Neuronal Cultures for CRISPR Screening

Primary mouse neuronal cultures were prepared as described^{3 (link),26 ,29} . Briefly, cortical neurons were dissected from E15.5 Ube3a^m+/pYFP embryos and plated in 384 well poly-D lysine coated plates. On days in vitro (DIV)3, each well was transfected with 50 ng CamKIIα:tdTomato and 50 ng of lentiCRISPR:gRNA plasmid using Lipofectamine 2000 (ThermoFisher). Each gRNA was transfected in quadruplicate. On DIV10, cells were fixed with 4% phosphate-buffered paraformaldehyde (PFA), and stained with primary rabbit anti-GFP antibody (Novus NB600–308), secondary donkey anti-rabbit IgG alexa 647 (ThermoFisher A31573) and 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired using the GE IN CELL Analyzer 2200 high-content imager. YFP expression was quantified in tdTomato⁺ nuclei using a custom Cell Profiler pipeline. The screen was performed in triplicate, presented as the average of the three replicates. All subsequent experiments using mouse primary cortical neuron cultures followed the same culture protocol and timeline.