Fig. 5

Generation of Cars2-deficient mice via the CRISPR/CAS9 system. a Schematic illustration of the mouse Cars2 gene structure and sequences of WT and mutant alleles around the target locus. Green and black letters indicate the first exon and intron of Cars2, respectively. The targeted locus of gRNA and protospacer-adjacent motif (PAM) sequence were indicated in the WT sequence are indicated by underlined and bold letters, respectively. A modified allele sequence obtained from the Cars2-edited mouse (line 1) is shown below. b Detection of mutations introduced by gRNA-Cas9 targeting Cars2 via PCR with genomic DNA from WT and Cars2 ^+/− mice. Cars2 ^+/−, Cars2 heterozygous KO mice, M: DNA molecular weight marker. c Western blotting of CARS2 and mitochondrial proteins, e.g., MTCO1 and SDHA, from mitochondria isolated from the liver. The lower panel shows the densitometric analysis of the western blot. Data are means ± s.d. (n = 3). ***P < 0.001. d CysSSH production in mitochondria isolated from the liver of WT and Cars2 ^+/− littermate mice. Various concentrations of isolated mitochondria were reacted with HPE-IAM for 1 h, followed by LC-MS/MS analysis (see Supplementary Methods for details). Mitochondria were obtained from line 2 Cars2 ^+/– mice (Supplementary Figs. 20 and 21). *P < 0.05, WT vs. Cars2 ^+/– mice (two-way ANOVA). e Western blotting of CARS1, CSE, CBS, and 3-MST with liver tissue obtained from WT and Cars2 ^+/– mice. Supplementary Fig. 19 provides full blot images. The right panels show the densitometric analysis of the CARS1 and CARS2 immunoblots. Data are means ± s.d. (n = 3). ***P < 0.001