Coupling endonucleases with end-processing enzymes drives high efficiency gene disruption

Abstract

The present disclosure relates to the co-expression of an endonuclease with an end-processing enzyme for the purpose of enhanced processing of the polynucleotide ends generated by endonuclease cleavage.

What is claimed is: 
     
         1 . A method of increasing the mutagenic activity of an endonuclease comprising: providing the endonuclease concurrently with an end-processing enzyme. 
     
     
         2 . The method of  claim 1  wherein the endonuclease and the end-processing enzyme are encoded by a single polynucleotide. 
     
     
         3 . The method of  claim 2  wherein the endonuclease and the end-processing enzyme are linked by a T2A linker sequence. 
     
     
         4 . The method of  claim 1  wherein the endonuclease and exonuclease are provided as a fusion protein. 
     
     
         5 . The method  claim 1  wherein the end-processing enzyme exhibits 5-3′ exonuclease, 5-3′ alkaline exonuclease, 3-5′exonuclease, 5′ flap endonuclease, helicase or template-independent DNA polymerases activity. 
     
     
         6 . A polypeptide comprising an endonuclease domain, and end-processing domain. 
     
     
         7 . The polypeptide of  claim 6 , further comprising a linker domain. 
     
     
         8 . The polypeptide of  claim 6 , wherein the endonuclease domain comprises an engineered homing endonuclease or a biologically active fragment thereof. 
     
     
         9 . The polypeptide of  claim 6 , wherein the end-processing domain exhibits 5-3′ exonuclease, 5-3′ alkaline exonuclease, 3-5′exonuclease, 5′ flap endonuclease, helicase, or template-independent DNA polymerases activity. 
     
     
         10 . The polypeptide of  claim 9 , wherein the end-processing domain comprises Trex2 or a biologically active fragment thereof. 
     
     
         11 . The polypeptide of  claim 6 , wherein the polypeptide is capable of cleaving CCR-5. 
     
     
         12 . A method of treating a disease in a subject comprising: contacting the cells of the subject with a polypeptide of  claim 6 ,
 wherein the polypeptide binds to a site within the cells involved in the proliferation of the disease.   
     
     
         13 . A method of treating a disease in a subject comprising: contacting one or more cells of the subject with one or more polypeptides having endonuclease activity and one or more polypeptides having end-processing activity,
 wherein the one or more polypeptides having endonuclease activity bind to a portion of a nucleotide sequence within the cells encoding a gene involved in the disease or a regulatory sequence thereof.   
     
     
         14 . The method of  claim 13  wherein the disease is HIV. 
     
     
         15 . The method of  claim 13  wherein the disease is hyper IGE syndrome. 
     
     
         16 . The method of  claim 13  wherein one or more polypeptides having endonuclease activity is an engineered homing endonuclease or a biologically active fragment thereof.