Fig 6. Generation of endogenous mouse renin mutant alleles by genome editing.

(A) Schematic representation of the 156-kb mRen Tg, endogenous 5'-large-del and endogenous pseudo-WT alleles. Positions of mdE and FRT sequences are indicated by gray rectangles. (B) Partial restriction enzyme maps of the mouse endogenous WT and mutant alleles. The Cas9 target sites for generating 5'-large-del and pseudo-WT alleles are shown by solid and open arrowheads, respectively. Targeting at two upstream sites removes 63-kb sequence from the 5'-upstream region of mRen gene, generating a 2.4-kb EcoRV restriction fragment in the mutant allele. Knock-in of the FRT sequence at a downstream site introduces artificial PvuII site, generating 4.0- and 3.6-kb PvuII restriction fragments in the mutant allele. Probes used for Southern blot analysis in C are indicated by gray rectangles. (C) DNAs of WT and mutant animals were digested with EcoRV or PvuII, separated by electrophoresis, and hybridized to the probes shown in B. Shown on the right of each panel are expected bands with their sizes (in kb). (D) Sequence alignment of WT (reference) and 5'-large-del alleles confirmed the 63-kb sequence deletion in the 5'-large-del allele of mRen gene. PAM and g(uide)RNA sequences are shaded and underlined, respectively. Cleavage sites predicted from PAM locations are indicated by arrowheads.