Protein translational control

Abstract

Provided herein are compositions and methods for regulating protein translation. The compositions include a Cas polypeptide and a capped-sgRNA that includes (i) an m7G cap or an analog thereof; (ii) a spacer capable of specifically hybridizing with a target sequence in an RNA molecule; and (iii) a direct repeat capable of binding to the Cas polypeptide. The disclosure further provides methods of regulating translation of an mRNA in a cell, the method comprising contacting the cell with a nucleic acid comprising (a) a sequence encoding a Cas polypeptide; and (b) a sequence encoding a capped-sgRNA comprising (i) an m7G cap or analog thereof; (ii) a spacer capable of specifically hybridizing with a target sequence in an RNA molecule; and (iii) a direct repeat capable of binding to the Cas polypeptide.

1 . A complex comprising:
 a Cas polypeptide; and   a capped-sgRNA comprising
 (i) an m7G cap or an analog thereof; 
 (ii) a spacer capable of specifically hybridizing with a target sequence in an RNA molecule; and 
 (iii) a direct repeat capable of binding to the Cas polypeptide. 
   
     
     
         2 . The complex of  claim 1 , wherein the RNA molecule is a messenger RNA (mRNA). 
     
     
         3 . The complex of  claim 2 , wherein the mRNA has an endogenous m7G cap. 
     
     
         4 . The complex of  claim 3 , wherein the target sequence is downstream of the endogenous m7G cap of the mRNA. 
     
     
         5 .- 12 . (canceled) 
     
     
         13 . The complex of  claim 1 , wherein the spacer is at least 80% complementary to the target sequence. 
     
     
         14 .- 17 . (canceled) 
     
     
         18 . The complex of  claim 1 , wherein the spacer is connected to the m7G cap or analog thereof via a linker. 
     
     
         19 .- 22 . (canceled) 
     
     
         23 . The complex of  claim 1 , wherein the Cas polypeptide is a nuclease-deficient Cas (dCas) polypeptide, wherein the dCas comprises an inactivated target cleavage domain and a retained guide cleavage domain. 
     
     
         24 . The complex of  claim 23 , wherein the nuclease-deficient Cas polypeptide is a nuclease-deficient Cas13 (dCas13) polypeptide, wherein the dCas13 is dCas13b or dCas13d. 
     
     
         25 . The complex of  claim 24 , wherein the direct repeat is capable of binding to a nuclease-deficient Cas13 (dCas13) polypeptide, wherein the dCas13 is dCas13b or dCas13d. 
     
     
         26 . The complex of  claim 23 , wherein the nuclease-deficient Cas polypeptide is a nuclease-deficient Cas9 (dCas9) polypeptide. 
     
     
         27 . (canceled) 
     
     
         28 . A nucleic acid comprising a sequence encoding the capped-sgRNA in the complex of  claim 1 . 
     
     
         29 . (canceled) 
     
     
         30 . A nucleic acid comprising a sequence encoding a capped-sgRNA, wherein the capped-sgRNA comprises:
 (i) an m7G cap or analog thereof;   (ii) a spacer capable of specifically hybridizing with a target sequence in an RNA molecule; and   (iii) a direct repeat capable of binding to a Cas polypeptide.   
     
     
         31 . The nucleic acid of  claim 30 , wherein the RNA molecule is an mRNA. 
     
     
         32 .- 54 . (canceled) 
     
     
         55 . The nucleic acid of  claim 30 , further comprising a sequence encoding the Cas polypeptide. 
     
     
         56 . The nucleic acid of  claim 55 , wherein the Cas polypeptide is a nuclease-deficient Cas polypeptide. 
     
     
         57 .- 60 . (canceled) 
     
     
         61 . The nucleic acid of  claim 55 , wherein the sequence encoding the capped-sgRNA and the sequence encoding the Cas polypeptide are expressed from the same promoter. 
     
     
         62 . The nucleic acid of  claim 55 , wherein the sequence encoding the capped-sgRNA and the sequence encoding the Cas polypeptide are expressed from different promoters. 
     
     
         63 . A vector comprising the nucleic acid of  claim 30 . 
     
     
         64 . (canceled) 
     
     
         65 . A cell comprising the nucleic acid of  claim 30 . 
     
     
         66 . A method of regulating translation of an mRNA in a cell, the method comprising contacting the cell with a nucleic acid comprising
 (a) a sequence encoding a Cas polypeptide; and   (b) a sequence encoding a capped-sgRNA comprising
 (i) an m7G cap or analog thereof; 
 (ii) a spacer capable of specifically hybridizing with a target sequence in an RNA molecule; and 
 (iii) a direct repeat capable of binding to the Cas polypeptide. 
   
     
     
         67 .- 70 . (canceled)