Modified crispr rna and modified single crispr rna and uses thereof

Abstract

The present disclosure provides compounds comprising modified oligonucleotides for use in CRISPR. In certain embodiments, such modified oligonucleotides provide improved properties of crRNA. In certain embodiments, such modified oligonucleotides provide improved properties of scrRNA.

1 .- 293 . (canceled) 
     
     
         294 . A modified crRNA consisting of 29 to 42 nucleosides, comprising a DNA recognition portion and tracrRNA recognition portion, wherein the DNA recognition portion is located 5′ of the tracrRNA recognition portion;
 the DNA recognition portion comprises at least one modified nucleoside comprising a linearly modified sugar moiety; and 
 the tracrRNA recognition portion comprises at least one modified nucleoside comprising a bicyclic modified sugar moiety. 
 
     
     
         295 . The modified crRNA of  claim 294 , wherein at least one linearly modified sugar moiety in the DNA recognition portion of the modified crRNA is selected from a 2′-F and a 2′-OMe modified sugar moiety. 
     
     
         296 . The modified crRNA of  claim 295 , wherein the at least one linearly modified sugar moiety in the DNA recognition portion of the modified crRNA is a 2′-F modified sugar moiety. 
     
     
         297 . The modified crRNA of  claim 294 , wherein the DNA recognition portion of the modified crRNA is at the 5′-terminal of the cRNA. 
     
     
         298 . The modified crRNA of  claim 294 , wherein the DNA recognition portion of the modified crRNA is at the 5′-terminal of the cRNA, and wherein each of the seven 5′-terminal nucleosides comprise linearly modified sugar moieties. 
     
     
         299 . The modified crRNA of  claim 294 , wherein each linearly modified sugar moiety in the DNA recognition portion of the modified crRNA is selected from a 2′-F and a 2′-OMe modified sugar moiety. 
     
     
         300 . The modified crRNA of  claim 299 , wherein the linearly modified sugar moieties of the seven 5′-terminal nucleosides are each 2′-F or 2′-OMe. 
     
     
         301 . The modified crRNA of  claim 299 , wherein the modified sugar moieties of the seven 5′-terminal nucleosides alternate between 2′-F and 2′-OMe. 
     
     
         302 . The modified crRNA of  claim 294 , wherein the DNA recognition portion of the modified cRNA comprises at least one nucleoside comprising an unmodified sugar moiety. 
     
     
         303 . The compound of  claim 302 , wherein the unmodified sugar moiety is a 2′-hydroxy sugar moiety. 
     
     
         304 . The modified cRNA of  claim 294 , wherein the DNA recognition portion of the modified cRNA comprises at least ten consecutive nucleosides each comprising unmodified 2′-hydroxy sugar moieties at the 3′-end of the DNA recognition portion. 
     
     
         305 . The modified crRNA of  claim 294 , wherein the tracrRNA recognition portion of the modified crRNA comprises at least four modified sugar moieties. 
     
     
         306 . The modified crRNA of  claim 294 , wherein the tracrRNA recognition portion of the modified crRNA comprises no more than five modified sugar moieties. 
     
     
         307 . The modified crRNA of  claim 294 , wherein the tracrRNA recognition portion of the modified crRNA comprises exactly five modified sugar moieties. 
     
     
         308 . The modified crRNA of  claim 294 , wherein the nucleoside at the 5′-end of the tracrRNA recognition portion of the crRNA comprises a modified sugar moiety. 
     
     
         309 . The modified crRNA of  claim 294 , wherein each modified sugar moiety within the tracrRNA recognition portion of the modified crRNA is a bicyclic sugar moiety. 
     
     
         310 . The modified crRNA of  claim 294 , wherein each bicyclic sugar moiety is selected from LNA or cEt. 
     
     
         311 . The modified crRNA of  claim 294 , wherein the crRNA consists of 42 linked nucleosides. 
     
     
         312 . The modified crRNA of  claim 294 , wherein the cRNA consists of 29-32 linked nucleosides. 
     
     
         313 . The modified crRNA of  claim 294 , wherein the DNA recognition portion of the crRNA consists of 17 or fewer linked nucleosides. 
     
     
         314 . The modified crRNA of  claim 294 , wherein the tracrRNA recognition portion of the crRNA consists of 12 or fewer linked nucleosides. 
     
     
         315 . The modified crRNA of  claim 294 , wherein the nucleobase sequence of the DNA recognition portion of the cRNA is at least 90%, at least 95%, or 100% complementary to a target DNA. 
     
     
         316 . The modified cRNA of  claim 294 , wherein each internucleoside linkage of the modified cRNA is selected from a phosphodiester internucleoside linkage or a phosphorothioate internucleoside linkage. 
     
     
         317 . The modified cRNA of  claim 294 , wherein each internucleoside linkage of the modified cRNA is a phosphorothioate internucleoside linkage. 
     
     
         318 . A method comprising contacting a cell with a modified cRNA of  claim 294 ,
 a tracrRNA or a plasmid encoding a tracrRNA, and   a Cas9 protein or a plasmid encoding a Cas9 protein.   
     
     
         319 . The method of  claim 318 , wherein the cell is in an animal. 
     
     
         320 . A pharmaceutical composition comprising the modified crRNA of  claim 294  and a pharmaceutically acceptable carrier or diluent.