High-throughput mapping of regulatory DNA

Abstract

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 non-functional genotypes at a genomic location and identify the basepair-resolution functional motifs of regulatory elements.

Improved regulatory element prediction based on tissue-specific local epigenomic signatures

“…2C and SI Methods). Furthermore, we found that REPTILE predictions recaptured the most distal regulatory DNA elements that were identified by multiplexed editing regulatory assay (MERA), a high-throughput genome mutation screening approach (38) (Fig. S2C and SI Methods).…”

CRISPR-based reagents to study the influence of the epigenome on gene expression

“…2a). These reagents can bypass the epigenetic constraints of gene expression within a cell and have been used for a variety of genomewide screens to efficiently silence either single or multiple genes or silence the influence of regulatory domains in the native genomic context 49, 50, 51, 52, 53, 54, 55, 56, 57, 58.…”

Re-engineered RNA-Guided FokI-Nucleases for Improved Genome Editing in Human Cells

“…This suggests that the RFN system might be better suited than SpCas9 for assays where larger deletions are beneficial, such as for interrogating gene-regulatory elements (e.g., distal enhancers and promoters), which might not be greatly influenced by single base deletions. 25,26 Finally, we analyzed whether changing the linker peptide had any influence on the cleavage site. To address this, we plotted the raw reads containing indels for our various on-target sites ( Figure S6).…”

“…Although they have tested a broad range of spacer distances (0-30 bp) to target GFP in a GFP stable cell line, actually only a few spacing distances showed activity (14-17 and 26 bp). In contrast, Guilinger et al 17 tested a set of variants with 12 different fusion linkers, but compared them only at a small set of gRNA spacing combinations (5,10,14,20,25,32, and 43 bp), of which most did not reveal any functionality. It remained unclear why most variants performed poorly and/or lacked activity, and also what influence the fusion linkers might have over the full spectrum of gRNA spacing possibilities.…”