Extended Data Fig. 9: Characterization and validation of in vivo CRISPR–Cas9 screening reveals key genes contributing to TRN bursting firing properties.

a, Representative rebound burst traces of recorded neurons after knocking out different individual genes from Pool3 via CRISPR–Cas9 gene editing. Knocking out of Kcnd2 recapitulates the effects of Pool#3. b, Radar plots for Pool3 individual gene. Top: Changes in Spp1⁺ like (‘Spp1’) neurons, pink line showing the effect of the Pool3 gene knock out and colour shades showing the effect produced by individual gene knock out. Bottom: Changes in Ecel1⁺ like (‘Ecel1’) neurons, green line showing the Pool3 gene radar plot and colour shades showing the changes produced by individual gene knockout. Kcnd2 knockout closely recapitulates the effect of Pool3 in both populations. c, Summary of the maximum number of rebound bursts of TRN neurons elicited by comparable protocols after individual genes from Pool3 were knockout in Spp1⁺ like (‘Spp1’) vs Ecel1⁺ like (‘Ecel1’) neurons (‘Spp1’ Kcnd2, P = 0.0095. ‘Ecel1’ Kcng1, P = 0.0088; Kcnd2, P = 0.019, two-sided unpaired t-test). Bars represent the mean ± s.e.m. For a−c, ‘Spp1’ Kcng1 n = 6, Kcnc3 n = 6, Kcng4 n = 5, Kcnip1 n = 7, Kcnd2 n = 7; ‘Ecel1’ Kcng1 n = 10, Kcnc3 n = 8, Kcng4 n = 9, Kcnip1 n = 11, Kcnd2 n = 11. d, Schematics of the analysis for on-target and off-target efficiency. Upper: analysis flowchart. WGA: whole genome amplification; NGS: next generation sequencing. Lower: schematics of sgRNA design and primers for on-target analysis for Kcnd2 knockout. Five sgRNA were designed in Exon2, Exon3, and Exon4. As the length spanned by the leftmost sgRNA and the rightmost sgRNA exceeds the NGS analysis limit, nested PCR combined with Sanger sequencing was used for on-target efficiency analysis. Primers for the nested PCR are shown as black arrows in Exon1 and Exon5. e, Bar chart showing the on-target efficiency (5 sgRNA pooled) analysed by nested PCR and Sanger sequencing (control: n = 96 nuclei, viral injected: n = 384 nuclei) and off-target rate for the top predicted (Methods) off-target loci of each sgRNA analysed by NGS (n = 1,600 cells and 72,000 nuclei). Predicted off-target sequences are shown with mismatched bases in lower case. Bar plots represent maximum likelihood estimation (MLE) and upper Wilson score intervals, no raw data point applicable⁶⁴ (Supplementary Information).