Extended Data Fig. 8: Validation of Spen and Mink1 knockdown.

a, Schematics of SPEN and MINK1 protein with domains annotated and patient mutations. RRM, RNA Recognition motif. MINT, Mxs2-interacting protein. SPOC, Spen paralogue and orthologue SPOC. CNH, Citron homology domain. b, Dorsal views of Xenopus laevis embryos subjected to in situ hybridization for Pax3 to visualize the neural folds in Spen morphants. c, RT-PCR confirmed that Spen MO reduced the amount of normally spliced Spen transcript. d. Dorsal views of Xenopus l. embryos subjected to in situ hybridization for Pax3 in Mink1 morphants. e, Validation of Mink1 morpholinos by RT-PCR. c,e, Each experiment was performed independently at least twice with similar results. f, Dorsal views of embryos injected with Spen gRNAs only or gRNAs with Cas9, with the accompanying chromatogram showing Sanger sequencing at the CRISPR target site. Control embryos injected with gRNAs only (#1 and #2) developed normally and exhibited an intact sequence, while embryos injected with Spen gRNAs and Cas9 (#3-#6) displayed neural tube defects and mosaic mutations at the CRISPR target site. g, Dorsal views of embryos injected with Mink1 gRNAs only or Mink1 gRNAs with Cas9, with the accompanying chromatogram showing Sanger sequencing at the CRISPR target site. Mink1 crispants (#2-#4) exhibited neural tube defect phenotypes and mosaic mutations at the CRISPR target site in both the L and S alleles of Mink1.