Document Type

Article

Publication Date

1-31-2020

Identifier

DOI: 10.1186/s12864-020-6497-0; PMCID: PMC6995077

Abstract

BACKGROUND: 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, silencers) that influence cellular processes but high-throughput methods for evaluating their essentiality have yet to be established.

RESULTS: 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.

CONCLUSIONS: This work diversifies the applications for pooled CRISPR screens and provides a framework for future functional studies focused on noncoding regulatory elements.

Journal Title

BMC genomics [electronic resource]

Volume

21

Issue

1

First Page

107

Last Page

107

MeSH Keywords

Binding Sites; CRISPR-Cas Systems; Cell Line, Tumor; Cell Proliferation; Gene Knockout Techniques; Genome, Human; Humans; Neoplasms; Regulatory Elements, Transcriptional; Tumor Suppressor Protein p53

Keywords

CRISPR; CRISPR screen; Enhancer; Regulatory element; p53

Comments

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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