High throughput methods for functionally determining rna interference efficiency

Abstract

Provided is a single construct combining a sequence encoding an RNAi molecule, a sequence encoding a reporter, and a target sequence specific for the RNAi molecule. The construct can be used to determine the potency of the encoded RNAi molecule in a direct and unbiased way. These results can be used to inform the design of potent RNAi molecules of various types and can be extended to several other applications, including: (1) generation of tiled libraries comprising every possible RNAi molecule-encoding sequence for a given gene target; (2) large-scale parallel validation of RNAi molecules targeting many genes to generate validated RNAi molecule-encoding libraries; (3) experimental comparison of design algorithms and strategies; and (4) investigation of RNAi biology in target site mutagenesis assays by screening pools containing single nucleotide changes in target sites and/or in the RNAi molecule to identify the most relevant sequence characteristics of potent RNAi-target site predictions.

We claim: 
     
         1 . A single construct comprising:
 (i) a promoter;   (ii) a sequence encoding an RNAi molecule, operably linked to the promoter, wherein the RNAi molecule comprises a guide strand;   (iii) a target sensor, operably linked to the promoter, the target sensor comprising: a sequence encoding a reporter and a target sequence that comprises from about 8 to about 29 contiguous nucleotides complementary to at least a portion of the guide strand of the RNAi molecule.   
     
     
         2 . The construct of  claim 1 , wherein the promoter is an inducible promoter. 
     
     
         3 . The construct of  claim 1 , wherein the promoter is a constitutive promoter. 
     
     
         4 . The construct of  claim 1 , wherein the promoter is ubiquitous. 
     
     
         5 . The construct of  claim 1 , wherein the promoter is cell-type specific or tissue specific. 
     
     
         6 . The construct of  claim 1 , wherein the promoter is a TRE promoter. 
     
     
         7 . The construct of  claim 1 , wherein the RNAi molecule is an shRNA molecule. 
     
     
         8 . The construct of  claim 1 , wherein the reporter is a fluorescent protein. 
     
     
         9 . The construct of  claim 1 , further comprising a sequence encoding an additional reporter, operably linked to the promoter and 5′ of the sequence encoding the RNAi molecule. 
     
     
         10 . The construct of  claim 9 , wherein the sequence encoding the additional reporter is a selection gene. 
     
     
         11 . The construct of  claim 1 , wherein the target sequence is located in an untranslated region of the sequence encoding the reporter. 
     
     
         12 . The construct of  claim 1 , wherein the target sequence comprises from about 16 to about 29 contiguous nucleotides complementary to at least a portion of the guide strand of the RNAi molecule. 
     
     
         13 . The construct of  claim 1 , wherein the target sequence comprises from about 19 to about 22 contiguous nucleotides complementary to at least a portion of the guide strand of the RNAi molecule. 
     
     
         14 . The construct of  claim 1 , wherein the target sequence is completely complementary to at least a portion of the guide strand of the RNAi molecule. 
     
     
         15 . A single construct comprising:
 (i) a first promoter;   (ii) a sequence encoding an RNAi molecule, operably linked to the first promoter, wherein the RNAi molecule comprises a guide strand;   (iii) a second promoter;   (iv) a target sensor, operably linked to the second promoter, the target sensor comprising: a sequence encoding a reporter and a target sequence that comprises from about 8 to about 29 contiguous nucleotides complementary to at least a portion of the guide strand of the RNAi molecule.   
     
     
         16 . The construct of  claim 15 , wherein the first promoter is an inducible promoter and the second promoter is a constitutive promoter. 
     
     
         17 . The construct of  claim 16 , wherein the first promoter is a TRE promoter. 
     
     
         18 . The construct of  claim 15 , wherein the first promoter or the second promoter or both promoters are ubiquitous. 
     
     
         19 . The construct of  claim 15 , wherein the first promoter or the second promoter or both promoters are cell-type specific or tissue specific. 
     
     
         20 . The construct of  claim 15 , wherein the RNAi molecule is an shRNA molecule. 
     
     
         21 . The construct of  claim 15 , wherein the reporter is a fluorescent protein. 
     
     
         22 . The construct of  claim 15 , further comprising a sequence encoding an additional reporter, operably linked to the first promoter. 
     
     
         23 . The construct of  claim 22 , wherein the sequence encoding the additional reporter is a selection gene. 
     
     
         24 . The construction of  claim 15 , wherein the target sequence is located in an untranslated region of the sequence encoding the reporter. 
     
     
         25 . The construct of  claim 15 , wherein the target sequence comprises from about 16 to about 29 contiguous nucleotides complementary to at least a portion of the guide strand of the RNAi molecule. 
     
     
         26 . The construct of  claim 15 , wherein the target sequence comprises from about 19 to about 22 contiguous nucleotides complementary to at least a portion of the guide strand of the RNAi molecule. 
     
     
         27 . The construct of  claim 15 , wherein the target sequence is completely complementary to at least a portion of the guide strand of the RNAi molecule. 
     
     
         28 . A single construct comprising:
 (i) a viral 5′LTR;   (ii) a viral packaging signal;   (iii) a promoter;   (iv) a sequence encoding an RNAi molecule, operably linked to the promoter, wherein the RNAi molecule comprises a guide strand;   (v) a target sensor, operably linked to the promoter, the target sensor comprising: a sequence encoding a reporter and a target sequence that comprises from about 8 to about 29 contiguous nucleotides complementary to at least a portion of the guide strand of the RNAi molecule; and   (vi) a viral 3′LTR.   
     
     
         29 . The construct of  claim 28 , wherein the viral 5′LTR, the viral packaging signal, and the viral 3′LTR are from a retrovirus, a baculovirus, or an avian virus. 
     
     
         30 . The construct of  claim 29 , wherein the retrovirus is a lentivirus. 
     
     
         31 . The construct of  claim 28 , wherein the promoter is an inducible promoter. 
     
     
         32 . The construct of  claim 28 , wherein the promoter is a constitutive promoter. 
     
     
         33 . The construct of  claim 28 , wherein the promoter is ubiquitous. 
     
     
         34 . The construct of  claim 28 , wherein the promoter is cell-type specific or tissue specific. 
     
     
         35 . The construct of  claim 28 , wherein the promoter is a TRE promoter. 
     
     
         36 . The construct of  claim 31 , wherein the 3′LTR is self-inactivating. 
     
     
         37 . The construct of  claim 28 , wherein the RNAi molecule is an shRNA molecule. 
     
     
         38 . The construct of  claim 28 , wherein the reporter is a fluorescent protein. 
     
     
         39 . The construct of  claim 28 , further comprising a sequence encoding an additional reporter, operably linked to the promoter and 5′ of the sequence encoding the RNAi molecule. 
     
     
         40 . The construct of  claim 39 , wherein the sequence encoding the additional reporter is a selection gene. 
     
     
         41 . The construct of  claim 28 , wherein the target sequence is located in an untranslated region of the sequence encoding the reporter. 
     
     
         42 . The construct of  claim 28 , wherein the target sequence comprises from about 16 to about 29 contiguous nucleotides complementary to at least a portion of the guide strand of the RNAi molecule. 
     
     
         43 . The construct of  claim 28 , wherein the target sequence comprises from about 19 to about 22 contiguous nucleotides complementary to at least a portion of the guide strand of the RNAi molecule. 
     
     
         44 . The construct of  claim 28 , wherein the target sequence is completely complementary to at least a portion of the guide strand of the RNAi molecule. 
     
     
         45 . A single construct comprising:
 (i) a viral 5′LTR;   (ii) a viral packaging signal;   (iii) a first promoter;   (iv) a sequence encoding an RNAi molecule, operably linked to the first promoter, wherein the RNAi molecule comprises a guide strand;   (v) a second promoter;   (vi) a target sensor, operably linked to the promoter, the target sensor comprising: a sequence encoding a reporter and a target sequence that comprises from about 8 to about 29 contiguous nucleotides complementary to at least a portion of the guide strand of the RNAi molecule; and   (vii) a viral 3′LTR.   
     
     
         46 . The construct of  claim 45 , wherein the viral 5′LTR, the viral packaging signal, and the viral 3′LTR are from a retrovirus, a baculovirus, or an avian virus. 
     
     
         47 . The construct of  claim 46 , wherein the retrovirus is a lentivirus. 
     
     
         48 . The construct of  claim 45 , wherein the first promoter is an inducible promoter and the second promoter is a constitutive promoter. 
     
     
         49 . The construct of  claim 48 , wherein the first promoter is a TRE promoter. 
     
     
         50 . The construct of  claim 48 , wherein the 3′LTR is self-inactivating. 
     
     
         51 . The construct of  claim 45 , wherein the first promoter or the second promoter or both promoters are ubiquitous. 
     
     
         52 . The construct of  claim 45 , wherein the first promoter or the second promoter or both promoters are cell-type specific or tissue specific. 
     
     
         53 . The construct of  claim 45 , wherein the RNAi molecule is an shRNA molecule. 
     
     
         54 . The construct of  claim 45 , wherein the reporter is a fluorescent protein. 
     
     
         55 . The construct of  claim 45 , further comprising a sequence encoding an additional reporter, operably linked to the first promoter. 
     
     
         56 . The construct of  claim 55 , wherein the sequence encoding the additional reporter is a selection gene. 
     
     
         57 . The construct of  claim 45 , wherein the target sequence is located in an untranslated region of the sequence encoding the reporter. 
     
     
         58 . The construct of  claim 45 , wherein the target sequence comprises from about 16 to about 29 contiguous nucleotides complementary to at least a portion of the guide strand of the RNAi molecule. 
     
     
         59 . The construct of  claim 45 , wherein the target sequence comprises from about 19 to about 22 contiguous nucleotides complementary to at least a portion of the guide strand of the RNAi molecule. 
     
     
         60 . The construct of  claim 45 , wherein the target sequence is completely complementary to at least a portion of the guide strand of the RNAi molecule. 
     
     
         61 . An RNAi library comprising a plurality of the construct of  claim 1 , wherein the sequence encoding the RNAi molecule is different in each construct. 
     
     
         62 . An RNAi library comprising a plurality of the construct of  claim 15 , wherein the sequence encoding the RNAi molecule is different in each construct. 
     
     
         63 . An RNAi library comprising a plurality of the construct of  claim 28 , wherein the sequence encoding the RNAi molecule is different in each construct. 
     
     
         64 . An RNAi library comprising a plurality of the construct of  claim 45 , wherein the sequence encoding the RNAi molecule is different in each construct. 
     
     
         65 . The RNAi library of any one of  claims 61  to  64 , wherein the RNAi library is a tiled library. 
     
     
         66 . A method for determining potency of an RNAi molecule, the method comprising:
 (a) introducing the construct of  claim 1  or  claim 15  into a cell; and   (b) determining the amount of reporter expression in the cell, wherein high reporter expression indicates a less potent RNAi molecule and low or no reporter expression indicates a more potent RNAi molecule.   
     
     
         67 . A method for determining potency of RNAi molecules, the method comprising:
 (a) introducing the RNAi library of  claim 61  or  claim 62  into cells; and   (b) determining the amount of reporter expression in the cells, wherein high reporter expression indicates less potent RNAi molecules and low or no reporter expression indicates more potent RNAi molecules.   
     
     
         68 . A method for determining potency of an RNAi molecule, the method comprising:
 (a) introducing the construct of  claim 28  or  claim 45  into a first cell, wherein the construct is packaged into a virion in the first cell;   (b) infecting a second cell with the virion;   (c) determining the amount of reporter expression in the cell, wherein high reporter expression indicates a less potent RNAi molecule and low or no reporter expression indicates a more potent RNAi molecule.   
     
     
         69 . A method for determining potency of RNAi molecules, the method comprising:
 (a) introducing the RNAi library of  claim 63  or  claim 64  into a first population of cells, wherein the constructs of the RNAi library are packaged into virions in the first population of cells;   (b) infecting a second population of cells with the virions;   (c) determining the amount of reporter expression in the cells, wherein high reporter expression indicates less potent RNAi molecules and low or no reporter expression indicates more potent RNAi molecules.   
     
     
         70 . A method for identifying potent RNAi molecules, the method comprising:
 (a) introducing the RNAi library of  claim 61  or  claim 62  into cells;   (b) sorting cells based on reporter expression; and   (c) determining the sequence of the RNAi molecules in cells exhibiting low or no reporter expression;   thereby identifying potent RNAi molecules.   
     
     
         71 . A method for identifying potent RNAi molecules, the method comprising:
 (a) introducing the RNAi library of  claim 63  or  claim 64  into a first population of cells, wherein the constructs of the RNAi library are packaged into virions in the first population of cells;   (b) infecting a second population of cells with the virions;   (b) sorting cells based on reporter expression; and   (c) determining the sequence of the RNAi molecules in cells exhibiting low or no reporter expression;   
       thereby identifying potent RNAi molecules. 
     
     
         72 . The method of  claim 70  or  claim 71 , wherein the cells are sorted by flow cytometry. 
     
     
         73 . The method of  claim 72 , wherein the flow cytometry is fluorescence activated cells sorting. 
     
     
         74 . The method of  claim 70  or  claim 71 , wherein cells are sorted based on reversibility of reporter expression, wherein greater reversible reduction of reporter expression indicates more potent RNAi molecules. 
     
     
         75 . A method for designing RNAi molecules, the method comprising:
 (a) introducing the RNAi library of  claim 65  into cells;   (b) sorting cells based on reporter expression; and   (c) determining the sequence of the RNAi molecules exhibiting greatest reversible reduction of reporter expression;   (d) designing an RNAi molecule with high potency based on step (c).   
     
     
         76 . The method of  claim 75 , wherein the cells are sorted by flow cytometry. 
     
     
         77 . The method of  claim 76 , wherein the flow cytometry is fluorescence activated cells sorting. 
     
     
         78 . A modified cell line comprising DF-1 chicken embryo fibroblasts (CEFs), wherein the DF-1 CEFs are genetically modified to express rtTA3 reverse tet-transactivator and EcoR ecotropic receptor, wherein the modified cell line enables single-copy genomic integration of tet-regulatable transgenes.