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. 2017 Jun;35(6):561-568.
doi: 10.1038/nbt.3853. Epub 2017 Apr 3.

CRISPR-Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome

Tyler S Klann  1   2 , Joshua B Black  1   2 , Malathi Chellappan  1   2 , Alexias Safi  2 , Lingyun Song  2 , Isaac B Hilton  1   2 , Gregory E Crawford  2   3 , Timothy E Reddy  1   2   4 , Charles A Gersbach  1   2   5
Affiliations

CRISPR-Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome

VSports注册入口 - Tyler S Klann et al. Nat Biotechnol. 2017 Jun.

Abstract

Large genome-mapping consortia and thousands of genome-wide association studies have identified non-protein-coding elements in the genome as having a central role in various biological processes. However, decoding the functions of the millions of putative regulatory elements discovered in these studies remains challenging. CRISPR-Cas9-based epigenome editing technologies have enabled precise perturbation of the activity of specific regulatory elements. Here we describe CRISPR-Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity in the native genomic context. Using dCas9KRAB repressor and dCas9p300 activator constructs and lentiviral single guide RNA libraries to target DNase I hypersensitive sites surrounding a gene of interest, we carried out both loss- and gain-of-function screens to identify regulatory elements for the β-globin and HER2 loci in human cells VSports手机版. CERES readily identified known and previously unidentified regulatory elements, some of which were dependent on cell type or direction of perturbation. This technology allows the high-throughput functional annotation of putative regulatory elements in their native chromosomal context. .

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Conflict of interest statement

CONFLICT OF INTEREST

TSK, JBB, IBH, GEC, TER, and CAG are named inventors on patent applications related to genome engineering. TSK, GEC, TER, and CAG are founders of Element Genomics V体育安卓版.

Figures

Figure 1
Figure 1
CRISPR–Cas9-based Epigenetic Regulatory Element Screening (CERES) identifies regulatory elements of the β-globin locus in a loss-of-function screen. (a) CERES involves the design and synthesis of libraries of gRNAs targeted to all candidate gene regulatory elements in a genomic region, in this case as defined by DNase I hypersensitive sites (DHS) identified by DNase-seq. Lentiviral vectors encoding the gRNA library are delivered to cell lines expressing the dCas9KRAB repressor, for loss-of-function screens, or the dCas9p300 activator, for gain-of-function screens. The cells can then be selected for changes in phenotype, such as gain or loss of expression of a target gene. Sequencing the gRNAs in the selected cell subpopulations and mapping them back to the genome reveals regulatory elements involved in controlling the selected phenotype. In the example shown here, a gRNA library was designed to all DHSs in a 4.5 Mb region surrounding the β-globin locus, and introduced into human K562 cells expressing dCas9KRAB and containing an mCherry reporter at the HBE1 gene. (b) Representative flow cytometry data of the HBE1 reporter cells containing the gRNA library, and expression levels of cells sorted for gRNA enrichment. (c) Manhattan plot of a high-throughput screen for regulatory elements in the 4.5 Mb surrounding the globin locus using the dCas9KRAB repressor. (d) Enriched DHSs following selection for decreased HBE1 expression were found only in the HBE1 promoter and enhancers (HS1-4), while the promoters of HBG1/2 were enriched in cells with increased HBE1 expression. Diamonds indicate adjusted p-value < 0.05 and gray circles represent adjusted p-value > 0.05. Red indicates DHS fold change < 0 and blue indicates DHS fold change > 0.
Figure 2
Figure 2
A dCas9KRAB loss-of-function screen in A431 cells identifies regulatory elements of HER2. (a) Flow cytometry for HER2 expression in A431 cells expressing dCas9KRAB and a gRNA library targeted to all DHSs in 4 Mb surrounding HER2 detected in the HER2-overexpressing SKRB3 breast cancer cell line. (b) Manhattan plot showing regulatory elements affecting HER2 expression. (c) A detailed view of the region around HER2. When comparing high and low HER2-expressing cell populations, gRNAs are enriched in the promoter and an intronic DHS of HER2, as well as surrounding DHSs including the promoter of GRB7, an adapter protein that associates with tyrosine kinases. (d) HER2 mRNA fold-change in response to the most enriched gRNAs from (b) relative to treatment with a control gRNA. * indicates samples are significantly different from control as determined by one-way analysis of variance followed by Dunnett’s Test; adjusted P < 0.05. (n = 3 biological replicates, mean ± SEM). (e) HER2 mRNA log2 fold-change in response to gRNA treatment versus log2 fold-change of the abundance of the corresponding gRNA in the HER2 dCas9KRAB screen (Spearman correlation ρ = 0.5175).
Figure 3
Figure 3
A dCas9p300 gain-of-function screen in HEK293T cells identifies regulatory elements of HER2. (a) Flow cytometry for HER2 expression in HEK293T cells expressing dCas9p300 and a gRNA library targeted to all the DHSs in a 4 Mb region surrounding HER2 found in the SKBR3 HER2-overexpressing breast cancer cell line. (b) Manhattan plot showing the results of a screen for regulatory elements affecting HER2 expression. (c) A detailed view of the region around HER2. When comparing high and low HER2-expressing cell populations, gRNAs are enriched in the promoter and three intronic DHSs of HER2 as well as several nearby DHSs. (d) HER2 mRNA fold-change in response to the most enriched gRNAs from (b) relative to treatment with a control gRNA. * indicates samples are significantly different from control as determined by one-way analysis of variance followed by Dunnett’s Test; adjusted P < 0.05 (n = 3 biological replicates, mean ± SEM). (e) HER2 mRNA log2 fold-change in response to gRNA treatment versus log2 fold-change of the abundance of the corresponding gRNA in the HER2 HEK293T dCas9p300 screen (Spearman correlation ρ =0.9429).
Figure 4
Figure 4
dCas9p300 and dCas9KRAB remodel epigenetic marks near novel regulatory elements identified from screens. (a) Genomic tracks displaying the qPCR amplicon regions for the H3K27ac ChIP-qPCR assay, DNase-seq signal, and DHS sites for SKBR3 cells near HER2. (b) dCas9p300 targeted to a DHS with a gRNA enriched in the screen (1548.4) in HEK293T cells leads to H3K27ac enrichment near the DHS, in contrast to gRNA 1548.5 targeting the same DHS but was not enriched in the screen. (c) Genomic tracks displaying the qPCR amplicon regions for the H3K9me3 ChIP-qPCR assay, DNase-seq signal, and DHS sites for SKBR3 cells near HER2 and GRB7. (d) dCas9KRAB targeted to a DHS with a gRNA enriched in the screen (1561.8) in A431 cells deposits H3K9me3 near the DHS. A gRNA that was not significantly enriched in the screen (1561.19) also deposits similar amounts of H3K9me3 near the DHS. * indicates samples are significantly different from control as determined by one-way analysis of variance followed by Dunnett’s Test; adjusted P < 0.05 (n = 3 biological replicates, mean ± SEM). All fold enrichments are relative to transduction of a control gRNA and normalized to a region of the ACTB locus.
Figure 5
Figure 5
Comparison of HER2 activation screens between different cell types. (a) Log2 fold-change gRNA abundance in the dCas9p300 screen in HEK293T cells versus A431 cells. (b and c) A detailed view of the region around HER2 for the dCas9p300 screens in (b) A431 cells or (c) HEK293T cells. Several of the same DHS contained enriched gRNAs in both screens, including the promoter region, but others, such as an intronic DHS highly enriched in the dCas9KRAB screen in A431 cells, were only enriched in a single screen and may be cell-type specific enhancers.
Figure 6
Figure 6
Comparisons between HER2 activation and repression screens. (a) Log2 fold-change of the gRNA abundance in the dCas9KRAB screen versus the dCas9p300 screen in A431 cells. (b and c) A detailed view of the region around HER2 for the (b) dCas9p300 or (c) dCas9KRAB screens in A431 cells. Several DHSs contain enriched gRNAs in both screens, including the promoter and an HER2-intronic DHSs. However, DHSs in the GRB7 promoter region are only enriched in the dCas9KRAB screen.
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