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Towards a comprehensive catalogue of validated and target-linked human enhancers


The human gene catalogue is essentially complete, but we lack an equivalently vetted inventory of bona fide human enhancers. Hundreds of thousands of candidate enhancers have been nominated via biochemical annotations; however, only a handful of these have been validated and confidently linked to their target genes. Here we review emerging technologies for discovering, characterizing and validating human enhancers at scale. We furthermore propose a new framework for operationally defining enhancers that accommodates the heterogeneous and complementary results that are emerging from reporter assays, biochemical measurements and CRISPR screens.

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Fig. 1: Approaches for identifying, validating and characterizing enhancers.
Fig. 2: CRISPR-based approaches for perturbing enhancers.
Fig. 3: CRISPR-based screens of enhancer–gene links.
Fig. 4: A tiered framework to describe the level of support for the enhancer candidacy of a non-coding sequence.
Fig. 5: The blind men and the elephant of human enhancer biology.


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The authors thank S. Kim, C. Trapnell and S. Domcke, as well as other members of the Shendure Lab, for helpful discussions. J.S. is an investigator for the Howard Hughes Medical Institute.

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M.G. and J.S. wrote the initial manuscript. M.G., J.T. and J.S. contributed to researching content for the article, discussing the content and reviewing/editing the manuscript before submission.

Corresponding author

Correspondence to Jay Shendure.

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Transcription factors

(TFs). Proteins that bind DNA, typically consisting of specific DNA sequences or motifs, and contribute to the regulation of RNA transcription.

Open chromatin

A nucleosome-loose packaging state of DNA that is permissive for transcription-factor binding.

Episomal reporter vector

Plasmid DNA that can be synthetically delivered, is autonomous from genomic DNA and includes a reporter gene, typically downstream of a candidate regulatory element (for example, an enhancer adjacent to a minimal promoter).

Expressed sequence tag

(EST). In the early days of genomics, shotgun sequencing of cDNA was used as an efficient strategy for discovering genes, and subsequently to quantify their relative abundance.

Open reading frame

The portion of a gene that is translatable by a ribosome; these are relatively straightforward to annotate by sequence alone, due to the required start and stop codons.

Regulatory element

A functional non-coding DNA sequence that regulates transcription; classes of regulatory elements include enhancers, promoters, silencers and insulators (further defined in Box 2).

Chromosome conformation capture

(3C). Methods that map the 3D positioning, looping and spatial organization of DNA within the nucleus, often relative to other segments of DNA.


Clustered regularly interspaced short palindromic repeats. A system that consists of the components of a bacterial immune system that have been adopted for synthetic genetic perturbation. The term is most often used in reference to the Type II Cas9 endonuclease version, which can introduce a double-stranded break into genomic DNA as directed by a synthetic guide RNA.

Topologically associating domains

(TADs). Broad regions of genomic DNA that are physically packaged together in the nucleus in 3D space, typically at a scale from hundreds of kilobases to several megabases.

Pioneer factor

A TF that can directly interact with compact, closed chromatin; this class of TFs are thought to initiate (‘pioneer’) chromatin remodelling events.

Linkage disequilibrium

The population genetics phenomenon by which genetic variants are nonrandomly associated within a population. Variants are said to be in ‘linkage disequilibrium’ if they are found to reside on a haplotype more frequently than one would expect by completely random assortment; variants in linkage disequilibrium are nearby on a genomic locus and hence are co-inherited because they are rarely separated through meiotic recombination.

Simpson’s paradox

A phenomenon in statistics in which different trends may exist in subgroups of a dataset but are undetectable when the groups are analysed as a whole.

Saturation mutagenesis

A molecular biology technique in which all possible sequence changes are generated from a parental sequence (for example, all possible amino acids in an open reading frame, or all possible single-nucleotide variants in an enhancer).

Protospacer-adjacent motif

(PAM). In the original CRISPR bacterial immune system, fragments of previously encountered viral DNA are preserved in the bacterial genome; these ‘remembered’ sequences are processed into RNAs that guide the CRISPR nuclease to destroy newly invading viral DNA. But, to prevent the nuclease from destroying the matching ‘remembered’ sequence in the bacteria’s own genome, a motif (the PAM) is required next to the target sequence in the viral genome. When genome editing is performed in eukaryotic cells, the presence of this sequence is still required by CRISPR nucleases.

Shadow enhancers

Redundant enhancers, often located far away from their target gene; enhancer redundancy is thought to enable robust buffered expression of the target gene and to provide a versatile platform for the evolution of new regulatory functions.


The fourth generation of projects funded by the Encyclopedia of DNA Elements (ENCODE) Consortium, begun in 2017 and including a new component focused on the implementation of high-throughput functional assays.

Human Cell Atlas

An international scientific community to coordinate the generation of human single-cell datasets, with the goal of generating a reference map of every cell type in the human body.

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Gasperini, M., Tome, J.M. & Shendure, J. Towards a comprehensive catalogue of validated and target-linked human enhancers. Nat Rev Genet 21, 292–310 (2020).

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