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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

Construction of mitochondria-targeted MTS-HA-Cas9. (a) Schematic illustration of mitochondria-targeting Cas9 (mitoCas9). Mitochondria-targeting sequence (MTS) and HA tag information are presented. (b) Expression of mitoCas9 in HEK-293T cells transiently transfected with MTS-HA-Cas9 construct determined by Western blots using HA antibody. β-actin was used as a loading control. (c) Subcellular localization of MTS-HA-Cas9 assessed in the cytosolic (Cyt), mitochondrial (Mit), and nuclear (Nu) fractions of HEK-293T cells transfected with lentiCRISPR-sgRNA-eGFP#2 and monitored by Western blot. GAPDH served as a cytosolic marker, PARP1 served as a nuclear marker, and SDHA served as a mitochondrial marker. (d) Representative immunofluorescence microscopic image of MTS-HA-Cas9 (HA, green) and CoxIV (red) subcellular distributions in HEK-293T cells transfected with MTS-HA-Cas9 construct. The merged image (yellow, right panel) shows colocalization of mitoCas9 and CoxIV. (e) Representative gel images of the PCR product of hU6-sgRMA-eGFP and hU6-sgRNA-Cox1 which were purified by gel extraction (bottom panel). Schematics of primers and lentiCRISPR-sgRNA templates used to amplify U6 promoter and respective sgRNA components for transfection (upper panel). (f) Quantification of copy numbers for Cox1, Cox3, and ND1 regions of mtDNA extracted from HEK-293T cells transiently transfected with indicated constructs (mitoCas9 is a plasmid, while U6-sgRNAs are PCR product.), determined by real-time quantitative PCR using primers listed in Table 2. GAPDH was used as an internal loading control (n = 3 per group). Quantified data (b) are expressed as mean ± s.e.m., ∗∗∗P < 0.001, analysis of variance (ANOVA) test followed by Student-Newman-Keuls post hoc analysis.
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fig3: Construction of mitochondria-targeted MTS-HA-Cas9. (a) Schematic illustration of mitochondria-targeting Cas9 (mitoCas9). Mitochondria-targeting sequence (MTS) and HA tag information are presented. (b) Expression of mitoCas9 in HEK-293T cells transiently transfected with MTS-HA-Cas9 construct determined by Western blots using HA antibody. β-actin was used as a loading control. (c) Subcellular localization of MTS-HA-Cas9 assessed in the cytosolic (Cyt), mitochondrial (Mit), and nuclear (Nu) fractions of HEK-293T cells transfected with lentiCRISPR-sgRNA-eGFP#2 and monitored by Western blot. GAPDH served as a cytosolic marker, PARP1 served as a nuclear marker, and SDHA served as a mitochondrial marker. (d) Representative immunofluorescence microscopic image of MTS-HA-Cas9 (HA, green) and CoxIV (red) subcellular distributions in HEK-293T cells transfected with MTS-HA-Cas9 construct. The merged image (yellow, right panel) shows colocalization of mitoCas9 and CoxIV. (e) Representative gel images of the PCR product of hU6-sgRMA-eGFP and hU6-sgRNA-Cox1 which were purified by gel extraction (bottom panel). Schematics of primers and lentiCRISPR-sgRNA templates used to amplify U6 promoter and respective sgRNA components for transfection (upper panel). (f) Quantification of copy numbers for Cox1, Cox3, and ND1 regions of mtDNA extracted from HEK-293T cells transiently transfected with indicated constructs (mitoCas9 is a plasmid, while U6-sgRNAs are PCR product.), determined by real-time quantitative PCR using primers listed in Table 2. GAPDH was used as an internal loading control (n = 3 per group). Quantified data (b) are expressed as mean ± s.e.m., ∗∗∗P < 0.001, analysis of variance (ANOVA) test followed by Student-Newman-Keuls post hoc analysis.

Mentions: Although we demonstrated that FLAG-Cas9 with NLS peptide can effectively localize to mitochondria for functional editing of mtDNA, Cas9, which can specifically localize to mitochondria, is required for safe application for mtDNA editing without affecting genomic DNA. Therefore, we aimed at creating a new version of Cas9 that can specifically target mtDNA. By removing FLAG and NLS sequences from lentiCRISPR-sgRNA-eGFP#2 and adding mitochondrial targeting sequence (MTS) and HA tag in the N-terminus of SpCas9, we synthesized mitochondria-targeting Cas9 (mitoCas9) (Figure 3(a)). MTS of cytochrome C (mitochondria matrix protein) was used to direct mitoCas9 into the matrix of mitochondria so that it can interact with mtDNA. MitoCas9 was expressed and detected with HA-specific antibody at the expected molecular weight (Figure 3(b)), and subcellular fractionation showed that mitoCas9 mainly localized to mitochondria (Figure 3(c)). In addition, immunofluorescence confirmed colocalization of HA-tagged mitoCas9 with mitochondria marker protein CoxIV (Figure 3(d)).


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)

Construction of mitochondria-targeted MTS-HA-Cas9. (a) Schematic illustration of mitochondria-targeting Cas9 (mitoCas9). Mitochondria-targeting sequence (MTS) and HA tag information are presented. (b) Expression of mitoCas9 in HEK-293T cells transiently transfected with MTS-HA-Cas9 construct determined by Western blots using HA antibody. β-actin was used as a loading control. (c) Subcellular localization of MTS-HA-Cas9 assessed in the cytosolic (Cyt), mitochondrial (Mit), and nuclear (Nu) fractions of HEK-293T cells transfected with lentiCRISPR-sgRNA-eGFP#2 and monitored by Western blot. GAPDH served as a cytosolic marker, PARP1 served as a nuclear marker, and SDHA served as a mitochondrial marker. (d) Representative immunofluorescence microscopic image of MTS-HA-Cas9 (HA, green) and CoxIV (red) subcellular distributions in HEK-293T cells transfected with MTS-HA-Cas9 construct. The merged image (yellow, right panel) shows colocalization of mitoCas9 and CoxIV. (e) Representative gel images of the PCR product of hU6-sgRMA-eGFP and hU6-sgRNA-Cox1 which were purified by gel extraction (bottom panel). Schematics of primers and lentiCRISPR-sgRNA templates used to amplify U6 promoter and respective sgRNA components for transfection (upper panel). (f) Quantification of copy numbers for Cox1, Cox3, and ND1 regions of mtDNA extracted from HEK-293T cells transiently transfected with indicated constructs (mitoCas9 is a plasmid, while U6-sgRNAs are PCR product.), determined by real-time quantitative PCR using primers listed in Table 2. GAPDH was used as an internal loading control (n = 3 per group). Quantified data (b) are expressed as mean ± s.e.m., ∗∗∗P < 0.001, analysis of variance (ANOVA) test followed by Student-Newman-Keuls post hoc analysis.
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fig3: Construction of mitochondria-targeted MTS-HA-Cas9. (a) Schematic illustration of mitochondria-targeting Cas9 (mitoCas9). Mitochondria-targeting sequence (MTS) and HA tag information are presented. (b) Expression of mitoCas9 in HEK-293T cells transiently transfected with MTS-HA-Cas9 construct determined by Western blots using HA antibody. β-actin was used as a loading control. (c) Subcellular localization of MTS-HA-Cas9 assessed in the cytosolic (Cyt), mitochondrial (Mit), and nuclear (Nu) fractions of HEK-293T cells transfected with lentiCRISPR-sgRNA-eGFP#2 and monitored by Western blot. GAPDH served as a cytosolic marker, PARP1 served as a nuclear marker, and SDHA served as a mitochondrial marker. (d) Representative immunofluorescence microscopic image of MTS-HA-Cas9 (HA, green) and CoxIV (red) subcellular distributions in HEK-293T cells transfected with MTS-HA-Cas9 construct. The merged image (yellow, right panel) shows colocalization of mitoCas9 and CoxIV. (e) Representative gel images of the PCR product of hU6-sgRMA-eGFP and hU6-sgRNA-Cox1 which were purified by gel extraction (bottom panel). Schematics of primers and lentiCRISPR-sgRNA templates used to amplify U6 promoter and respective sgRNA components for transfection (upper panel). (f) Quantification of copy numbers for Cox1, Cox3, and ND1 regions of mtDNA extracted from HEK-293T cells transiently transfected with indicated constructs (mitoCas9 is a plasmid, while U6-sgRNAs are PCR product.), determined by real-time quantitative PCR using primers listed in Table 2. GAPDH was used as an internal loading control (n = 3 per group). Quantified data (b) are expressed as mean ± s.e.m., ∗∗∗P < 0.001, analysis of variance (ANOVA) test followed by Student-Newman-Keuls post hoc analysis.
Mentions: Although we demonstrated that FLAG-Cas9 with NLS peptide can effectively localize to mitochondria for functional editing of mtDNA, Cas9, which can specifically localize to mitochondria, is required for safe application for mtDNA editing without affecting genomic DNA. Therefore, we aimed at creating a new version of Cas9 that can specifically target mtDNA. By removing FLAG and NLS sequences from lentiCRISPR-sgRNA-eGFP#2 and adding mitochondrial targeting sequence (MTS) and HA tag in the N-terminus of SpCas9, we synthesized mitochondria-targeting Cas9 (mitoCas9) (Figure 3(a)). MTS of cytochrome C (mitochondria matrix protein) was used to direct mitoCas9 into the matrix of mitochondria so that it can interact with mtDNA. MitoCas9 was expressed and detected with HA-specific antibody at the expected molecular weight (Figure 3(b)), and subcellular fractionation showed that mitoCas9 mainly localized to mitochondria (Figure 3(c)). In addition, immunofluorescence confirmed colocalization of HA-tagged mitoCas9 with mitochondria marker protein CoxIV (Figure 3(d)).

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