CRISPR/Cas9 system-mediated impairment of synaptobrevin/VAMP function in postmitotic hippocampal neurons
Patricia M Horvath1, Ege T Kavalali2, Lisa M Monteggia1
1Department of Neuroscience, UT Southwestern Medical Center, Dallas, TX 75390, USA.
Background:
The use of the CRISPR/Cas9 system is becoming widespread, however current studies have predominantly focused on dividing cells. It is currently unknown if CRISPR/Cas9 can be used in a postmitotic setting to examine non-cell autonomous/presynaptic phenotypes in the resulting genetically heterogeneous cell population.
New Method:
A single CRISPR/Cas9 lentivirus was used to transfect a high percentage of primary cultured neurons and target synaptobrevin 2 (Syb2, also called VAMP2).
Results:
Primary hippocampal cultures infected with the Syb2 targeting virus displayed dramatic reductions in Syb2 protein and immunocytochemical staining. In many boutons Syb2 was completely undetected. These cultures recapitulated the known functional phenotypes of Syb2 knockout neurons, which are non-cell autonomous and presynaptic in origin, indicating that Syb2 was knocked out in a large fraction of neurons.
Comparison With Existing Method(S):
Previous methods used multiple viruses or sparse transfection methods and only examined cell autonomous or postsynaptic phenotypes. The current method demonstrates that the CRISPR/Cas9 system can be used to alter network dynamics by removing or lowering the target gene from a majority of cells in the culture.
Conclusions:
A combination of CRISPR/Cas9 system and single high efficiency lentivirus infection can be used to examine non-cell autonomous and presynaptic phenotypes in postmitotic neurons.
Insights
CRISPR/Cas9 gene editing effectively targets synaptobrevin 2 in postmitotic neurons. This method enables the study of presynaptic phenotypes in non-dividing cells, advancing neuroscience research.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- CRISPR/Cas9 applications are expanding, but primarily studied in dividing cells.
- Its utility in postmitotic (non-dividing) cells for studying presynaptic phenotypes remains largely unexplored.
Purpose of the Study:
- To investigate the efficacy of CRISPR/Cas9 in postmitotic neurons.
- To assess the system's ability to examine non-cell autonomous and presynaptic phenotypes.
Main Methods:
- Utilized a single CRISPR/Cas9 lentivirus for high-efficiency transfection of primary cultured neurons.
- Targeted the synaptobrevin 2 (Syb2) gene, also known as VAMP2.
Main Results:
- Achieved significant reductions in Syb2 protein levels and staining in targeted neurons.
- Observed complete Syb2 absence in numerous boutons, mirroring Syb2 knockout phenotypes.
- Demonstrated non-cell autonomous and presynaptic functional consequences.
Conclusions:
- CRISPR/Cas9 combined with lentiviral delivery is effective in postmitotic neurons.
- This approach allows for the study of non-cell autonomous and presynaptic phenotypes.
- The method enables gene alteration in a majority of cells within a culture.
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