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

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.