Compositions and methods of nucleic acid-targeting nucleic acids

Abstract

This disclosure provides for compositions and methods for the use of nucleic acid-targeting nucleic acids and complexes thereof.

1 - 26 . (canceled) 
     
     
         27 . An engineered nucleic acid targeting nucleic acid, comprising:
 an engineered CRISPR RNA (crRNA), an engineered trans-activating CRISPR RNA (tracrRNA), or a combination thereof, wherein (i) the engineered nucleic acid targeting nucleic acid can associate with a Cas9 protein to form a nucleic acid targeting nucleic acid-Cas9 complex capable of binding a target DNA, and (ii) the engineered crRNA, the engineered tracrRNA, or the combination thereof is altered relative to a corresponding wild-type crRNA, tracrRNA, or both; and   the engineered nucleic acid targeting nucleic acid comprises at least one of the following: a duplex variant, a spacer extension, a mid-tracr modification, or a tracr extension.   
     
     
         28 . The engineered nucleic acid targeting nucleic acid of  claim 27 , wherein the duplex variant comprises at least one of the following modifications: a modification of a first stem, a modification of a bulge, a modification of a first duplex, or an affinity tag. 
     
     
         29 . The engineered nucleic acid targeting nucleic acid of  claim 28 , wherein the modification of the first stem comprises a Csy4 binding sequence to which a Csy4 protein binds, a Cas5 binding sequence to which a Cas5 protein binds, or a Cas6 binding sequence to which a Cas6 protein binds. 
     
     
         30 . The engineered nucleic acid targeting nucleic acid of  claim 27 , wherein the spacer extension comprises at least one of the following: a spacer extension less than 10 nucleotides in length, a 5′ cap, a riboswitch sequence, a sequence that forms a hairpin, a sequence that targets the nucleic acid targeting nucleic acid to a subcellular location, a sequence that provides for tracking, a sequence that provides a protein binding site for a protein, a primer binding site, or an affinity tag. 
     
     
         31 . The engineered nucleic acid targeting nucleic acid of  claim 30 , wherein the sequence that provides the protein binding site for the protein comprises a Csy4 binding sequence to which a Csy4 protein binds, a Cas5 binding sequence to which a Cas5 protein binds, or a Cas6 binding sequence to which a Cas6 protein binds. 
     
     
         32 . The engineered nucleic acid targeting nucleic acid of  claim 27 , wherein the mid-tracr modification comprises at least one of the following modifications: a modification of a P domain, a modification of a second stem, a modification of a third stem, an added nucleic acid binding protein binding site, or an affinity tag. 
     
     
         33 . The engineered nucleic acid targeting nucleic acid of  claim 32 , wherein the added nucleic acid binding protein binding site comprises a Csy4 binding sequence to which a Csy4 protein binds, a Cas5 binding sequence to which a Cas5 protein binds, or a Cas6 binding sequence to which a Cas6 protein binds. 
     
     
         34 . The engineered nucleic acid targeting nucleic acid of  claim 27 , wherein the tracr extension comprises at least one of the following: a 3′ hybridizing extension, an added nucleic acid binding protein binding site, or an affinity tag. 
     
     
         35 . The engineered nucleic acid targeting nucleic acid of  claim 34 , wherein the added nucleic acid binding protein binding site comprises a Csy4 binding sequence to which a Csy4 protein binds, a Cas5 binding sequence to which a Cas5 protein binds, or a Cas6 binding sequence to which a Cas6 protein binds. 
     
     
         36 . The engineered nucleic acid targeting nucleic acid of  claim 34 , wherein a donor polynucleotide is hybridized to the 3′ hybridizing extension. 
     
     
         37 . The engineered nucleic acid targeting nucleic acid of  claim 27 , wherein the tracr extension comprises at least one of the following: a tracr extension having a length of from 1 to 400 nucleotides, a 3′ polyadenylated tail, a riboswitch sequence, a sequence that forms a hairpin, a sequence that targets the nucleic acid targeting nucleic acid to a subcellular location, a sequence that provides for tracking, or a primer binding site. 
     
     
         38 . The engineered nucleic acid targeting nucleic acid of  claim 29 , wherein the Csy4 protein binds to the Csy4 binding sequence with a Kd ˜50 pM. 
     
     
         39 . The engineered nucleic acid targeting nucleic acid of  claim 27 , wherein the engineered nucleic acid targeting nucleic acid is a double guide nucleic acid structure. 
     
     
         40 . The engineered nucleic acid targeting nucleic acid of  claim 27 , wherein the engineered nucleic acid targeting nucleic acid is a single guide nucleic acid structure. 
     
     
         41 . The engineered nucleic acid targeting nucleic acid of  claim 27 , wherein the engineered nucleic acid targeting nucleic acid comprises DNA. 
     
     
         42 . The engineered nucleic acid targeting nucleic acid of  claim 27 , wherein the engineered nucleic acid targeting nucleic acid comprises RNA. 
     
     
         43 . The engineered nucleic acid targeting nucleic acid of  claim 41 , wherein the engineered nucleic acid targeting nucleic acid comprises RNA.