Figure 4. CRISPR base editing to generate ATP5G1^L32P AGS NPCs results in a partial loss of AGS metabolic resilient phenotypes.

(A) AGS ATP5G1 CRISPR base editing strategy. To create AGS cells with the human amino acid substitution at leucine-32, AGS cells transiently expressing ABEmax were nucleofected with an sgRNA (blue underline) directed toward a PAM site (green underline) on the (-) strand to target conversion of adenine to guanine, which on the (+) strand is a cytosine-to-thymine (*). (B) Sequencing data from a successfully edited clonal AGS cell line demonstrating the cytosine-to- thymine base edit resulting in the desired leucine to proline knock-in cell line. (C) AGS ATP5G1^L32P (ABE KI) NPCs exhibit decreased cell survival compared to unedited AGS cells (ABE WT) when exposed to hypoxia (1%, 24 hr), hypothermia (31°C, 72 hr), or rotenone (10 μM, 16 hr). Bar graphs represent the mean ± SEM of three independent experiments with three replicates/condition. (D) Seahorse XF analyzer assay of cultured ABE KI and WT cells sequentially exposed to (i) oligomycin (1 μM), (ii) FCCP (2 μM), and (iii) rotenone/antimycin (0.5 μM) showing enhanced FCCP-stimulated oxygen consumption (spare respiratory capacity). Data represents the mean ± SEM of 3 independent experiments with 4–6 replicates/species. (E and F) Representative confocal images of ABE WT (E) and ABE KI (F) NPCs expressing the mitochondrial marker mCherry-mito7 to demonstrate mitochondrial morphology one hour following treatment with FCCP. Scale bar represents 5 μm. (G) Percent of mitochondria ± SEM with fragmented morphology, data obtained from 50 to 60 cells/genotype. (H) Relative fluorescence ± SEM of 3 independent experiments in triplicate each of cultured ABE AGS NPCs loaded with TMRE (50 nM) and exposed to vehicle or FCCP (1 μM). (I) Complex V enzymatic activity normalized to protein content of ABE WT and KI mitochondrial extracts normalized to protein content and treated with vehicle or oligomycin (1 μM). Data are the mean ± SEM of 3 independent experiments expressed as a fraction of ABE WT enzymatic activity. (J) Representative immunoblots for ATP5G (left), ATP5A (right), or citrate synthase (CS, left, input control) of clear-native gel electrophoresis of mitochondrial extracts from ABE WT and ABE demonstrate ATP synthase dimers (D) and monomers (M). (K) Quantification of ATP5G demonstrates a reduction in ATP synthase dimers relative to total ATP synthase protein (D:(D+M) ratio) in ABE KI. Data are mean ± SEM of 3 independent blots. *p<0.05; **p<0.01.

Figure 4—figure supplement 1. Additional data for ABE cell lines.

(A) Sequencing data from an unsuccessfully edited clonal AGS cell line demonstrating the preservation of the wild-type sequence (* indicates wild-type thymidine). (B) qRT-PCR for ATP5G1, ATP5G2, and ATP5G3 demonstrating the same relative abundance of ATP5G1 in ABE WT and ABE KI NPCs. Data are means ± SEM from three independent experiments performed in triplicate. (C) Mean branch length of mitochondrial networks of ABE AGS NPCs expressing mCherry-mito7. Data obtained from 30 cells/genotype. (D) Representative western blot images and (E) quantification demonstrating the relative abundance of ATP5G and ATP5A proteins are similar in ABE WT and ABE KI cells. Quantification of western blots from four independent experiments with 2–3 replicates each. **p<0.01.