An Alternative Gene Editing Strategy Using a Single AAV Vector.

Area of Science:

• Neuroscience
• Molecular Biology
• Genetics

Background:

• CRISPR/Cas9 gene editing allows precise DNA modification.
• Adeno-associated viruses (AAVs) are effective vectors for gene delivery in the nervous system.
• Simultaneous gene editing and transgene expression in specific cell types is challenging.

Purpose of the Study:

• To present a protocol for designing and generating single-vector plasmids and AAVs for cell type-specific CRISPR/Cas9 editing and transgene expression.
• To enable the co-expression of standard neuroscience tools in edited cells.
• To facilitate the study of gene function in the central and peripheral nervous systems.

Main Methods:

• Guide RNA (gRNA) design for targeting specific genes.
• Ligation and cloning of CRISPR-competent AAV vectors.
• Production and viral titer quantification of AAVs.
• Compatibility with Cre-dependent Cas9 mouse lines for selective co-expression.

Main Results:

• A protocol for generating single-vector CRISPR/Cas9-compatible AAVs is described.
• The AAVs can be produced in any serotype for versatile delivery.
• The system allows for cell type-specific editing and co-expression of tools like fluorescent proteins, optogenetic, and chemogenetic tools.
• The vectors are suitable for use in both the central and peripheral nervous systems.

Conclusions:

• This single-vector approach simplifies gene editing and tool delivery in the mouse nervous system.
• The protocol provides a flexible method for investigating gene function in neural circuits.
• This technique can accelerate the discovery and functional testing of novel genes involved in synaptic transmission, circuit activity, and morphology.