A Neuron-Optimized CRISPR/dCas9 Activation System for Robust and Specific Gene Regulation.

Area of Science:

• Neuroscience
• Molecular Biology
• Gene Editing

Background:

• CRISPR technology offers gene function interrogation but faces challenges in post-mitotic neuron transgene expression.
• Efficient gene regulation tools are needed for studying the central nervous system (CNS).

Purpose of the Study:

• To develop and validate a neuron-optimized CRISPR-based transcriptional activation (CRISPRa) system for robust gene induction in CNS research.
• To assess the efficiency, selectivity, and applicability of CRISPRa in primary neurons and *in vivo*.

Main Methods:

• Developed a dual lentiviral CRISPRa system optimized for neuron-specific gene expression.
• Applied CRISPRa to regulate the brain-derived neurotrophic factor (Bdnf) gene in primary rodent neuron cultures.
• Validated CRISPRa efficiency and target gene protein level increases *in vivo* in various brain structures.

Main Results:

• Demonstrated a highly efficient, modular, and tunable CRISPRa system for gene induction in primary neurons.
• Achieved transcript- and genome-level selectivity when targeting the Bdnf gene.
• Confirmed robust CRISPRa *in vivo* with increased target gene protein levels in diverse brain regions.

Conclusions:

• CRISPRa is an efficient and selective method for studying gene expression in the CNS.
• This system facilitates research into gene regulation in brain health and disease.
• The developed CRISPRa tool overcomes previous limitations in neuronal gene manipulation.