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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

Alterations in mitochondria-associated proteins following CRISPR/Cas9-mediated mtDNA editing. (a) Mitochondrial proteins in HEK-293T cells after CRISPR/Cas9-mediated cleavage of mtDNA at Cox1 and Cox3 loci as determined by Western blots using indicated antibodies. β-actin was used as a loading control. (b) Quantification of mitochondria proteins in HEK-293T cells transfected with lentiCRISPR-sgRNA-Cox1 + Cox3 or lentiCRISPR-sgRNA-eGFP#2 control as shown in panel (a) normalized to β-actin. (c) Illustration of human mtDNA. Specific loci targeted by lentiCRISPR-sgRNAs (Cox1, Cox2, Cox3, and ATP8/6) are indicated with filled triangles. (d) Representative Western blots showing differential alteration of mitochondrial proteins following cleavage of specific mtDNA loci mediated by indicated sgRNAs in HEK-293T cells. Quantified data (b) are expressed as mean ± s.e.m., ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, unpaired two-tailed Student's t-test.
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fig2: Alterations in mitochondria-associated proteins following CRISPR/Cas9-mediated mtDNA editing. (a) Mitochondrial proteins in HEK-293T cells after CRISPR/Cas9-mediated cleavage of mtDNA at Cox1 and Cox3 loci as determined by Western blots using indicated antibodies. β-actin was used as a loading control. (b) Quantification of mitochondria proteins in HEK-293T cells transfected with lentiCRISPR-sgRNA-Cox1 + Cox3 or lentiCRISPR-sgRNA-eGFP#2 control as shown in panel (a) normalized to β-actin. (c) Illustration of human mtDNA. Specific loci targeted by lentiCRISPR-sgRNAs (Cox1, Cox2, Cox3, and ATP8/6) are indicated with filled triangles. (d) Representative Western blots showing differential alteration of mitochondrial proteins following cleavage of specific mtDNA loci mediated by indicated sgRNAs in HEK-293T cells. Quantified data (b) 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: Next, we examined alterations in mitochondria-associated proteins as an indication of disturbance of mitochondria that could be induced by CRISPR/Cas9-mediated mtDNA cleavage. When mtDNA was truncated at the Cox1 and Cox3 loci by CRISPR/Cas9, transcription of mtDNA heavy strands and thus expression of downstream genes from Cox1 locus could be affected (Figure 1(b)). When we monitored several mitochondrial marker proteins, there were alterations in several proteins: the levels of SDHA, heat shock protein 60 (HSP60), and prohibitin 1 (PHB1) decreased, whereas pyruvate dehydrogenase (PDH) and superoxide dismutase 1 (SOD1) levels increased (Figures 2(a) and 2(b)). However, no significant changes were observed in CoxIV or Cytochrome C (CytC) (Figures 2(a) and 2(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)

Alterations in mitochondria-associated proteins following CRISPR/Cas9-mediated mtDNA editing. (a) Mitochondrial proteins in HEK-293T cells after CRISPR/Cas9-mediated cleavage of mtDNA at Cox1 and Cox3 loci as determined by Western blots using indicated antibodies. β-actin was used as a loading control. (b) Quantification of mitochondria proteins in HEK-293T cells transfected with lentiCRISPR-sgRNA-Cox1 + Cox3 or lentiCRISPR-sgRNA-eGFP#2 control as shown in panel (a) normalized to β-actin. (c) Illustration of human mtDNA. Specific loci targeted by lentiCRISPR-sgRNAs (Cox1, Cox2, Cox3, and ATP8/6) are indicated with filled triangles. (d) Representative Western blots showing differential alteration of mitochondrial proteins following cleavage of specific mtDNA loci mediated by indicated sgRNAs in HEK-293T cells. Quantified data (b) 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

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fig2: Alterations in mitochondria-associated proteins following CRISPR/Cas9-mediated mtDNA editing. (a) Mitochondrial proteins in HEK-293T cells after CRISPR/Cas9-mediated cleavage of mtDNA at Cox1 and Cox3 loci as determined by Western blots using indicated antibodies. β-actin was used as a loading control. (b) Quantification of mitochondria proteins in HEK-293T cells transfected with lentiCRISPR-sgRNA-Cox1 + Cox3 or lentiCRISPR-sgRNA-eGFP#2 control as shown in panel (a) normalized to β-actin. (c) Illustration of human mtDNA. Specific loci targeted by lentiCRISPR-sgRNAs (Cox1, Cox2, Cox3, and ATP8/6) are indicated with filled triangles. (d) Representative Western blots showing differential alteration of mitochondrial proteins following cleavage of specific mtDNA loci mediated by indicated sgRNAs in HEK-293T cells. Quantified data (b) 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: Next, we examined alterations in mitochondria-associated proteins as an indication of disturbance of mitochondria that could be induced by CRISPR/Cas9-mediated mtDNA cleavage. When mtDNA was truncated at the Cox1 and Cox3 loci by CRISPR/Cas9, transcription of mtDNA heavy strands and thus expression of downstream genes from Cox1 locus could be affected (Figure 1(b)). When we monitored several mitochondrial marker proteins, there were alterations in several proteins: the levels of SDHA, heat shock protein 60 (HSP60), and prohibitin 1 (PHB1) decreased, whereas pyruvate dehydrogenase (PDH) and superoxide dismutase 1 (SOD1) levels increased (Figures 2(a) and 2(b)). However, no significant changes were observed in CoxIV or Cytochrome C (CytC) (Figures 2(a) and 2(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