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

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.
Copyright © 2025 The Author(s). Published by Elsevier Inc. All rights reserved.

Authors

Leonardo A Parra-Rivas, Rohan Sharma, Trinity E Rust, Hannah O Bazick, Jared Carlson-Stevermer, Mark J Zylka, Yuki Ogawa, Subhojit Roy

Department of Pathology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, USA; Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA; Department of Neuroscience, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, USA. Electronic address: sroy@ucsd.edu.