Rapid and Quantitative Functional Interrogation of Human Enhancer Variant Activity in Live Mice.

Autor:	Hollingsworth EW; Department of Developmental and Cell Biology, University of California, Irvine, CA 92697, USA.; Medical Scientist Training Program, University of California, Irvine School of Medicine, Irvine, CA 92697, USA., Liu TA; Department of Developmental and Cell Biology, University of California, Irvine, CA 92697, USA., Jacinto SH; Department of Developmental and Cell Biology, University of California, Irvine, CA 92697, USA., Chen CX; Department of Developmental and Cell Biology, University of California, Irvine, CA 92697, USA., Alcantara JA; Department of Developmental and Cell Biology, University of California, Irvine, CA 92697, USA., Kvon EZ; Department of Developmental and Cell Biology, University of California, Irvine, CA 92697, USA.
Jazyk:	angličtina
Zdroj:	BioRxiv : the preprint server for biology [bioRxiv] 2023 Dec 10. Date of Electronic Publication: 2023 Dec 10.
DOI:	10.1101/2023.12.10.570890
Abstrakt:	Functional analysis of non-coding variants associated with human congenital disorders remains challenging due to the lack of efficient in vivo models. Here we introduce dual-enSERT, a robust Cas9-based two-color fluorescent reporter system which enables rapid, quantitative comparison of enhancer allele activities in live mice of any genetic background. We use this new technology to examine and measure the gain- and loss-of-function effects of enhancer variants linked to limb polydactyly, autism, and craniofacial malformation. By combining dual-enSERT with single-cell transcriptomics, we characterize variant enhancer alleles at cellular resolution, thereby implicating candidate molecular pathways in pathogenic enhancer misregulation. We further show that independent, polydactyly-linked enhancer variants lead to ectopic expression in the same cell populations, indicating shared genetic mechanisms underlying non-coding variant pathogenesis. Finally, we streamline dual-enSERT for analysis in F0 animals by placing both reporters on the same transgene separated by a synthetic insulator. Dual-enSERT allows researchers to go from identifying candidate enhancer variants to analysis of comparative enhancer activity in live embryos in under two weeks. Competing Interests: Competing interests: The authors declare no competing interests.
Databáze:	MEDLINE
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