Abstract
BackgroundGenome-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.ResultsHere, 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.ConclusionsThis work diversifies the applications for pooled CRISPR screens and provides a framework for future functional studies focused on noncoding regulatory elements.
Highlights
Genome-scale pooled Clustered regularly interspaced short palindromic repeats (CRISPR) screens are powerful tools for identifying genetic dependencies across varied cellular processes
We demonstrate that pooled CRISPR screens are capable of distinguishing p53-bound regulatory elements that influence cell proliferation and/or cell cycle arrest in response to DNA damage
In addition to identifying p53-bound regulatory elements that influence cell proliferation and/or cell cycle arrest in response to DNA damage we explore a variety of practical considerations for the use of pooled CRISPR screens to profile the function of regulatory elements
Summary
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. The majority of CRISPR screens reported to date have focused exclusively on the function of protein-coding genes. Many of the studies that have utilized pooled screens to characterize regulatory elements have designed dense tiling CRISPR libraries with genomic target sites that are restricted to sequences immediately adjacent to a gene of interest [15,16,17]. Isolated reports have described pooled CRISPR screens that target regulatory elements dispersed throughout the genome. A pooled CRISPR screen targeting 685 p53-bound regions was able to identify a functional enhancer element upstream of CDKN1A [18]. A pooled CRISPR screen targeting 398 AP1bound regions was able to identify an enhancer element
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