Identification of functional regulatory elements in the human genome using pooled CRISPR screens
Open Access
- 31 January 2020
- journal article
- research article
- Published by Springer Science and Business Media LLC in BMC Genomics
- Vol. 21 (1), 1-15
- https://doi.org/10.1186/s12864-020-6497-0
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.Keywords
This publication has 36 references indexed in Scilit:
- Functional CRISPR screen identifies AP1-associated enhancer regulating FOXF1 to modulate oncogene-induced senescenceGenome Biology, 2018
- Computational correction of copy number effect improves specificity of CRISPR–Cas9 essentiality screens in cancer cellsNature Genetics, 2017
- p53 regulates enhancer accessibility and activity in response to DNA damageNucleic Acids Research, 2017
- TP53Variations in Human Cancers: New Lessons from the IARC TP53 Database and Genomics DataHuman Mutation, 2016
- Genome-Scale CRISPR-Cas9 Knockout Screening in Human CellsScience, 2014
- CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in EukaryotesCell, 2013
- Multiplex Genome Engineering Using CRISPR/Cas SystemsScience, 2013
- Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteriaProceedings of the National Academy of Sciences of the United States of America, 2012
- The p53 Tumor Suppressor Network Is a Key Responder to Microenvironmental Components of Chronic Inflammatory StressCancer Research, 2005
- Differential cell cycle checkpoint response in normal human keratinocytes and fibroblasts.1998