Rapid and Functional Quantitative Interrogation of Human Enhancer Variants in Live Mice

Functional characterisation of noncoding variants linked to human congenital disorders remains challenging due to the lack of efficient in vivo models. Here we introduce dual-enSERT, a robust two-color fluorescent reporter system which enables rapid comparative assessment of human enhancer variant activities in live mice. We use this new technology to examine the gain- and loss-of-function effects of enhancer variants previously linked to limb polydactyly, autism, and craniofacial malformation. By combining dual-enSERT with single-cell transcriptomics, we characterize variant enhancer allele activity at cellular resolution, revealing candidate molecular pathways implicated in pathogenic enhancer misregulation. We also use dual-enSERT to show that independent polydactyly-linked enhancer variants lead to ectopic expression in the same cell populations, indicating shared genetic mechanisms underlying noncoding variant pathogenesis. Finally, we streamline dual-enSERT for F0 analysis by placing both reporters on the same allele separated by an insulator. Our F0 dual-enSERT cuts the experimental time of noncoding variant selection to corresponding comparative in vivo activity in live embryos from three months to two weeks. Overall design: Hindlimbs of two E11.5 transgenic embryos with two ZRS-reporters from dual-enSERT-1 and dual-enSERT-2 were independently dissected, dissociated into single cells, and scRNA-seq was performed.