Plasmid construction

Single-guide RNAs targeting the human SOX10 locus (exon 1 or exon 2; Fig. ​Fig.1A)1A) were designed using E-CRISP tools. Three pairs of annealed guide oligos were chosen and cloned into the CRISPR-Cas9 expression vector pSpCas9 (BB)-2A-GFP (PX458) (Addgene, Cambridge, MA, USA; #48138) by T4 DNA ligase (TaKaRa Bio, Kusatsu, Japan). To evaluate the targeting efficiency, three different plasmids were separately transfected into 293FT cells using Lipofectamine 2000 transfection reagent (Invitrogen). Two days after transfection, genomic DNA was extracted from transfected 293FT cells, and the targeting efficiency was verified by a T7EN1 assay (New England Biolabs, Ipswich, MA, USA) and genome sequencing. The most efficient sgRNA (sgRNA2) was selected for SOX10 targeting in hiPSCs.

A Schematic diagram of sgRNAs targeting at exons 1 and 2 of the human SOX10 locus. B Detection of sgRNA:Cas9-mediated cleavage of SOX10 by PCR and T7EN1 cleavage assay (M, DNA marker). C Sanger sequencing was performed on PCR products amplifed from SOX10-knockout hiPSCs. Deletions (−). N/N indicates positive colonies out of total sequenced. WT wild-type sample, KO1 and KO2 the two mutant samples. D The protein expression level of SOX10 in WT and KO cells was detected by western blotting. E The protein expression level of SOX10 in WT and KO cells was further confirmed by immunofluorescence assay. Scale bar, 50 μm. F Cell morphology was observed by phase-contrast microscope. Scale bar, 250 μm.