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Related Concept Videos

CRISPR01:59

CRISPR

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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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Related Experiment Video

Updated: Jun 21, 2025

CRISPR Epigenome Editing in Human Cells using Plasmid DNA Transfection and mRNA Nucleofection Delivery
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CRISPR Epigenome Editing in Human Cells using Plasmid DNA Transfection and mRNA Nucleofection Delivery

Published on: May 30, 2025

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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
PubMed
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.

Keywords:
CRISPR/dCas9Epigenetic editingMCF-7 cellsSingle-cell analysisTransfection efficiency

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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.