Assays to identify genetic elements affecting phenotype

Abstract

The present invention comprises generally applicable methods for identifying endogenous physiologically relevant genetic elements that affects a intracellular phenotype of interest. In the methods, non-living cells that have been subjected to a mutagenesis treatment are sorted based on phenotype and analyzed to identify the genetic element. By use of these methods, elements previously unknown to be involved in a phenotype can be identified, for example in relationship to health conditions, external stress or drug response, in particular in cancer.

What is claimed is: 
     
         1 . A method for identifying a genetic element that affects a phenotype of a cell, preferably wherein said phenotype is manifested intracellularly, the method comprising the steps of:
 (a) Subjecting a pool of cells to mutagenesis treatment;   (b) Fixating the pool of cells, preferably with a fixation reagent, and optionally with a cross-linking agent; and permeabilizing the pool of cells, preferably with a permeabilization reagent;   (c) Treating the pool of cells with one or more detectable probe(s), preferably an antibody or a RNA probe, to detect the affected phenotype;   (d) Sorting the cells based on the detection of at least one of the one or more detectable probe(s) to obtain one or more population of cells;   (e) Optionally, de-crosslinking the cells in each of the obtained populations of cells; and   (f) Sequencing at least part of the cells of at least part of the obtained populations of cells to identify a genetic element that affects the phenotype of the cell.   
     
     
         2 . The method of any of the previous claims wherein in step (d) at least two population of cells are obtained with the sorting of the cells based on the detection of at least one of the one or more datable probes(s) and wherein in step (f) at least two population of cells are sequenced and compared to identify a genetic element that affects the phenotype of the cell. 
     
     
         3 . The method of any of the previous claims, wherein the genetic element is selected from the group consisting of a gene, an intron, an exon, a promoter and a noncoding RNA. 
     
     
         4 . The method of any of the previous claims wherein the cell is selected from the group consisting of a eukaryotic cell, an animal cell, a plant cell, a yeast cell, a mammalian cell, a human cell, or a stem cell. 
     
     
         5 . The method of any of the previous claims wherein the cell is a near-haploid cell or fully haploid cell, preferably a near-haploid cell or fully haploid mammalian cell, more preferably a near-haploid cell or fully haploid cell human cell. 
     
     
         6 . The method of any of the previous claims wherein the mutagenesis is random mutagenesis. 
     
     
         7 . The method of any of the previous claims wherein the mutagenesis involves the use of radiation, mutagenic chemicals, preferably ethyl methanesulfonate, nitrous acid, or ethyl nitrosourea, insertion mutagenesis, preferably transposon-based insertional mutagenesis or retrovirus-based (random) insertional mutagenesis, a CRISPR library of guide RNA sequences. 
     
     
         8 . The method of any of the previous claims wherein the cells are exposed to a stress condition or growth condition and/or wherein the cells are treated with a compound, preferably a drug before the cells are fixed and permeabilized in step (b), preferably between performing step (a) and step (b). 
     
     
         9 . The method of any of the previous claims wherein the fixation reagent to fix the cells is selected from the group consisting of crosslinking reagents, preferably formaldehyde, paraformaldehyde, formalin and glutaraldehyde or non-crosslinking reagents, preferably mercuric chloride-based fixatives, ethanol, methanol or acetone and/or wherein the permeabilization reagent is selected from the group consisting of solvents, preferably methanol and acetone, or detergents, preferably saponin, digitonin, Triton X-100, NP-40, Leucoperm and Tween-20. 
     
     
         10 . The method of any of the previous claims wherein the detectable probe binds to a protein, a post-translation modified protein, a lipid, DNA, RNA, or binds or detects a metabolite or cellular element. 
     
     
         11 . The method of any of the previous claims wherein sorting involves flow cytometry, FACS analysis, mass cytometry, and/or magnetic sorting. 
     
     
         12 . The method of any of the previous claims wherein the probe comprises a detectable moiety, preferably selected from a fluorescent moiety, a radioactive moiety, magnetic moiety, or label that can be measured using mass-spectrometry. 
     
     
         13 . The method of any of the previous claims wherein de phenotype is manifested in the cytosol, inside an organelle, in the membrane of an organelle or in the cell membrane. 
     
     
         14 . The method of any of the previous claims wherein the phenotype is increased protein abundance, decreased protein abundance, increased protein activity, decreased protein activity, increased post-translational modification of a protein, decreased posttranslational expression of a protein, increased mRNA abundance or decreased mRNA abundance. 
     
     
         15 . The method of any of the previous claims wherein sequencing involves high-throughput sequencing. 
     
     
         16 . A method for identifying a modulator of a gene product encoded by a candidate gene that affects a phenotype of a cell, preferably wherein said phenotype is manifested intracellularly, the method comprising the steps of:
 (a) Subjecting a pool of cells to mutagenesis treatment;   (b) Fixating of the pool of cells, preferably with a fixation reagent, and optionally with a cross-linking agent, and permeabilizing the pool of cells, preferably with a permeabilization reagent;   (c) Treating the pool of cells with one or more detectable probe(s), preferably an antibody or a RNA probe, to detect the affected phenotype;   (d) Sorting the cells based on the detection of at least one of the one or more detectable probe(s) to obtain one or more population of cells,   (e) Optionally, de-crosslinking the cells in each of the obtained populations of cells;   (f) Sequencing at least part of the cells of at least part of the obtained populations of cells to identify a genetic element that affects the phenotype of the cell, wherein the genetic element is a candidate gene.   (g) Identifying a modulator that affects expression or activity of an expression product of said identified candidate gene that affects the phenotype of the cell.   
     
     
         17 . The method of any of the previous  claim 17  wherein in step (d) at least two population of cells are obtained with the sorting of the cells based on the detection of at least one of the one or more datable probes(s) and wherein in step (f) at least two population of cells are sequenced and compared to identify a genetic elements that affects the phenotype of the cell. 
     
     
         18 . The method of any of the previous claims wherein the method is used for establishing or analyzing biological pathways, for identifying genes involved in disease, preferably in cancer, for studying drug-target interaction, for studying drug-drug interaction, or to analyze suppression or modulation of a phenotype, preferably wherein the phenotype is a phenotype associated with a disease. 
     
     
         19 . A method according to any of the previous claims further comprising identifying a cellular element that is related to the genetic element identified, preferably wherein the cellular element is a protein.