Generation and skeletal phenotype of Hgsnat ^P304L mice. (A) Schema showing the Cas9/sgRNA-targeting site in Hgsnat exon 9. The sgRNA-targeting sequence is underlined, and the protospacer-adjacent motif (PAM) sequence is shown in green. The c.911C>T mutation is shown in red and marked with an arrow. The C>T substitution disrupts the NcoI restriction site (shown in bold). The exon sequence is capitalized. (B) Sanger sequencing of single allele fragment obtained by PCR amplification of genomic DNA from the tail clips of the Hgsnat^P304L founder mouse showing the presence of the c.911C>T mutation. (C) Genotyping of Hgsnat^P304L mice. The DNA was extracted from clipped mouse tails and a 988-bp product amplified using a forward primer 5′-ATG​GAG​TGC​CTG​ATG​GGA​GG-3′ and a reverse primer 5′-GAT​CTA​GAA​ACG​GCC​CGA​AGA-3′. The PCR products were further digested with NcoI and analyzed on a 2% agarose gel. The 688- and 300-bp fragments are detected for the WT allele, and an undigested 988-bp fragment, for the targeted Hgsnat^P304L allele. (D) A 763-bp fragment of the Spg7 gene, containing the potential off-target sequence 5′-CTG​TGG​GAA​GAC​GCT​GTT​GGC​CA-3′, was amplified by PCR from DNA extracted from tail clips of Hgsnat^P304L founder mice (KI-1 and KI-2) and a control WT mouse. (E) Sanger sequencing of a PCR product confirms the absence of mutations in the Spg7 gene fragment adjacent to the 5′-CTG​TGG​GAA​GAC​GCT​GTT​GGC​CA-3′ fragment homologous to the PAM sequence. (F) A high-resolution in vivo micro-CT scanner (SkyScan 1176) was used to evaluate skeletal deformities in 4-mo-old Hgsnat-Geo and Hgsnat^P304L mice. The mice were anesthetized by isoflurane flow and the images were taken from the dorsal side. Both Hgsnat-Geo and Hgsnat^P304L mice do not develop abnormalities of skull bones. Panels show typical images of three mice analyzed per genotype.