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2016 ; 34
(2
): 167-74
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High-throughput mapping of regulatory DNA
#MMPMID26807528
Rajagopal N
; Srinivasan S
; Kooshesh K
; Guo Y
; Edwards MD
; Banerjee B
; Syed T
; Emons BJ
; Gifford DK
; Sherwood RI
Nat Biotechnol
2016[Feb]; 34
(2
): 167-74
PMID26807528
show ga
Quantifying the effects of cis-regulatory DNA on gene expression is a major
challenge. Here, we present the multiplexed editing regulatory assay (MERA), a
high-throughput CRISPR-Cas9-based approach that analyzes the functional impact of
the regulatory genome in its native context. MERA tiles thousands of mutations
across ?40 kb of cis-regulatory genomic space and uses knock-in green fluorescent
protein (GFP) reporters to read out gene activity. Using this approach, we obtain
quantitative information on the contribution of cis-regulatory regions to gene
expression. We identify proximal and distal regulatory elements necessary for
expression of four embryonic stem cell-specific genes. We show a consistent
contribution of neighboring gene promoters to gene expression and identify
unmarked regulatory elements (UREs) that control gene expression but do not have
typical enhancer epigenetic or chromatin features. We compare thousands of
functional and nonfunctional genotypes at a genomic location and identify the
base pair-resolution functional motifs of regulatory elements.
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