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Experimental and Computational Approaches for Single-Cell Enhancer Perturbation Assay
1Department of Obstetrics and Gynecology, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Methods in Molecular Biology (Clifton, N.J.)
|February 14, 2019
Summary
We improved Mosaic-seq2, a method for high-throughput enhancer activity testing. This method uses CRISPRi-based epigenetic suppression and single-cell transcriptomics to reveal enhancer function and target genes.
Area of Science:
- Genomics
- Molecular Biology
- Epigenetics
Background:
- Transcriptional enhancers regulate cell-type-specific gene expression, crucial for development and disease.
- Over one million putative human enhancers are cataloged, but their functions are largely uncharacterized.
- Determining enhancer-target gene interactions and contribution to gene expression is a significant challenge.
Purpose of the Study:
- To describe Mosaic-seq2, an optimized method for high-throughput interrogation of enhancer activity.
- To simplify library preparation and increase the sensitivity and throughput of enhancer function assays.
Main Methods:
- Utilizes pooled CRISPRi-based epigenetic suppression of enhancers.
- Employs single-cell transcriptomic readout to assess enhancer function.
- Mosaic-seq2 offers improvements over the original Mosaic-seq method.
Main Results:
- Mosaic-seq2 significantly simplifies the library preparation process.
- The optimized method demonstrates increased overall sensitivity for detecting enhancer activity.
- Mosaic-seq2 enhances the throughput of functional enhancer interrogation.
Conclusions:
- Mosaic-seq2 is a powerful tool for dissecting the functional roles of enhancers.
- The method facilitates large-scale functional characterization of putative enhancers.
- This advancement aids in understanding gene regulation in development and disease.
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