Extended Data Fig. 6: Knock-in strategy to introduce the mutations into the endogenous mouse alleles of hs122 (uc468+uc469) and hs121 (uc467).

a, e, The mutant enhancer alleles were knocked-in via homologous recombination of the reference allele via a CRISPR/Cas9 microinjection protocol. A sgRNA for hs122 was designed to target the reference allele sequence that overlaps mutations introduced into the mutant allele. The target sequence of a sgRNA for hs121 was 115 bp upstream of the ultraconserved sequence, thus hs121 repair templates carried a point mutation altering the PAM sequence for the sgRNA (schematically indicated by the pink lines) to prevent their cleavage by the CRISPR/Cas9 complex. b, f, The genomic region between the Arx and Pola1 genes contains hs122, and hs121 is located in the first intron of the Pola1 gene. Conservation tracks (green) show phastCons scores between mouse and other vertebrate genomes. c, g, Positions of the ultraconserved sequences within enhancers (uc468+uc469 OR uc467), the sequences tested in the transgenic assay, the homologous recombination templates, and genotyping primers are shown schematically in black. Position of the sgRNA target sites shown in pink. d, h, Males hemizygous for the mutant alleles of the hs122 and hs121 enhancers were genotyped with PCR amplification (positions of genotyping primers shown as black triangles in c and g) and Sanger sequencing. Top: alignment of reads from the knock-in alleles and wild-type alleles to the reference mouse genome (mm10) is shown in grey, with mismatches indicating the location of the introduced mutations represented by red lines. Bottom: Sanger sequencing traces for mutant knock-in and wild-type alleles are shown for the region within the dotted lines. Introduced mutations are highlighted in red. See Supplementary Tables 4, 5 for more information on coordinates and sequences.