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

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CRISPR Based Programmable RNA Editing in Primary Hippocampal Neurons.

Karthick Ravichandran¹, Tulika Khargonkar¹, Sarbani Samaddar¹

¹National Brain Research Centre, Manesar, Haryana, India.

Current Protocols

|September 27, 2023

Summary

Researchers adapted a CRISPR-based RNA editing tool to visualize RNA editing in neurons. This new method allows for precise manipulation of RNA function without altering the genome, paving the way for studying RNA-protein interactions.

Keywords:

CRISPR Cas13b Dendra2 RNA-editing crRNA mouse primary hippocampal culture

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Area of Science:

Neuroscience
Molecular Biology
Genetics

Background:

Understanding RNA regulation is crucial for studying neuronal function and RNA-protein interactions.
Existing tools for analyzing RNA-protein interactions in physiological environments are limited.
CRISPR-based RNA editing tools offer potential for precise RNA manipulation.

Purpose of the Study:

To explore the targeted RNA sequence editing capabilities of the dCas13b-ADAR2DD system in primary hippocampal neurons.
To adapt and visualize RNA editing for manipulating RNA function in a neuronal context.
To establish a method for studying RNA-protein interactions without genomic alterations.

Main Methods:

Utilized a two-component CRISPR-based system (dCas13b-ADAR2DD) with a programmable guide RNA (gRNA).
Targeted the Dendra2 mRNA transcript, which was engineered with a nonsense mutation to abolish fluorescence.
Visualized RNA editing efficacy by restoring Dendra2 fluorescence through targeted nucleotide editing.

Main Results:

Successfully demonstrated targeted RNA editing in primary hippocampal neurons using the dCas13b-ADAR2DD system.
Restored Dendra2 fluorescence by editing specific nucleotides in the Dendra2 mRNA, proving the system's efficacy.
Visualized RNA editing at the single-cell level in neurons.

Conclusions:

The dCas13b-ADAR2DD system provides a powerful tool for targeted RNA manipulation and visualization in neurons.
This methodology facilitates the study of RNA-protein interactions and dynamics in neurons without genomic modification.
The approach can be extended to manipulate endogenous RNA in various neuronal subtypes and offers insights into spatial and temporal RNA-protein interactions.