Efficient Mitochondrial Genome Editing by CRISPR/Cas9

Figure 4

MitoCas9-induced mtDNA damage leads to mitochondria dysfunction. (a) Quantification of copy numbers for ND1 region of mtDNA extracted from HEK-293T cells transiently transfected with indicated constructs (gRNA control, mitoCas9 and U6-sgRNA to eGFP; gRNA Cox1, Cox3, mitoCas9, and U6-sgRNA to Cox1 and Cox3), determined by real-time quantitative PCR using primers listed in Table 2 at the indicated time points (2 days and 5 days after transfection). GAPDH was used as an internal loading control ( per group). (b) Quantification of messenger RNAs for ND1, Cox1, and Cox2 genes in HEK-293T cells 5 days following transient transfection with lentiCRISPR-sgRNA-Cox1 + Cox3 or lentiCRISPR-sgRNA-eGFP#2 control determined by real-time PCR and normalized to GAPDH ( per group). (c) Representative images of mitotracker Red staining for functional mitochondria in HEK-293T cells transfected with the indicated constructs (upper panel). Quantification of relative mitotracker Red staining intensities in two groups was shown in the bottom panel ( mitochondria from three independent cultures per group). (d) Cell counts demonstrating proliferation rate of the HEK-293T cells following cleavage of specific mtDNA loci ( individual measurements per group). Days indicate the time intervals proceeded after cell seeding following 5 days of transient transfectin. Quantified data are expressed as mean ± s.e.m., , , and , unpaired two-tailed Student’s t-test.

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