Figure 1
From: Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system
Introduction of the C>T substitution in Sf-1 locus.
(A) A schematic illustration showing the locations of the gRNAs and ssODNs, along with the mouse Sf-1 locus. Blue bars and letters indicate the position of the gRNA targets with red bars highlight the PAM sequences. ssODNs are shown in green letters at the corresponding position to the Sf-1 locus. Red letters indicate the substitution target site in the Sf-1 locus and corresponding mismatched nucleotides in the ssODNs. The deduced amino acid sequences from wild type (WT) and substituted sequences are shown at the bottom, with red letters indicating the target and the results of the substitution. (B) The results of the sequence analysis of the Sf-1 locus of a representative mouse, with a monoallelic C>T substitution. The Sf-1 locus around the gRNA target site was PCR amplified and directly sequenced (Upper panel), or cloned into the plasmid and sequenced independently (Lower panel). The upper panel shows the electropherogram of the direct sequencing results, in which one allele received a C>T substitution. WT and donor DNA sequences are shown on the top, with red letters indicating the mutated nucleotide. The red arrow indicates the overlapping peak caused by the monoallelic substitution. The lower panel shows the sequence alignment of eight independent clones. The eight sequences are separated into two types: the WT sequence and substituted sequences. (C) The results of the direct sequencing of the Sf-1 locus around the gRNA target site of another representative mouse obtained in experiment 4 shown in Table 1, in which one alleles received C>T substitutions, followed by additional mutation(s) that caused continuous overlapping peaks.
