Crispr-cas based system for targeting single-stranded sequences

Abstract

The present invention relates to a CRISPR-Cas based system for targeting nucleic acid sequences. In part, the invention relates to synthetic guiding components for targeting single-stranded sequences, as well as design principles for constructing such components. Also described herein are methods of employing such components, e.g., to repress or activate a desired target within the subject.

1 . A synthetic guide component comprising a structure having the formula (I):
 W-X-Y-L-Z or salt thereof, wherein:   W is an optional third portion comprising a nucleic acid sequence of from about 1 to 20 nucleic acids;   X is a targeting portion comprising a nucleic acid sequence configured to bind to a target site of a single-stranded target sequence;   Y is a first portion comprising a nucleic acid sequence configured to interact with a nuclease configured to bind and/or cleave the single-stranded target sequence;   L is a linker; and   Z is a second portion comprising a nucleic acid sequence configured to interact with the nuclease and the first portion;   wherein the synthetic guiding component is configured to bind to the single-stranded target sequence without aid of a proto-spacer adjacent motif (PAM) sequence (PAMmer); and   X is configured to bind to the target site of a single-stranded mRNA target sequence or a single-stranded pathogen target sequence.   
     
     
         2 . The synthetic guide component of  claim 1 , wherein:
 Y comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:150-155, 179-186, 213-225, 239-243, 251-253, 256, 257, 260, and 262-264; and   Z comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 162-178, 192-212, 232-238, 244-250, 254, 255, 258, 259, 261, and 270-273.   
     
     
         3 . The synthetic guide component of  claim 1 , wherein:
 L comprises a bond or a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 156-161, 187-191, 226-231, and 265-269.   
     
     
         4 . The synthetic guide component of  claim 1 , having a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 274-293. 
     
     
         5 . The synthetic guide component of  claim 1 , wherein W and/or Y comprises one or more modified nucleic acids or bulges. 
     
     
         6 . The synthetic guide component of  claim 1 , wherein X has a length of from about 15 to about 30 nucleotides. 
     
     
         7 . The synthetic guide component of  claim 1 , wherein single-stranded target sequence is the single-stranded mRNA target sequence. 
     
     
         8 . The synthetic guide component of  claim 1 , wherein the nuclease is a Cas protein or a modified form thereof. 
     
     
         9 . The synthetic guide component of  claim 1 , wherein the nuclease is a SauCas9 protein or a CjeCas9 protein. 
     
     
         10 . The synthetic guide component of  claim 1 , wherein
 Y comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 150-155, or a complement of any of these;   L is a bond or comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:156-161, or a complement of any of these;   Z comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 162-178, or a complement of any of these; and   the nuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 100, 101, and 110.   
     
     
         11 . The synthetic guide component of  claim 10 , wherein the nuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 100, 101, 104, and 110-121. 
     
     
         12 . A substrate comprising:
 a synthetic guiding component comprising a structure having the formula (I):
   W-X-Y-L-Z or salt thereof;  (I)
 
   wherein:
 W is an optional third portion comprising a nucleic acid sequence of from about 1 to 20 nucleic acids; 
 X is a targeting portion comprising a nucleic acid sequence configured to bind to a target site of a single-stranded target sequence; 
 Y is a first portion comprising a nucleic acid sequence configured to interact with a nuclease configured to bind and/or cleave the single-stranded target sequence; 
 L is a linker; and 
 Z is a second portion comprising a nucleic acid sequence configured to interact with the nuclease and the first portion; 
 wherein the synthetic guiding component is configured to bind and cleave single-stranded RNA sequences without aid of a proto-spacer adjacent motif (PAM) sequence (PAMmer); and 
 X is configured to bind to the target site of a single-stranded mRNA target sequence or a single-stranded pathogen target sequence; and 
   a mismatched nucleic acid sequence comprising a plurality of base pair mismatches with the targeting portion of the synthetic guiding component.   
     
     
         13 . The substrate of  claim 12 , wherein X has a length of from about 15 to about 30 nucleotides. 
     
     
         14 . The substrate of  claim 12 , wherein single-stranded target sequence is the single-stranded mRNA target sequence. 
     
     
         15 . The substrate of  claim 12 , wherein:
 Y comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:150-155, 179-186, 213-225, 239-243, 251-253, 256, 257, 260, and 262-264; and   Z comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:162-178, 192-212, 232-238, 244-250, 254, 255, 258, 259, 261, and 270-273.   
     
     
         16 . A nucleoprotein complex comprising:
 a synthetic guiding component comprising a structure having the formula (I):
   W-X-Y-L-Z or salt thereof; and  (I)
 
   a nuclease configured to bind and/or cleave a single-stranded target sequence,
 wherein:
 W is an optional third portion comprising a nucleic acid sequence of from about 1 to 20 nucleic acids; 
 X is a targeting portion comprising a nucleic acid sequence configured to bind to a target site of a single-stranded target sequence; 
 Y is a first portion comprising a nucleic acid sequence configured to interact with the nuclease configured to bind and/or cleave the single-stranded target sequence; 
 L is a linker; and 
 Z is a second portion comprising a nucleic acid sequence configured to interact with the nuclease and the first portion; 
 
 wherein the synthetic guiding component is configured to bind to the target site of the single-stranded target sequence, without aid of a proto-spacer adjacent motif (PAM) sequence (PAMmer); and 
 the single-stranded target sequence is a single-stranded mRNA target sequence or a single-stranded pathogen target sequence. 
   
     
     
         17 . The nucleoprotein complex of  claim 16 , wherein the nuclease is a Cas protein or a modified form thereof. 
     
     
         18 . The nucleoprotein complex of  claim 17 , wherein the Cas protein is SauCas9 or CjeCas9. 
     
     
         19 . The nucleoprotein complex of  claim 17 , wherein the nuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 100, 101, 104, and 110-121. 
     
     
         20 . The nucleoprotein complex of  claim 17 , further comprising:
 one or more divalent ions associated with the complex.