Targeted disruption of the Cep55 gene in mice, and phenotypes of the face and limb from Cep55^+/+ and Cep55^−/− mice, related to Fig. 1. (A) Generation of Cep55^−/− mice. Strategy used to target the mouse Cep55 locus with CRISPR-Cas9 system. Schematic representation of mouse Cep55 protein (1–462 aa) and Cep55 genomic locus. The correlation between Cep55 functional domains and their coding regions is also indicated (CC1, CC2, coiled-coil domain [green]; EABR [ESCRT and ALIX-binding domain, red]; and C-terminal region involved in localization [orange]). Modified Cep55 locus indicated excision of the genomic region from exon3 to exon5 and formation of a frameshift. Red arrows indicate PCR primers used to verify the null allele. UTR, untranslated region; CDS, coding sequence. (B) Genotype analysis of mouse tails from Cep55 wild-type, heterozygous, and null pups at P0.5. Primers F1 and R1 amplify a 300-bp fragment in Cep55^+/+ and Cep55^+/− pups. Primers F2 and R2 amplify a 244-bp fragment in Cep55^+/− and Cep55^−/− pups. (C and D) Immunoblot analysis of protein extracts from Cep55^+/+, Cep55^+/−, and Cep55^−/− primary MEFs by using anti-Cep55 antibodies produced by Cell Signaling Technologies (C) and Abnova (D). GAPDH was used as a loading control. (E and F) Sections of E14.5 faces (E) or E11.5 limbs (F) were stained with hematoxylin and eosin. Scale bars, 100 µm. (G and H) Sections of E14.5 faces (G) or E11.5 limbs (H) from Cep55^+/+ and Cep55^−/− mice were stained with a ciliary marker (ARL13B, red) and DNA (blue). Scale bars, 20 µm.