the incubated cells in medium containing a counterselecting agent; and

measuring cell growth of the selected cells, wherein increased levels of cell growth are correlated with increased zinc finger nuclease-induced cleavage of the target sequence.

8. A method of ranking a panel of zinc finger nuclease pairs for their activity in inducing cleavage at a specific target site, the method comprising the steps of:

introducing one or more expression constructs encoding the zinc finger nuclease pairs of the panel into separate host cells according to claim 1, wherein the reporter construct in the host cell comprises a target sequence recognized by the zinc finger nuclease pair;

incubating the cells under conditions such that the zinc finger nuclease pairs are expressed;

selecting the incubated cells in medium containing a counterselecting agent;

measuring cell growth of the selected cells, wherein increased levels of cell growth are correlated with increased zinc finger nuclease-induced cleavage of the target sequence; and

ranking the nucleases according to levels of cell growth.

9. A method of predicting in vivo cleavage activity of a pair of zinc finger nucleases, the method comprising the steps of:

introducing an expression construct encoding the pair of zinc finger into a host cell according to claim 1, wherein the reporter construct comprises a target sequence recognized by the pair of zinc finger nucleases;

incubating the cells under conditions such that the pair of zinc finger nucleases is expressed;

selecting the incubated cells in medium containing a counterselecting agent; and

measuring cell growth of the selected cells, wherein increased levels of cell growth are correlated with increased zinc finger nuclease-induced cleavage of the target sequence;

wherein cell growth is predictive of a pair of zinc finger nucleases that will be active in vivo.

10. The method of any one of claim 7, 8 or 9, wherein the cell growth is determined by spectrophotometry.