Extended Data Fig. 5: Scn2a-CRISPRa off-target analysis in the mouse neocortex.
From: CRISPR activation for SCN2A-related neurodevelopmental disorders
a. RNA-seq expression levels of Scn2a TAD-domain genes from Scn2a-rAAV-CRISPRa treated Neuro-2a cells compared to VP64-only. Circles are individual mice, box plots are medians, quartiles and 100% tails. b. RNA-seq expression levels of sodium channels from Scn2a-rAAV-CRISPRa treated Neuro-2a cells compared to VP64-only. Circles are individual mice, box plots are medians, quartiles and 100% tails. c. Volcano plot representing Log2 fold-change in expression levels for each gene for WT vs Scn2a+/− “Het”. Wald test. P-values noted above each comparison. Grey dots represent no significant DEGs for CRISPRa, purple dots signify Scn2a and nearby genes, and orange dots denote upregulated genes. d. Same as c, but for CRISPRa-treated Scn2a+/− “CRISPRa” vs WT. e. Same as c, but for CRISPRa-treated Scn2a+/− “CRISPRa” vs Scn2a+/− “Het”. f. Analysis of sgRNA sequence off-targeting using Cas-OFFinder. g. Data representation of NeuN-positive neuronal nuclei isolated from cortical tissue for FACS sorting. Representative FACS plots visualized with FlowJo V10 with percentage of parent gates for each population. Singlets were selected based on FSC-H versus FSC-A. h. From singlets, DAPI-positive events were gated to identify nuclei. i. NeuN-positive neuronal nuclei were selected using an anti-NeuN antibody conjugated to Alexa Fluor 488.
