Fig. 2: CRISPR-mediated conversion of Lphn3 alternative splicing from E31 to E32 impairs neuronal network activity.

a, The CRISPR strategy to produce either an acute deletion of Lphn3 expression (Lphn3 knockout (KO)) or a selective deletion of Lphn3 E31 (E31 KO) of Lphn3. b, LPHN3 immunoblots from hippocampal neurons showing that the Lphn3-E31-specific KO does not change LPHN3 protein levels, whereas Lphn3 KO ablates LPHN3 expression. Statistical analysis from n = 6 independent cultures was performed using two-sided t-tests. c, RT–qPCR analysis demonstrating that the Lphn3-E31 KO and the Lphn3 KO similarly ablate the expression of E31-containing Lphn3 mRNAs but have the opposite effect on E32-containing Lphn3 mRNAs. Statistical analysis from n = 8 independent cultures was performed using two-sided t-tests. d, RNA-seq analyses of differentially expressed genes, comparing Lphn3 KO with the control (left) or Lphn3-E31-specific KO with the control (right). Statistical analysis from n = 3 biologically independent cultures was performed using two-sided Wald tests (in DESeq2); P values are shown. e, Representative Ca²⁺ imaging experiment of hippocampal neurons expressing gRNA, eGFP and jRGECO1a³⁸. Cells expressing the gRNA with nuclear eGFP are shown in green. Red fluorescence from the soma corresponds to the jRGECO1a peak signal. Scale bar, 20 μm. f, Representative illustration of the extraction of jRGECO1a signals (Ca²⁺ imaging traces) from individual neurons (grey, top) of which the average is the synchronous firing trace for one field of view (orange, bottom). g, Representative traces of synchronous firing in control, Lphn3-KO and Lphn3-E31-KO neurons. h, Quantification of the synchronous firing rate (left) and amplitude (right). Statistical analysis from n = 3 independent cultures was performed using two-sided t-tests. For b, c and h, statistically significant P values are shown (P < 0.05).