Protocol for CRISPR-based manipulation and visualization of endogenous α-synuclein in cultured mouse hippocampal neurons
Summary: 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...
Saved in:
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-09-01
|
| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S266616672500351X |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849731866647592960 |
|---|---|
| author | Leonardo A. Parra-Rivas Rohan Sharma Trinity E. Rust Hannah O. Bazick Jared Carlson-Stevermer Mark J. Zylka Yuki Ogawa Subhojit Roy |
| author_facet | Leonardo A. Parra-Rivas Rohan Sharma Trinity E. Rust Hannah O. Bazick Jared Carlson-Stevermer Mark J. Zylka Yuki Ogawa Subhojit Roy |
| author_sort | Leonardo A. Parra-Rivas |
| collection | DOAJ |
| description | Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| format | Article |
| id | doaj-art-7e72691c5ecf449f8d158aec754b5892 |
| institution | DOAJ |
| issn | 2666-1667 |
| language | English |
| publishDate | 2025-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj-art-7e72691c5ecf449f8d158aec754b58922025-08-20T03:08:24ZengElsevierSTAR Protocols2666-16672025-09-016310394510.1016/j.xpro.2025.103945Protocol for CRISPR-based manipulation and visualization of endogenous α-synuclein in cultured mouse hippocampal neuronsLeonardo A. Parra-Rivas0Rohan Sharma1Trinity E. Rust2Hannah O. Bazick3Jared Carlson-Stevermer4Mark J. Zylka5Yuki Ogawa6Subhojit Roy7Department 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, USADepartment of Pathology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, USADepartment of Cell Biology and Physiology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USAUNC Neuroscience Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USASerotiny Inc., 329 Oyster Point Boulevard, 3rd Floor, South San Francisco, CA 94080, USADepartment of Cell Biology and Physiology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; UNC Neuroscience Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Carolina Institute for Developmental Disabilities, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USADepartment of Neuroscience, Baylor College of Medicine, Houston, TX, USADepartment 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; Corresponding authorSummary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S266616672500351XCell BiologyCell cultureMicroscopyMolecular BiologyCRISPRNeuroscience |
| spellingShingle | Leonardo A. Parra-Rivas Rohan Sharma Trinity E. Rust Hannah O. Bazick Jared Carlson-Stevermer Mark J. Zylka Yuki Ogawa Subhojit Roy Protocol for CRISPR-based manipulation and visualization of endogenous α-synuclein in cultured mouse hippocampal neurons STAR Protocols Cell Biology Cell culture Microscopy Molecular Biology CRISPR Neuroscience |
| title | Protocol for CRISPR-based manipulation and visualization of endogenous α-synuclein in cultured mouse hippocampal neurons |
| title_full | Protocol for CRISPR-based manipulation and visualization of endogenous α-synuclein in cultured mouse hippocampal neurons |
| title_fullStr | Protocol for CRISPR-based manipulation and visualization of endogenous α-synuclein in cultured mouse hippocampal neurons |
| title_full_unstemmed | Protocol for CRISPR-based manipulation and visualization of endogenous α-synuclein in cultured mouse hippocampal neurons |
| title_short | Protocol for CRISPR-based manipulation and visualization of endogenous α-synuclein in cultured mouse hippocampal neurons |
| title_sort | protocol for crispr based manipulation and visualization of endogenous α synuclein in cultured mouse hippocampal neurons |
| topic | Cell Biology Cell culture Microscopy Molecular Biology CRISPR Neuroscience |
| url | http://www.sciencedirect.com/science/article/pii/S266616672500351X |
| work_keys_str_mv | AT leonardoaparrarivas protocolforcrisprbasedmanipulationandvisualizationofendogenousasynucleininculturedmousehippocampalneurons AT rohansharma protocolforcrisprbasedmanipulationandvisualizationofendogenousasynucleininculturedmousehippocampalneurons AT trinityerust protocolforcrisprbasedmanipulationandvisualizationofendogenousasynucleininculturedmousehippocampalneurons AT hannahobazick protocolforcrisprbasedmanipulationandvisualizationofendogenousasynucleininculturedmousehippocampalneurons AT jaredcarlsonstevermer protocolforcrisprbasedmanipulationandvisualizationofendogenousasynucleininculturedmousehippocampalneurons AT markjzylka protocolforcrisprbasedmanipulationandvisualizationofendogenousasynucleininculturedmousehippocampalneurons AT yukiogawa protocolforcrisprbasedmanipulationandvisualizationofendogenousasynucleininculturedmousehippocampalneurons AT subhojitroy protocolforcrisprbasedmanipulationandvisualizationofendogenousasynucleininculturedmousehippocampalneurons |