Limits...
Efficient Mitochondrial Genome Editing by CRISPR/Cas9.

Jo A, Ham S, Lee GH, Lee YI, Kim S, Lee YS, Shin JH, Lee Y - Biomed Res Int (2015)

Bottom Line: MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition.In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9.Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmacology, Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi-do 440-746, Republic of Korea.

ABSTRACT
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been widely used for nuclear DNA editing to generate mutations or correct specific disease alleles. Despite its flexible application, it has not been determined if CRISPR/Cas9, originally identified as a bacterial defense system against virus, can be targeted to mitochondria for mtDNA editing. Here, we show that regular FLAG-Cas9 can localize to mitochondria to edit mitochondrial DNA with sgRNAs targeting specific loci of the mitochondrial genome. Expression of FLAG-Cas9 together with gRNA targeting Cox1 and Cox3 leads to cleavage of the specific mtDNA loci. In addition, we observed disruption of mitochondrial protein homeostasis following mtDNA truncation or cleavage by CRISPR/Cas9. To overcome nonspecific distribution of FLAG-Cas9, we also created a mitochondria-targeted Cas9 (mitoCas9). This new version of Cas9 localizes only to mitochondria; together with expression of gRNA targeting mtDNA, there is specific cleavage of mtDNA. MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition. This mitoCas9 could be applied to edit mtDNA together with gRNA expression vectors without affecting genomic DNA. In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9. Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing.

No MeSH data available.


Related in: MedlinePlus

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 (n = 3 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 (n = 3 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 (n = 50 mitochondria from three independent cultures per group). (d) Cell counts demonstrating proliferation rate of the HEK-293T cells following cleavage of specific mtDNA loci (n = 6 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., ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, unpaired two-tailed Student's t-test.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4581504&req=5

fig4: 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 (n = 3 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 (n = 3 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 (n = 50 mitochondria from three independent cultures per group). (d) Cell counts demonstrating proliferation rate of the HEK-293T cells following cleavage of specific mtDNA loci (n = 6 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., ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, unpaired two-tailed Student's t-test.

Mentions: Mitochondria targeting of certain restriction enzymes such as EcoRI or PstI has been employed to destroy mitochondrial DNA [13, 14] together with chemical induction of mtDNA elimination. To determine whether mitoCas9-mediated site-specific mtDNA cleavage leads to functional disruption of mitochondria, we first examined mtDNA copy number and several mitochondrial gene expressions. 5 days following transient transfection of HEK-293T cells with mitoCas9 and sgRNA targeting Cox1 and Cox3 regions, real-time PCR quantification of mtDNA copy number was performed using ND1 primers. Cell division for 5 days with site-specific mitoCas9 cleavage at Cox1 and Cox3 regions leads to approximate 30% reduction of mtDNA copy numbers (Figure 4(a)). Consistent with the fact that mtDNA was cleaved and steady state mtDNA copy number was reduced by CRISPR/Cas9 system, the amounts of messenger RNAs encoding mitochondrial ND1, Cox1, and Cox2 genes were reduced by about 50% compared to those of sgRNA control HEK-293T cells (Figure 4(b)).


Efficient Mitochondrial Genome Editing by CRISPR/Cas9.

Jo A, Ham S, Lee GH, Lee YI, Kim S, Lee YS, Shin JH, Lee Y - Biomed Res Int (2015)

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 (n = 3 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 (n = 3 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 (n = 50 mitochondria from three independent cultures per group). (d) Cell counts demonstrating proliferation rate of the HEK-293T cells following cleavage of specific mtDNA loci (n = 6 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., ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, unpaired two-tailed Student's t-test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581504&req=5

fig4: 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 (n = 3 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 (n = 3 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 (n = 50 mitochondria from three independent cultures per group). (d) Cell counts demonstrating proliferation rate of the HEK-293T cells following cleavage of specific mtDNA loci (n = 6 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., ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, unpaired two-tailed Student's t-test.
Mentions: Mitochondria targeting of certain restriction enzymes such as EcoRI or PstI has been employed to destroy mitochondrial DNA [13, 14] together with chemical induction of mtDNA elimination. To determine whether mitoCas9-mediated site-specific mtDNA cleavage leads to functional disruption of mitochondria, we first examined mtDNA copy number and several mitochondrial gene expressions. 5 days following transient transfection of HEK-293T cells with mitoCas9 and sgRNA targeting Cox1 and Cox3 regions, real-time PCR quantification of mtDNA copy number was performed using ND1 primers. Cell division for 5 days with site-specific mitoCas9 cleavage at Cox1 and Cox3 regions leads to approximate 30% reduction of mtDNA copy numbers (Figure 4(a)). Consistent with the fact that mtDNA was cleaved and steady state mtDNA copy number was reduced by CRISPR/Cas9 system, the amounts of messenger RNAs encoding mitochondrial ND1, Cox1, and Cox2 genes were reduced by about 50% compared to those of sgRNA control HEK-293T cells (Figure 4(b)).

Bottom Line: MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition.In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9.Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmacology, Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi-do 440-746, Republic of Korea.

ABSTRACT
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been widely used for nuclear DNA editing to generate mutations or correct specific disease alleles. Despite its flexible application, it has not been determined if CRISPR/Cas9, originally identified as a bacterial defense system against virus, can be targeted to mitochondria for mtDNA editing. Here, we show that regular FLAG-Cas9 can localize to mitochondria to edit mitochondrial DNA with sgRNAs targeting specific loci of the mitochondrial genome. Expression of FLAG-Cas9 together with gRNA targeting Cox1 and Cox3 leads to cleavage of the specific mtDNA loci. In addition, we observed disruption of mitochondrial protein homeostasis following mtDNA truncation or cleavage by CRISPR/Cas9. To overcome nonspecific distribution of FLAG-Cas9, we also created a mitochondria-targeted Cas9 (mitoCas9). This new version of Cas9 localizes only to mitochondria; together with expression of gRNA targeting mtDNA, there is specific cleavage of mtDNA. MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition. This mitoCas9 could be applied to edit mtDNA together with gRNA expression vectors without affecting genomic DNA. In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9. Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing.

No MeSH data available.


Related in: MedlinePlus