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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

FLAG-NLS-Cas9 localizes to mitochondria. (a) Subcellular localization of FLAG-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. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a cytosolic marker, poly(ADP-ribose) polymerase 1 (PARP1) served as a nuclear marker, and succinate dehydrogenase complex subunit A (SDHA) served as a mitochondrial marker. Ponceau staining of the blotted nitrocellulose membrane was presented in the bottom lane to visualize relative protein loading amounts. (b) Immunofluorescence images of FLAG-NLS-Cas9 demonstrating its localization to nucleus, cytoplasm, and mitochondria. HEK-293T cells were immunostained with mouse antibody to FLAG (green) 24 hours following transient transfection with FLAG-NLS-Cas9 construct. Mitochondria or nucleus was stained with mitotracker Red or DAPI, respectively. (c) Illustration of human mitochondrial DNA (mtDNA). Simplified view of mtDNA is presented to depict regions encoding peptides or tRNA for indicated amino acids. Filled triangles indicate sites targeted by gRNAs against Cox1 or Cox3. Arrows indicate primer annealing sites used for real-time PCR of Cox1 or Cox3 regions. (d) Quantification of copy numbers for Cox1, Cox3, and ND1 regions of mtDNA extracted from HEK-293T cells transiently transfected with lentiCRISPR-sgRNA-Cox1 and lentiCRISPR-sgRNA-Cox3 or lentiCRISPR-sgRNA-eGFP#2 as a control, 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 (d) are expressed as mean ± s.e.m., ∗∗∗P < 0.001, unpaired two-tailed Student's t-test.
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fig1: FLAG-NLS-Cas9 localizes to mitochondria. (a) Subcellular localization of FLAG-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. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a cytosolic marker, poly(ADP-ribose) polymerase 1 (PARP1) served as a nuclear marker, and succinate dehydrogenase complex subunit A (SDHA) served as a mitochondrial marker. Ponceau staining of the blotted nitrocellulose membrane was presented in the bottom lane to visualize relative protein loading amounts. (b) Immunofluorescence images of FLAG-NLS-Cas9 demonstrating its localization to nucleus, cytoplasm, and mitochondria. HEK-293T cells were immunostained with mouse antibody to FLAG (green) 24 hours following transient transfection with FLAG-NLS-Cas9 construct. Mitochondria or nucleus was stained with mitotracker Red or DAPI, respectively. (c) Illustration of human mitochondrial DNA (mtDNA). Simplified view of mtDNA is presented to depict regions encoding peptides or tRNA for indicated amino acids. Filled triangles indicate sites targeted by gRNAs against Cox1 or Cox3. Arrows indicate primer annealing sites used for real-time PCR of Cox1 or Cox3 regions. (d) Quantification of copy numbers for Cox1, Cox3, and ND1 regions of mtDNA extracted from HEK-293T cells transiently transfected with lentiCRISPR-sgRNA-Cox1 and lentiCRISPR-sgRNA-Cox3 or lentiCRISPR-sgRNA-eGFP#2 as a control, 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 (d) are expressed as mean ± s.e.m., ∗∗∗P < 0.001, unpaired two-tailed Student's t-test.

Mentions: The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) defense system in bacteria has been successfully applied to edit specific genomic loci in many species [11]. CRISPR-associated (Cas) genes together with a single guide RNA (sgRNA) enable sequence-specific cleavage and editing of DNA. The currently available CRISPR/Cas9 system uses nuclear localization sequence- (NLS-) tagged SpCas9 to facilitate nuclear transfer of SpCas9, where genome editing should occur [12]. However, the precise subcellular distribution has not been examined. To determine in which subcellular compartment overexpressed FLAG-tagged SpCas9 is present, we performed subcellular fractionation into cytoplasmic, mitochondrial, and nuclear fractions for HEK-293T cells transiently transfected with lentiCRISPR-sgRNA-eGFP#2 construct, which should express FLAG-tagged Cas9 and sgRNA against eGFP. As expected, FLAG-Cas9 was detected in the nuclear fraction (Figure 1(a)). However, overexpression of FLAG-Cas9 showed substantial distribution to the other compartments, cytoplasm and mitochondria (Figure 1(a)). Consistent with the results from subcellular fractionation, immunofluorescence for overexpressed FLAG-Cas9 demonstrates its colocalization with mitotracker red-labeled mitochondria and DAPI-stained nucleus (Figure 1(a)).


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)

FLAG-NLS-Cas9 localizes to mitochondria. (a) Subcellular localization of FLAG-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. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a cytosolic marker, poly(ADP-ribose) polymerase 1 (PARP1) served as a nuclear marker, and succinate dehydrogenase complex subunit A (SDHA) served as a mitochondrial marker. Ponceau staining of the blotted nitrocellulose membrane was presented in the bottom lane to visualize relative protein loading amounts. (b) Immunofluorescence images of FLAG-NLS-Cas9 demonstrating its localization to nucleus, cytoplasm, and mitochondria. HEK-293T cells were immunostained with mouse antibody to FLAG (green) 24 hours following transient transfection with FLAG-NLS-Cas9 construct. Mitochondria or nucleus was stained with mitotracker Red or DAPI, respectively. (c) Illustration of human mitochondrial DNA (mtDNA). Simplified view of mtDNA is presented to depict regions encoding peptides or tRNA for indicated amino acids. Filled triangles indicate sites targeted by gRNAs against Cox1 or Cox3. Arrows indicate primer annealing sites used for real-time PCR of Cox1 or Cox3 regions. (d) Quantification of copy numbers for Cox1, Cox3, and ND1 regions of mtDNA extracted from HEK-293T cells transiently transfected with lentiCRISPR-sgRNA-Cox1 and lentiCRISPR-sgRNA-Cox3 or lentiCRISPR-sgRNA-eGFP#2 as a control, 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 (d) are expressed as mean ± s.e.m., ∗∗∗P < 0.001, unpaired two-tailed Student's t-test.
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fig1: FLAG-NLS-Cas9 localizes to mitochondria. (a) Subcellular localization of FLAG-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. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a cytosolic marker, poly(ADP-ribose) polymerase 1 (PARP1) served as a nuclear marker, and succinate dehydrogenase complex subunit A (SDHA) served as a mitochondrial marker. Ponceau staining of the blotted nitrocellulose membrane was presented in the bottom lane to visualize relative protein loading amounts. (b) Immunofluorescence images of FLAG-NLS-Cas9 demonstrating its localization to nucleus, cytoplasm, and mitochondria. HEK-293T cells were immunostained with mouse antibody to FLAG (green) 24 hours following transient transfection with FLAG-NLS-Cas9 construct. Mitochondria or nucleus was stained with mitotracker Red or DAPI, respectively. (c) Illustration of human mitochondrial DNA (mtDNA). Simplified view of mtDNA is presented to depict regions encoding peptides or tRNA for indicated amino acids. Filled triangles indicate sites targeted by gRNAs against Cox1 or Cox3. Arrows indicate primer annealing sites used for real-time PCR of Cox1 or Cox3 regions. (d) Quantification of copy numbers for Cox1, Cox3, and ND1 regions of mtDNA extracted from HEK-293T cells transiently transfected with lentiCRISPR-sgRNA-Cox1 and lentiCRISPR-sgRNA-Cox3 or lentiCRISPR-sgRNA-eGFP#2 as a control, 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 (d) are expressed as mean ± s.e.m., ∗∗∗P < 0.001, unpaired two-tailed Student's t-test.
Mentions: The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) defense system in bacteria has been successfully applied to edit specific genomic loci in many species [11]. CRISPR-associated (Cas) genes together with a single guide RNA (sgRNA) enable sequence-specific cleavage and editing of DNA. The currently available CRISPR/Cas9 system uses nuclear localization sequence- (NLS-) tagged SpCas9 to facilitate nuclear transfer of SpCas9, where genome editing should occur [12]. However, the precise subcellular distribution has not been examined. To determine in which subcellular compartment overexpressed FLAG-tagged SpCas9 is present, we performed subcellular fractionation into cytoplasmic, mitochondrial, and nuclear fractions for HEK-293T cells transiently transfected with lentiCRISPR-sgRNA-eGFP#2 construct, which should express FLAG-tagged Cas9 and sgRNA against eGFP. As expected, FLAG-Cas9 was detected in the nuclear fraction (Figure 1(a)). However, overexpression of FLAG-Cas9 showed substantial distribution to the other compartments, cytoplasm and mitochondria (Figure 1(a)). Consistent with the results from subcellular fractionation, immunofluorescence for overexpressed FLAG-Cas9 demonstrates its colocalization with mitotracker red-labeled mitochondria and DAPI-stained nucleus (Figure 1(a)).

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