Comprehensive In Vivo Interrogation Reveals Phenotypic Impact of Human Enhancer Variants

Abstract

Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal led to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knock-in mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.

The contribution of non-coding regulatory elements to cardiovascular disease

“…The complex nature of many CVDs is also calling for improved strategies to evaluate tissue-specific contributions experimentally, such as the development of engineered human tissues [ 141 ]. Lastly, novel high-throughput approaches to screen gene expression contributions [ 105 ] and tissue-specificity [ 142 ] of regulatory elements (and the non-coding variants within) should be pivotal in gleaning mechanistic insights into how gene regulation contributes to onset and progression of CVDs.…”

Analysis of zebrafish periderm enhancers facilitates identification of a regulatory variant near humanKRT8/18

“…We considered the possibility that, against our prediction, the elements are enhancers in mammals but not zebrafish. We engineered all four elements (separately) into reporter vectors with a minimal Shh promoter and the LacZ gene and carried out transgenic reporter assays in F0 mouse embryos using site-directed transgene integration 63 . Across 18 transgenic embryos injected with SNP1 element, 11 with non-risk allele, 7 with risk allele, we did not observe reproducible reporter expression, defined as expression in the same anatomical structure in at least two embryos injected with the same construct, and the majority of transgenic embryos did not show any reporter staining (Figure 6-figure supplement 3A).…”

“…All elements were subcloned into the cFos-GFP plasmid (a gift from Shannon Fisher) 37 or cFos-tdTomato, a derivative of cFos-GFP, or cFos-GFP; Cry-GFP, a derivative that includes a lens-specific promoter (cloning details available upon request). For each reporter construct, at least 100 embryos at the 1-cell to 2-cell stage were injected (20 pg reporter construct plus 20 pg tol2 mRNA); three replicates were performed, each on a different day 37 63 . In this assay, the reporter cassette is flanked by homology arms targeting the H11 safe harbor locus 97 .…”

“…In this assay, the reporter cassette is flanked by homology arms targeting the H11 safe harbor locus 97 . Cas9 protein and a sgRNA targeting H11 were co-injected into the pronucleus of FVB single cell stage mouse embryos (E0.5) together with the reporter vector 63 .…”

Parallel genetics of regulatory sequencesin vivo

“…Although parallel interrogation of regulatory sequences has been developed in cell lines and yeast [8][9][10][11][12] , only a few in vivo approaches have been achieved in animal models. These use integration of reporters 13,14 or injection of RNA libraries 15,16 and therefore do not evaluate endogenous phenotypes, or are restricted to one stage of the animal life cycle. Classical genome editing by injection, now widely accessible due to CRISPR-Cas 17,18 , has enabled endogenous functional tests, but is still work-intensive and limited in scalability 19,20 .…”