(A) Schematic representation of IUE and the CRISPR validation methods. FACS-sorted GFP-labeled cells are processed for next-generation sequencing to identify specific mutations. Brain sections are immunostained to visualize the in vivo qualitative change in αII spectrin immunostaining. Immunostaining on dissociated cortical neurons is performed to quantify the efficiency of Sptan1 CRISPR knockout. (B) Representative confocal image from a P1 brain section after prior transfection with Sptan1 CRISPR A at E13–14. Sptan1 CRISPR–transfected cells are marked by GFP (green) in the cerebral cortex (CTX, between the dashed lines). These cells also project axons to the contralateral hemisphere through white matter (WM). Red, αII spectrin; blue, bisbenzimide (BB) nuclear stain. (C) The area outlined by the dotted square (same image as in B) with a high density of GFP labeling has a marked reduction in αII spectrin immunoreactivity. Another area with a cluster of GFP⁺ cells in the WM (arrow) also demonstrates a clear lack of αII spectrin staining compared with the surrounding nontransfected (GFP^–) area. Note that these regions do not have homogeneous loss of αII spectrin owing to the mosaic mutation introduced by CRISPR IUE. (D) Quantification of fluorescence intensity across the transfected regions (yellow box) shows that the intensities of αII spectrin and GFP signals are inversely correlated. (E) Representative confocal image of αII spectrin (magenta) and RFP expression (white) in rat cortical neurons dissociated from brains 4 days after IUE with Sptan1 CRISPR gRNA and RFP (to label the cells) at E14–15, then cultured for 10 days. Three RFP⁺ cells lack αII spectrin immunoreactivity, whereas other RFP^– cells are αII spectrin positive. Scale bars: 100 μm in B (for B–D), 50 μm in E.