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

Area of Science:

• Molecular Biology
• Genetics
• Epigenetics

Background:

• Epigenetics involves chemical modifications regulating gene expression, crucial for cell differentiation and aging.
• Aberrant epigenome patterns are linked to various diseases.
• CRISPR technology has enabled programmable tools for targeted epigenetic modifications.

Purpose of the Study:

• To present protocols for dCas9-mediated epigenome editing in human cell lines.
• To demonstrate transient gene repression using CRISPR interference (CRISPRi).
• To showcase durable gene silencing for weeks using CRISPRoff.

Main Methods:

• Utilized plasmid DNA transfections and mRNA nucleofection for CRISPR epigenome editor delivery.
• Employed catalytically dead Cas9 (dCas9) fused with epigenetic modifiers.
• Applied CRISPR interference (CRISPRi) for transient gene repression and CRISPRoff for durable silencing.

Main Results:

• Successfully demonstrated programmable epigenome editing for gene repression and silencing.
• Achieved transient gene repression via CRISPRi.
• Achieved durable gene silencing for extended periods (weeks) using CRISPRoff.

Conclusions:

• CRISPR-based epigenome editing provides a safe alternative to genome editing for controlling gene expression without DNA alteration.
• The presented methods allow precise, programmable manipulation of the epigenome.
• Guidance is provided for quantitative assessment and tool selection based on experimental needs.