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

Abstract

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

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.