Protocol for CRISPR-based manipulation and visualization of endogenous α-synuclein in cultured mouse hippocampal neurons

TL;DR

This paper provides a detailed protocol for using CRISPR-Cas9 to study alpha-synuclein in mouse hippocampal neurons.

Contribution

A new protocol for CRISPR-based α-synuclein manipulation and visualization in cultured hippocampal neurons.

Findings

CRISPR-Cas9 enables α-synuclein depletion or tagging in hippocampal neurons.

AAVs and lentiviruses are used for gene delivery and validation steps are described.

The method allows studying protein function and trafficking without overexpression.

Abstract

CRISPR-Cas9 technology enables acute gene knockdown and endogenous tagging to study single-synapse function. Here, we present a protocol for depleting alpha-synuclein (α-syn) or visualizing native α-syn with an endogenously inserted fluorescent tag in cultured mouse hippocampal neurons. We describe detailed steps, including CRISPR design, virus packaging/transduction (delivery), and validation of on-/off-target editing. This protocol should be useful for assigning precise function to contentious synaptic proteins and for visualizing protein trafficking without overexpression in cultured hippocampal neurons—an established model system for synaptic biology.

For complete details on the use and execution of this protocol, please refer to Parra-Rivas et al.1

•CRISPR-Cas9 knockdown of α-synuclein in hippocampal neurons via AAV or lentivirus•Endogenous C-terminal tagging of α-synuclein in hippocampal neurons using AAVs•Step-by-step validation for CRISPR-Cas9 knockdown and endogenous tagging

CRISPR-Cas9 knockdown of α-synuclein in hippocampal neurons via AAV or lentivirus

Endogenous C-terminal tagging of α-synuclein in hippocampal neurons using AAVs

Step-by-step validation for CRISPR-Cas9 knockdown and endogenous tagging

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

CRISPR-Cas9 technology enables acute gene knockdown and endogenous tagging to study single-synapse function. Here, we present a protocol for depleting alpha-synuclein (α-syn) or visualizing native α-syn with an endogenously inserted fluorescent tag in cultured mouse hippocampal neurons. We describe detailed steps, including CRISPR design, virus packaging/transduction (delivery), and validation of on-/off-target editing. This protocol should be useful for assigning precise function to contentious synaptic proteins and for visualizing protein trafficking without overexpression in cultured hippocampal neurons—an established model system for synaptic biology.

Figures