Fig. 3.

Generation of single-amino-acid-substituted mice at the Spp1 locus. (A) Schematic illustration to generate a three-base substituted allele at the Spp1 locus. A serine residue in exon 5 was replaced with an aspartic acid (TCA to GAC; red letters). A gRNA was designed to cut in the close vicinity of the serine residue (underlined in black and red). An ssODN was designed to carry the three-base substitution. A black box indicates the PAM sequence. Arrows indicate the primer sets for PCR. Blue underline indicates the recognition site of restriction enzymes in the RFLP analysis. (B,C) The sequencing analyses of Spp1-targeted mice. (A) Example of a three-base substitution detected with the wild-type allele. (B) Example of a three-base substitution detected with the wild-type allele and indel mutation. The target sequence of gRNA is indicated by black underline. The PAM sequence is enclosed in a black box.