Rapid and quantitative functional interrogation of human enhancer variant activity in live mice
Abstract Functional analysis of non-coding variants associated with 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...
Saved in:
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2025-01-01
|
| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-024-55500-7 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850119485770432512 |
|---|---|
| author | Ethan W. Hollingsworth Taryn A. Liu Joshua A. Alcantara Cindy X. Chen Sandra H. Jacinto Evgeny Z. Kvon |
| author_facet | Ethan W. Hollingsworth Taryn A. Liu Joshua A. Alcantara Cindy X. Chen Sandra H. Jacinto Evgeny Z. Kvon |
| author_sort | Ethan W. Hollingsworth |
| collection | DOAJ |
| description | Abstract Functional analysis of non-coding variants associated with 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 in less than two weeks. We use this technology to examine and measure the gain- and loss-of-function effects of enhancer variants previously linked to limb polydactyly, autism spectrum disorder, and craniofacial malformation. By combining dual-enSERT with single-cell transcriptomics, we characterise gene expression in cells where the enhancer is normally and ectopically active, revealing candidate pathways that may lead to enhancer misregulation. Finally, we demonstrate the widespread utility of dual-enSERT by testing the effects of fifteen previously uncharacterised rare and common non-coding variants linked to neurodevelopmental disorders. In doing so we identify variants that reproducibly alter the in vivo activity of OTX2 and MIR9-2 brain enhancers, implicating them in autism. Dual-enSERT thus allows researchers to go from identifying candidate enhancer variants to analysis of comparative enhancer activity in live embryos in under two weeks. |
| format | Article |
| id | doaj-art-87bde904a13b44098115099208b5ea6b |
| institution | OA Journals |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-87bde904a13b44098115099208b5ea6b2025-08-20T02:35:37ZengNature PortfolioNature Communications2041-17232025-01-0116111510.1038/s41467-024-55500-7Rapid and quantitative functional interrogation of human enhancer variant activity in live miceEthan W. Hollingsworth0Taryn A. Liu1Joshua A. Alcantara2Cindy X. Chen3Sandra H. Jacinto4Evgeny Z. Kvon5Department of Developmental and Cell Biology, University of CaliforniaDepartment of Developmental and Cell Biology, University of CaliforniaDepartment of Developmental and Cell Biology, University of CaliforniaDepartment of Developmental and Cell Biology, University of CaliforniaDepartment of Developmental and Cell Biology, University of CaliforniaDepartment of Developmental and Cell Biology, University of CaliforniaAbstract Functional analysis of non-coding variants associated with 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 in less than two weeks. We use this technology to examine and measure the gain- and loss-of-function effects of enhancer variants previously linked to limb polydactyly, autism spectrum disorder, and craniofacial malformation. By combining dual-enSERT with single-cell transcriptomics, we characterise gene expression in cells where the enhancer is normally and ectopically active, revealing candidate pathways that may lead to enhancer misregulation. Finally, we demonstrate the widespread utility of dual-enSERT by testing the effects of fifteen previously uncharacterised rare and common non-coding variants linked to neurodevelopmental disorders. In doing so we identify variants that reproducibly alter the in vivo activity of OTX2 and MIR9-2 brain enhancers, implicating them in autism. Dual-enSERT thus allows researchers to go from identifying candidate enhancer variants to analysis of comparative enhancer activity in live embryos in under two weeks.https://doi.org/10.1038/s41467-024-55500-7 |
| spellingShingle | Ethan W. Hollingsworth Taryn A. Liu Joshua A. Alcantara Cindy X. Chen Sandra H. Jacinto Evgeny Z. Kvon Rapid and quantitative functional interrogation of human enhancer variant activity in live mice Nature Communications |
| title | Rapid and quantitative functional interrogation of human enhancer variant activity in live mice |
| title_full | Rapid and quantitative functional interrogation of human enhancer variant activity in live mice |
| title_fullStr | Rapid and quantitative functional interrogation of human enhancer variant activity in live mice |
| title_full_unstemmed | Rapid and quantitative functional interrogation of human enhancer variant activity in live mice |
| title_short | Rapid and quantitative functional interrogation of human enhancer variant activity in live mice |
| title_sort | rapid and quantitative functional interrogation of human enhancer variant activity in live mice |
| url | https://doi.org/10.1038/s41467-024-55500-7 |
| work_keys_str_mv | AT ethanwhollingsworth rapidandquantitativefunctionalinterrogationofhumanenhancervariantactivityinlivemice AT tarynaliu rapidandquantitativefunctionalinterrogationofhumanenhancervariantactivityinlivemice AT joshuaaalcantara rapidandquantitativefunctionalinterrogationofhumanenhancervariantactivityinlivemice AT cindyxchen rapidandquantitativefunctionalinterrogationofhumanenhancervariantactivityinlivemice AT sandrahjacinto rapidandquantitativefunctionalinterrogationofhumanenhancervariantactivityinlivemice AT evgenyzkvon rapidandquantitativefunctionalinterrogationofhumanenhancervariantactivityinlivemice |