Nuclease systems for genetic engineering

Abstract

Fusion constructs encoding RNase-H-like domain containing compositions are disclosed. Disclosed are also compositions and methods utilizing RNase-H-like domain containing compositions for the treatment of cancer. Also disclosed are the methods of making and using the RNase-H-like domain containing compositions in treating various diseases, conditions, and cancer.

1 - 143 . (canceled) 
     
     
         144 . A nucleic acid editing system comprising:
 (a) a polypeptide construct or a nucleic acid encoding the polypeptide construct, wherein the polypeptide construct comprises an Argonaute polypeptide sequence from a mesophilic organism; and   (b) a guide nucleic acid that binds to a target nucleic acid and the Argonaute polypeptide sequence,   wherein upon contacting the target nucleic acid with the nucleic acid editing system at a mesophilic temperature, the nucleic acid editing system introduces a single stranded break or a double stranded break in the target nucleic acid.   
     
     
         145 . The nucleic acid editing system of  claim 144 , wherein the nucleic acid editing system introduces the single stranded break or the double stranded break in the target nucleic acid at a temperature from about 19° C. to 40° C. 
     
     
         146 . The nucleic acid editing system of  claim 144 , wherein the nucleic acid editing system introduces the single stranded break or the double stranded break in the target nucleic acid at about 37° C. 
     
     
         147 . The nucleic acid editing system of  claim 144 , wherein the polypeptide construct is a synthetic fusion polypeptide. 
     
     
         148 . The nucleic acid editing system of  claim 144 , wherein the polypeptide construct comprises a nuclease, a nickase, an RNase, a recombinase, a flippase, a transposase, or a combination thereof. 
     
     
         149 . The nucleic acid editing system of  claim 144 , wherein the Argonaute polypeptide sequence is from a  Clostridium.    
     
     
         150 . The nucleic acid editing system of  claim 144 , wherein the Argonaute polypeptide sequence is from an organism selected from the group consisting of:  Paenibacillus borealis, Deinococcus  sp. YIM 77859 , Calothrix  sp. PCC 7103, Clostridiales bacterium NK3B98,  Thermosynechococcus elongatus  BP-1,  Hyphomonas  sp. T16B2 , Chroococcidiopsis thermalis, Clostridium disporicum, Rhodopirellula maiorica  SM1,  Clostridium perfringens, Clostridium saudiense, Clostridium bolteae, Clostridium sartagoforme , and  Clostridium perfringens.    
     
     
         151 . The nucleic acid editing system of  claim 144 , wherein the target nucleic acid is of prokaryotic origin or of eukaryotic origin. 
     
     
         152 . The nucleic acid editing system of  claim 144 , wherein the target nucleic acid is a single-stranded DNA (ssDNA). 
     
     
         153 . The nucleic acid editing system of  claim 144 , wherein the target nucleic acid is a double-stranded DNA (dsDNA). 
     
     
         154 . The nucleic acid editing system of  claim 144 , wherein the guide nucleic acid is a DNA or an RNA. 
     
     
         155 . The nucleic acid editing system of  claim 144 , wherein the guide nucleic acid is at least partially complementary to the target nucleic acid. 
     
     
         156 . A method of modifying a target nucleic acid sequence in a eukaryotic cell, the method comprising:
 contacting the eukaryotic cell with a nucleic acid editing system or a nucleic acid encoding the nucleic acid editing system, wherein the nucleic acid editing system comprises:
 (i) a polypeptide construct, wherein the polypeptide construct comprises an Argonaute polypeptide sequence from a mesophilic organism; and 
 (ii) a guide nucleic acid that binds to a target nucleic acid and the Argonaute polypeptide sequence, 
   wherein upon contacting the target nucleic acid with the nucleic acid editing system at a mesophilic temperature, the Argonaute polypeptide sequence from the mesophilic organism cleaves the target nucleic acid within the eukaryotic cell, thereby modifying the target nucleic acid sequence.   
     
     
         157 . The method of  claim 156 , wherein the eukaryotic cell is a human cell. 
     
     
         158 . The method of  claim 156 , wherein the nucleic acid editing system introduces a single stranded break or a double stranded break in the target nucleic acid at a temperature from about 19° C. to 40° C. 
     
     
         159 . The method of  claim 156 , wherein the polypeptide construct is a synthetic fusion polypeptide. 
     
     
         160 . The method of  claim 156 , wherein the polypeptide construct comprises a nuclease, a nickase, an RNase, a recombinase, a flippase, a transposase, or a combination thereof. 
     
     
         161 . The method of  claim 156 , wherein the guide nucleic acid is a DNA or an RNA.