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. Author manuscript; available in PMC: 2021 Jan 27.
Published in final edited form as: Cell Rep Phys Sci. 2020 Dec 9;1(12):100270. doi: 10.1016/j.xcrp.2020.100270

Figure 3. Perimitochondrial ENS Delivers CRISPR/Cas9 Components into the Mitochondria of Cancer Cells for Mitochondrial Genome Editing.

Figure 3.

(A) Fluorescent images of HeLa cells incubated with plasmid encoding CRISPR-Cas9-mCherry and siRNA for MT-CO1. The red fluorescence from mCherry in mitochondria suggests the gene delivery and expression in mitochondria.

(B) Western blot analysis of MT-CO1 for HeLa cells treated by solvent control (PBS), free CRISPR-MT-CO1 plasmid, Mito-FLAG, and the mixture of Mito-FLAG and CRISPR-MT-CO1 plasmid. The reduced level of MT-CO1 protein in the combination treatment suggest the KO of the MT-CO1 gene.

(C) PCR analysis of the MT-CO1 gene for HeLa cells treated by solvent control (PBS), free CRISPR-MT-CO1 plasmid, Mito-FLAG, and the mixture of Mito-FLAG and CRISPR-MT-CO1 plasmid. The band of MT-CO1 gene in the combination group is hardly observed.

(D) ATP (red) visualization in HeLa cells incubated with solvent control (PBS), free CRISPR-MT-CO1 plasmid, Mito-FLAG, and the mixture of Mito-FLAG and CRISPR-MT-CO1 plasmid. The cells in the combination group exhibit little fluorescence from ATP-Red in mitochondria, indicating the disfunction of OXPHOS (MT-CO1 is the catalytic site of cytochrome c oxidase).

(E–G) The KO of MT-CO1 (E) leads to reduced efficiency in MDR, (F) sensitizes the cancer cell to cisplatin, and (G) reduces mitochondrial membrane potential. Data are presented as mean ± standard deviation.