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Assaying for RNA abundance and protein phosphorylation in pooled CRISPR screens

ABSTRACT: Assaying for RNA abundance and protein phosphorylation in pooled CRISPR screens

PROVIDER: PRJNA605978 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11066863.fastq.gz	Fastqsanger.gz
	SRR11066864.fastq.gz	Fastqsanger.gz
	SRR11066865.fastq.gz	Fastqsanger.gz
	SRR11066866.fastq.gz	Fastqsanger.gz
	SRR11066867.fastq.gz	Fastqsanger.gz

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Assaying for RNA abundance and protein phosphorylation in pooled CRISPR screens

Project description:Pooled CRISPR screens are a powerful tool to identify regulators of a biological process, but have been limited to phenotypes that affect viability or can be monitored with fluorescent protein reporters. We evaluate two additional strategies for phenotypic enrichment based on quantification of RNA using a Flow-FISH assay or protein phosphorylation through intracellular phospho-staining. These tools will greatly expand the applicability of CRISPR screens since they increase the number of possible molecular phenotypes and assay designs.

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Project description:Genome-scale pooled CRISPR screens are powerful tools for identifying genetic dependencies across varied cellular processes. The vast majority of CRISPR screens reported to date have focused exclusively on the perturbation of protein-coding gene function. However, protein-coding genes comprise <2% of the sequence space in the human genome leaving a substantial portion of the genome uninterrogated. Noncoding regions of the genome harbor important regulatory elements (e.g. promoters, enhancers, suppressors) that influence cellular processes but high-throughput methods for evaluating their essentiality have yet to be established. Here, we describe a CRISPR-based screening approach that facilitates the functional profiling of thousands of noncoding regulatory elements in parallel. We selected the tumor suppressor p53 as a model system and designed a pooled CRISPR library targeting thousands of p53 binding sites throughout the genome. Following transduction into dCas9-KRAB-expressing cells we identified several regulatory elements that influence cell proliferation. Moreover, we uncovered multiple elements that are required for the p53-mediated DNA damage response. Surprisingly, many of these elements are located deep within intergenic regions of the genome that have no prior functional annotations. This work diversifies the applications for pooled CRISPR screens and provides a framework for future functional studies focused on noncoding regulatory elements.

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