Identification of functional regulatory elements in the human genome using pooled CRISPR screens
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...
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doaj-0e84aa26e45147cfa4c561f52f324ad62021-01-31T16:12:03ZengBMCBMC Genomics1471-21642020-01-0121111510.1186/s12864-020-6497-0Identification of functional regulatory elements in the human genome using pooled CRISPR screensSamantha M. Borys0Scott T. Younger1Broad Institute of MIT and HarvardBroad Institute of MIT and HarvardAbstract 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.https://doi.org/10.1186/s12864-020-6497-0CRISPRCRISPR screenRegulatory elementEnhancerp53 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Samantha M. Borys Scott T. Younger |
spellingShingle |
Samantha M. Borys Scott T. Younger Identification of functional regulatory elements in the human genome using pooled CRISPR screens BMC Genomics CRISPR CRISPR screen Regulatory element Enhancer p53 |
author_facet |
Samantha M. Borys Scott T. Younger |
author_sort |
Samantha M. Borys |
title |
Identification of functional regulatory elements in the human genome using pooled CRISPR screens |
title_short |
Identification of functional regulatory elements in the human genome using pooled CRISPR screens |
title_full |
Identification of functional regulatory elements in the human genome using pooled CRISPR screens |
title_fullStr |
Identification of functional regulatory elements in the human genome using pooled CRISPR screens |
title_full_unstemmed |
Identification of functional regulatory elements in the human genome using pooled CRISPR screens |
title_sort |
identification of functional regulatory elements in the human genome using pooled crispr screens |
publisher |
BMC |
series |
BMC Genomics |
issn |
1471-2164 |
publishDate |
2020-01-01 |
description |
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. |
topic |
CRISPR CRISPR screen Regulatory element Enhancer p53 |
url |
https://doi.org/10.1186/s12864-020-6497-0 |
work_keys_str_mv |
AT samanthamborys identificationoffunctionalregulatoryelementsinthehumangenomeusingpooledcrisprscreens AT scotttyounger identificationoffunctionalregulatoryelementsinthehumangenomeusingpooledcrisprscreens |
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