ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons
Fig 4
Validation of ORANGE labeling efficiency.
(A) Representative images of dendrites transfected with soluble GFP, PSD95-GFP knock-in (KI) construct, or a PSD95-GFP overexpression construct (green) stained with anti-PSD95 (magenta, Alexa568). DIV 21. Scale bar, 5 μm. (B) Correlation between PSD95-GFP KI and anti-PSD95 staining intensity. (C) Quantification of synaptic PSD95 levels, (D) synapse area, and I PSD95 synapse/shaft intensity. (F) Representative images of dendrites coexpressing Homer1c-mCherry (green) and either the empty pORANGE template vector or PSD95-GFP KI construct (blue) stained with anti-PSD95 (magenta, Alexa647). DIV 21. Scale bar, 5 μm. (G) Quantification of PSD95 levels in transfected but KI-negative neurons. Data are represented as means ± SEM. * P < 0.05, **P < 0.01, *** P < 0.001, ANOVA or Student t test. Underlying data can be found in S1 Data. DIV, day in vitro; GFP, green fluorescent protein; HA, hemagglutinin; KI, knock-in; ns, not significant; OE, overexpression; ORANGE, Open Resource for the Application of Neuronal Genome Editing; PSD95, postsynaptic protein 95; RIM1, Rab3-interacting molecule 1.