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Updated: Jun 21, 2025
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CRISPR Epigenome Editing in Human Cells using Plasmid DNA Transfection and mRNA Nucleofection Delivery
Published on: May 30, 2025
769
Plasmid Delivery and Single-Cell Plasmid Expression Analysis for CRISPR/dCas9-Based Epigenetic Editing.
Anna C H van den Berg van Saparoea1, Quint C van Loosen1, Federica Sarno2
1Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands.
Methods in Molecular Biology (Clifton, N.J.)
|July 16, 2024
Summary
Optimizing transfection methods is key for CRISPR/dCas9 epigenetic editing. This study streamlines protocols and uses flow cytometry to enhance plasmid delivery and expression analysis in mammalian cells.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- CRISPR/dCas9 systems offer powerful epigenetic editing capabilities.
- Efficient delivery of large constructs is a challenge for epigenetic reprogramming.
- Optimized transfection protocols are essential for diverse biological applications.
Purpose of the Study:
- To develop and optimize transfection protocols for enhanced plasmid delivery in mammalian cells.
- To streamline the process of optimizing transfection efficiency for specific experimental needs.
- To enable accurate assessment of both transient and sustained epigenetic reprogramming effects.
Main Methods:
- Comparison of various transfection reagents and methods.
- Utilization of flow cytometry for assessing transfection efficiency and construct expression.
- Incorporation of heat shock treatment for specific cell lines (e.g., MCF-7).
- Testing protocols in HEK293T Lenti-X™ and MCF-7 cell lines.
Main Results:
- A streamlined protocol for transfection efficiency optimization was established.
- Flow cytometry effectively quantifies transfection rates and single-cell expression levels.
- Heat shock significantly improved transfection efficiency in MCF-7 cells.
- The protocol facilitates reliable assessment of epigenetic reprogramming.
Conclusions:
- Optimized transfection protocols are critical for maximizing the potential of CRISPR/dCas9 epigenetic editing.
- The developed protocol provides a robust framework for efficient plasmid delivery and expression analysis.
- This work supports advancements in epigenetic editing research across various cell types and applications.
