LAUSR

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.