High-throughput screening of regulatory element function with epigenome editing technologies
Disclosed herein are methods of using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) 9-based epigenomic editing systems for high-throughput screening of regulatory element function.
1. A method of high-throughput screening for one or more putative gene regulatory elements in a genome that modulate a phenotype, the method comprising:
a) contacting a plurality of modified target cells with a library of single guide RNAs (sgRNAs) that target a plurality of gene regulatory elements within the genome, thereby generating a plurality of test cells,
b) selecting a population of test cells or an organism having a modulated phenotype;
c) quantitating the frequency of the sgRNAs within the population of selected cells or the organism, wherein the sgRNAs that target gene regulatory elements that modulate the phenotype are overrepresented or underrepresented in the selected cells; and
d) identifying and characterizing the sgRNAs within the population of selected test cells or the organism thereby identifying the gene regulatory elements that modulate the phenotype,
wherein the modified target cell or organism comprises a fusion protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 and a second polypeptide domain having an activity selected from the group consisting of transcription activation activity, transcription repression activity, nuclease activity, transcription release factor activity, histone modification activity, nucleic acid association activity, DNA methylase activity, and direct or indirect DNA demethylase activity.
2. The method of claim 1 , wherein the modulated phenotype is a change in gene expression of at least one target gene or a change in cell or organismal phenotype and the gene regulatory element modulates the gene expression or the cell or organismal phenotype.
3. The method of claim 2 , wherein the cell phenotype is T-cell phenotype, cell differentiation, oncogenesis, immunomodulation, cell response to stimuli, cell death, cell growth, cell motility, cell metabolism, cell immunogenicity, drug resistance, or drug sensitivity.
4. The method of claim 1 , wherein the library of sgRNAs targets a plurality of target sites in the genome, one or more gene regulatory elements in a genomic region of a target gene, or one or more gene regulatory elements in the entire genome.
5. The method of claim 1 , wherein between 5 and 50 gRNAs are generated per gene regulatory element.
6. The method of claim 1 , wherein the modified target cell comprises an intracellular or cell-surface marker or the modified target cell comprises at least one target gene comprising a reporter gene inserted within the coding region of the target gene.
7. The method of claim 1 , further comprising enriching cells expressing differential levels of the reporter gene by fluorescence-activated cell sorting or magnetic-activated cell sorting.
8. The method of claim 1 , further comprising comparing the gene expression of at least one target gene in the test cells with the gene expression of the target gene in a plurality of control cells.
9. The method of claim 1 , wherein the modulated phenotype is a change in gene expression of at least one target gene.
10. The method of claim 1 , wherein the change in gene expression is determined by a change in protein expression, RNA expression, or protein activity.
11. The method of claim 1 , wherein identifying and characterizing the sgRNAs within the population of selected test cells comprises high-throughput sequencing, qRT-PCR, or RNA-seq.
12. The method of claim 4 , wherein the genomic region is between about 0 bp to about 150 Mb upstream and/or downstream of the transcription start site of at least one target gene.
13. The method of claim 1 , wherein the fusion protein comprises a dCas9 domain and a transcriptional activator, a dCas9 domain and a transcriptional repressor, or a dCas9 domain and a site-specific nuclease.
14. The method of claim 13 , wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:3.
15. The method of claim 1 , wherein the sgRNAs are selected to inhibit transcription of the target gene, activate transcription of the target gene, or knockout at least one target gene.
16. The method of claim 1 , wherein identifying and characterizing the sgRNAs within the population of selected test cells comprises deep sequencing of the genomic DNA of the test cell, and/or wherein selecting the test cells for a change in gene expression of the target gene further comprises culturing the cells and selecting the cells on the basis of cellular proliferation or survival.
17. The method of claim 1 , wherein the sgRNA is encoded by a polynucleotide sequence and packaged into a lentiviral vector, thereby generating a gRNA library lentiviral pool, and optionally, wherein the target cells are transduced with the gRNA library lentiviral pool at a low multiplicity of infection such that >90% of the target cells express a single gRNA or the target cells are transduced with the gRNA library lentiviral pool at a high multiplicity of infection such that >90% of the target cells expression more than one gRNAs.