Crispr/cas dropout screening platform to reveal genetic vulnerabilities associated with tau aggregation

Abstract

Cas-protein-ready tau biosensor cells, CRISPR/Cas synergistic activation mediator (SAM)-ready tau biosensor cells, and methods of making and using such cells to screen for genetic vulnerability associated with tau aggregation are provided.

We claim: 
     
         1 . A method of screening for genetic vulnerabilities associated with tau aggregation, comprising:
 (a) providing an aggregation-positive population of cells and an aggregation-negative population of cells, wherein each population of cells comprises a chimeric Cas protein comprising a nuclease-inactive Cas protein fused to one or more transcriptional activation domains, a chimeric adaptor protein comprising an adaptor protein fused to one or more transcriptional activation domains, a first tau repeat domain linked to a first reporter, and a second tau repeat domain linked to a second reporter,   wherein the cells are mammalian cells,   wherein the first reporter and the second reporter are fluorescent proteins, and wherein the first reporter and the second reporter are a fluorescence resonance energy transfer (FRET) pair,   wherein in the aggregation-positive population of cells the first tau repeat domain linked to the first reporter and the second tau repeat domain linked to the second reporter stably present in an aggregated state, and   wherein in the aggregation-negative population of cells the first tau repeat domain linked to the first reporter and the second tau repeat domain linked to the second reporter do not stably present in an aggregated state;   (b) introducing into each population of cells a library comprising a plurality of unique guide RNAs that target a plurality of genes, wherein the plurality of unique guide RNAs form complexes with the chimeric Cas protein and the chimeric adaptor protein, and the complexes activate transcription of the plurality of genes resulting in increased gene expression; and   (c) determining abundance of each of the plurality of unique guide RNAs at a plurality of time points over a time course in each population of cells,   wherein the plurality of time points comprises at least three time points, wherein there is more than 1 day between each time point,   wherein depletion of a guide RNA in the aggregation-positive population of cells but not in the aggregation-negative population of cells or a more dramatic depletion pattern of a guide RNA over the time course in the aggregation-positive population of cells relative to the aggregation-negative population of cells indicates that activation of the gene targeted by the guide RNA exhibits synthetic lethality with tau protein aggregates and is a genetic vulnerability associated with tau aggregation.   
     
     
         2 . The method of  claim 1 , wherein the Cas protein is a Cas9 protein. 
     
     
         3 . The method of  claim 2 , wherein the Cas protein is  Streptococcus pyogenes  Cas9. 
     
     
         4 . The method of  claim 1 , wherein the chimeric Cas protein comprises the nuclease-inactive Cas protein fused to a VP64 transcriptional activation domain, wherein the chimeric Cas protein comprises from N-terminus to C-terminus: the nuclease-inactive Cas protein; a nuclear localization signal; and the VP64 transcriptional activator domain. 
     
     
         5 . The method of  claim 1 , wherein the adaptor protein is an MS2 coat protein, and wherein the one or more transcriptional activation domains in the chimeric adaptor protein comprise a p65 transcriptional activation domain and an HSF1 transcriptional activation domain, wherein the chimeric adaptor protein comprises from N-terminus to C-terminus: the MS2 coat protein; a nuclear localization signal; the p65 transcriptional activation domain; and the HSF1 transcriptional activation domain. 
     
     
         6 . The method of  claim 1 , wherein the chimeric Cas protein comprises SEQ ID NO: 36, or wherein the chimeric Cas protein is encoded by a coding sequence comprising the sequence set forth in SEQ ID NO: 38. 
     
     
         7 . The method of  claim 1 , wherein the chimeric adaptor protein comprises SEQ ID NO: 37, or wherein the chimeric adaptor protein is encoded by a coding sequence comprising the sequence set forth in SEQ ID NO: 39. 
     
     
         8 . The method of  claim 1 , wherein the chimeric Cas protein, the chimeric adaptor protein, the first tau repeat domain linked to the first reporter, and the second tau repeat domain linked to the second reporter are stably expressed in the population of cells and are genomically integrated in the population of cells. 
     
     
         9 . The method of  claim 1 , wherein each guide RNA targets a guide RNA target sequence within 200 bp upstream of a transcription start site. 
     
     
         10 . The method of  claim 1 , wherein each guide RNA comprises two adaptor-binding elements to which the chimeric adaptor protein can specifically bind, wherein a first adaptor-binding element is within a first loop of each of the guide RNAs, and a second adaptor-binding element is within a second loop of each of the guide RNAs, wherein the adaptor-binding element comprises the sequence set forth in SEQ ID NO: 33, and
 wherein each of guide RNAs is a single guide RNA comprising a CRISPR RNA (crRNA) portion fused to a transactivating CRISPR RNA (tracrRNA) portion, and the first loop is the tetraloop corresponding to residues 13-16 of SEQ ID NO: 17, and the second loop is the stem loop 2 corresponding to residues 53-56 of SEQ ID NO: 17.   
     
     
         11 . The method of  claim 1 , wherein the first tau repeat domain and/or the second tau repeat domain is a human tau repeat domain. 
     
     
         12 . The method of  claim 1 , wherein the first tau repeat domain and/or the second tau repeat domain comprises a pro-aggregation mutation, wherein the pro-aggregation mutation is a tau P301S mutation. 
     
     
         13 . The method of  claim 1 , wherein the first tau repeat domain and/or the second tau repeat domain comprises a tau four-repeat domain. 
     
     
         14 . The method of  claim 1 , wherein the first tau repeat domain and/or the second tau repeat domain comprises SEQ ID NO: 11. 
     
     
         15 . The method of  claim 1 , wherein the first tau repeat domain and the second tau repeat domain are the same and each comprises a tau four-repeat domain comprising a tau P301S mutation. 
     
     
         16 . The method of  claim 1 , wherein the first reporter is cyan fluorescent protein (CFP) and the second reporter is yellow fluorescent protein (YFP). 
     
     
         17 . The method of  claim 1 , wherein the cells are human cells. 
     
     
         18 . The method of  claim 17 , wherein the cells are HEK293T cells. 
     
     
         19 . The method of  claim 1 , wherein the plurality of unique guide RNAs are introduced at a concentration selected such that a majority of the cells receive only one of the unique guide RNAs. 
     
     
         20 . The method of  claim 1 , wherein the plurality of unique guide RNAs target 100 or more genes, 1000 or more genes, or 10000 or more genes. 
     
     
         21 . The method of  claim 1 , wherein the library is a genome-wide library. 
     
     
         22 . The method of  claim 1 , wherein a plurality of target sequences are targeted on average in each of the targeted plurality of genes. 
     
     
         23 . The method of  claim 22 , wherein at least three target sequences are targeted on average in each of the targeted plurality of genes or wherein about three to about six target sequences are targeted on average in each of the targeted plurality of genes. 
     
     
         24 . The method of  claim 1 , wherein the plurality of unique guide RNAs are introduced into the populations of cells by lentiviral transduction, wherein each of the plurality of unique guide RNAs is in a separate viral vector. 
     
     
         25 . The method of  claim 24 , wherein the populations of cells are infected at a multiplicity of infection of less than about 0.3. 
     
     
         26 . The method of  claim 1 , wherein the plurality of unique guide RNAs are introduced into the populations of cells together with a selection marker that imparts resistance to a drug, and step (b) further comprises selecting cells that comprise the selection marker. 
     
     
         27 . The method of  claim 1 , wherein the populations of cells into which the plurality of unique guide RNAs are introduced in step (b) each comprise greater than about 500 cells per unique guide RNA. 
     
     
         28 . The method of  claim 1 , wherein the time course in step (c) is more than about 1 week. 
     
     
         29 . The method of  claim 28 , wherein the time course in step (c) is more than about 2 weeks. 
     
     
         30 . The method of  claim 1 , wherein the time course in step (c) comprises about 10 to about 15 cell doublings. 
     
     
         31 . The method of  claim 1 , wherein the plurality of time points in step (c) comprises about four time points or about six time points. 
     
     
         32 . The method of  claim 1 , wherein there is more than about 2 days between each time point in step (c). 
     
     
         33 . The method of  claim 32 , wherein there is between about 3 to about 4 days between each time point in step (c). 
     
     
         34 . The method of  claim 1 , wherein a gene is considered to exhibit synthetic lethality with tau protein aggregates in step (c) if a guide RNA targeting the gene is depleted in the aggregation-positive population of cells but not in the aggregation-negative population of cells. 
     
     
         35 . The method of  claim 34 , wherein a gene is considered to exhibit synthetic lethality with tau protein aggregates in step (c) if a guide RNA targeting the gene has a more dramatic depletion pattern over the time course in the aggregation-positive population of cells relative to the aggregation-negative population of cells. 
     
     
         36 . The method of  claim 1 , wherein a guide RNA is considered depleted in step (c) if the abundance of the guide RNA at each time point is less than or equal to the abundance of the guide RNA at the preceding time point. 
     
     
         37 . The method of  claim 1 , wherein a guide RNA is considered depleted in step (c) if the abundance of the guide RNA at each time point after the second time point is less than or equal to the abundance of the time point two time points prior. 
     
     
         38 . The method of  claim 1 , wherein a gene is considered to exhibit synthetic lethality with tau protein aggregates in step (c) if more than about 30% of the guide RNAs in the library that target the gene are depleted in the aggregation-positive population of cells but not in the aggregation-negative population of cells. 
     
     
         39 . The method of  claim 38 , wherein a gene is considered to exhibit synthetic lethality with tau protein aggregates in step (c) in any one of the following situations:
 (1) there is one guide RNA in the library that targets the gene, and the one guide RNA is depleted in the aggregation-positive population of cells but not in the aggregation-negative population of cells;   (2) there are two guide RNAs in the library that target the gene, and at least one of the two guide RNAs is depleted in the aggregation-positive population of cells but not in the aggregation-negative population of cells;   (3) there are three guide RNAs in the library that target the gene, and at least one of the three guide RNAs is depleted in the aggregation-positive population of cells but not in the aggregation-negative population of cells;   (4) there are four guide RNAs in the library that target the gene, and at least two of the four guide RNAs is depleted in the aggregation-positive population of cells but not in the aggregation-negative population of cells;   (5) there are five guide RNAs in the library that target the gene, and at least two of the five guide RNAs is depleted in the aggregation-positive population of cells but not in the aggregation-negative population of cells; and   (6) there are six guide RNAs in the library that target the gene, and at least three of the six guide RNAs is depleted in the aggregation-positive population of cells but not in the aggregation-negative population of cells.   
     
     
         40 . The method of  claim 1 , wherein the method is repeated at least three times in at least three different experiments, and a gene is considered to exhibit synthetic lethality with tau protein aggregates if it is considered to exhibit synthetic lethality with tau protein aggregates in more than about 50% of the at least three different experiments. 
     
     
         41 . The method of  claim 1 , wherein the time course in step (c) is more than about 2 weeks,
 wherein the plurality of time points in step (c) comprises about six time points,   wherein there is between about 3 to about 4 days between each time point in step (c),   wherein a guide RNA is considered depleted in step (c) if the abundance of the guide RNA at each time point after the second time point is less than or equal to the abundance of the time point two time points prior, and   wherein a gene is considered to exhibit synthetic lethality with tau protein aggregates in step (c) if more than about 30% of the guide RNAs in the library that target the gene are depleted in the aggregation-positive population of cells but not in the aggregation-negative population of cells.