Conditional Gene Editing in Presynaptic Extinction-ensemble Cells via the CRISPR-SaCas9 System

Area of Science:

• Neuroscience
• Molecular Biology
• Genetics

Background:

• CRISPR-Cas9 technology allows gene editing in diverse cell types, including neurons.
• Neuronal populations within the same brain region exhibit heterogeneity, necessitating targeted gene editing.
• Existing methods lack specificity for functionally or projection-defined neuronal ensembles.

Purpose of the Study:

• To develop and detail a method for function- and projection-specific gene editing in the mammalian brain.
• To demonstrate the efficacy of this technique in perturbing specific neuronal populations.
• To investigate the role of CREB-binding protein (CBP) in fear extinction learning.

Main Methods:

• A CRISPR-SaCas9 system was combined with activity-dependent cell-labeling and adeno-associated virus (AAV) vectors.
• Stereotaxic injection of AAV vectors carrying guide RNA (gRNA) into specific brain regions (amygdala-projecting infralimbic cortex).
• Gene perturbation of CBP in presynaptic extinction-ensemble neurons in adult rats, followed by contextual fear conditioning.

Main Results:

• The developed CRISPR-SaCas9 technique achieved highly specific gene editing in targeted neuronal ensembles.
• Perturbation of CBP in extinction-ensemble neurons significantly impaired fear extinction learning.
• The study provides a detailed protocol for implementing this technique in rodent models.

Conclusions:

• The CRISPR-SaCas9 system, when combined with cell-labeling and AAV vectors, enables precise gene editing in specific neuronal populations.
• This approach is effective for studying the role of specific genes in neural circuits and behavior, such as fear extinction.
• The technique offers broad applicability for future neural circuitry research.