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, 36 (12), 3926-38

Development of a Single-Chain, Quasi-Dimeric Zinc-Finger Nuclease for the Selective Degradation of Mutated Human Mitochondrial DNA

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Development of a Single-Chain, Quasi-Dimeric Zinc-Finger Nuclease for the Selective Degradation of Mutated Human Mitochondrial DNA

Michal Minczuk et al. Nucleic Acids Res.

Abstract

The selective degradation of mutated mitochondrial DNA (mtDNA) molecules is a potential strategy to re-populate cells with wild-type (wt) mtDNA molecules and thereby alleviate the defective mitochondrial function that underlies mtDNA diseases. Zinc finger nucleases (ZFNs), which are nucleases conjugated to a zinc-finger peptide (ZFP) engineered to bind a specific DNA sequence, could be useful for the selective degradation of particular mtDNA sequences. Typically, pairs of complementary ZFNs are used that heterodimerize on the target DNA sequence; however, conventional ZFNs were ineffective in our system. To overcome this, we created single-chain ZFNs by conjugating two FokI nuclease domains, connected by a flexible linker, to a ZFP with an N-terminal mitochondrial targeting sequence. Here we show that these ZFNs are efficiently transported into mitochondria in cells and bind mtDNA in a sequence-specific manner discriminating between two 12-bp long sequences that differ by a single base pair. Due to their selective binding they cleave dsDNA at predicted sites adjacent to the mutation. When expressed in heteroplasmic cells containing a mixture of mutated and wt mtDNA these ZFNs selectively degrade mutated mtDNA, thereby increasing the proportion of wt mtDNA molecules in the cell. Therefore, mitochondria-targeted single-chain ZFNs are a promising candidate approach for the treatment of mtDNA diseases.

Figures

Figure 1.
Figure 1.
Interactions of ZFNs with wt and mutated mtDNA. The sequence surrounding the m.8993T>G mutation in mtDNA was used to exemplify targeting of pathogenic mitochondrial mutations by ZFNs. The changed base at position 8993 is indicated in yellow. DNA cleavage is marked by the green scissors symbol. (A) Schematic diagram of ZFN heterodimer in the standard configuration bound to the mutated mtDNA target (upper). Each of the monomeric ZFN consists of the FokI nuclease domain linked to a ZFP. One of the ZFNs (red) was designed to bind to the mutated mtDNA site, whereas its companion ZFN binds a native sequence on the opposite DNA strand (blue). In the case of wt mtDNA (lower) mutation-specific ZFN does not bind the target therefore precluding a formation of a heterodimer and DNA cleavage. (B) Schematic diagram of the partner ZFN (blue) bound to the wt sequence and producing DNA cleavage by dimerization with another copy of the same construct non-specifically bound to mtDNA when the mutation-specific ZFN (red) is not bound. This outcome is undesirable when a mutation is targeted in heteroplasmic population of mtDNA molecules.(C) Schematic diagram of ‘single-chain ZFN’ consisting of two FokI nuclease domains tethered together by a long protein linker and fused to a ZFP. The ZFP is designed to bind exclusively to the mutated mtDNA site; therefore, only mtDNA molecules harbouring this mutation are cleaved (upper) while the wt copies are spared (lower).
Figure 2.
Figure 2.
Designing a single-chain ZFN to target a mitochondrial point mutation. (A) Schematic structure of mitochondrially targeted single-chain nucleases. The mitochondrial targeting sequence of F1β subunit of mitochondrial ATP synthase (MTS F) was fused to the N-terminus of the ZFP. The HA epitope tag and the NES facilitating mitochondrial targeting were added to the C-terminus of the ZFP. Two FokI domains were fused on the C-terminus of the ZFP and linked together via flexible linkers of various lengths denoted L(n), where n is the length of the linker in amino acids. (B) Optimization of the single-chain ZFN based on the NARPd construct (see Figure 1C). Variants of F-NARPd-Fok-L(n)-Fok were subjected to the in vitro assay as described in Materials and methods section using the specific target DNA in order to determine the optimal length of the linker between the two tethered FokI domains. The plot shows the results of the assay performed three times. (C) In vitro cleavage assay testing the specificity of the F-NARPd-Fok-L(n)-Fok constructs. The assay was performed as described in Materials and methods section. The following probes have been used: pCR4-NARP-G—containing the m.8993T>G substitution, pCR4-NARP-T —wt (i.e ‘T’) at the 8993 position and pCR4-NARP-C— containing the m.8993T>C substitution. The f.p denotes free probe. Specific digestion at the m.8993T>G mutation site results in the formation of 2.7- and 1.5-kb DNA fragments. The percentage of cleavage of the pCR4-NARP-G probe is given in the top panel. (D) Western blot illustrating that the constructs used in the test above are produced with the same efficiency in the in vitro transcription/translation system. ZFNs were detected with anti-HA antibody.
Figure 3.
Figure 3.
The specificity of single-chain ZFNs designed to target the mitochondrial point mutation. (A) In vitro assay verifying the specificity of the F-NARPd-Fok-L35-Fok (line 3) and F-NARPd-Fok-L40-Fok (line 4) constructs to a single site in human mtDNA. The assay was performed similarly to the experiment shown in Figure 2. The DNA probes used here though were generated in a long-range PCR reaction so that the two resulting products represented the entire mtDNA molecule harbouring the m.8993T>G substitution (grey curved bars with the mtDNA coordinates given on each end). The PCR products were radioactively labelled using T4 Kinase. (B) Mapping the main cleavage sites of F-NARPd-Fok-L35-Fok and F-NARPd-Fok-L40-Fok constructs by primer extension. Unlabelled DNA probes (as described above) were subjected to the in vitro digestion assay with F-NARPd-Fok-L35-Fok (line 3) or F-NARPd-Fok-L40-Fok (line 4). Next the digested DNA served as a template for the extension reaction of the 5' labelled primer that anneals 100-bp upstream from the NARPd-binding site. DNA strand breaks introduced by F-NARPd-Fok-L35-Fok or F-NARPd-Fok-L40-Fok map to several sites 2–7-bp upstream from the NARPd-binding site.
Figure 4.
Figure 4.
ZFNs are localized in mitochondria in cells. (A) and (B) The F-NARPd-Fok and F-NARPd-Fok-L35-Fok ZNFs localize inside mitochondria. The 143B cells harbouring wt mtDNA were transiently transfected with monomeric F-NARPd-Fok (A) or single-chain ZFN—F-NARPd-Fok-L35-Fok (B), fractionated 48 h post-transfection and the protein fractions were analysed by western blotting using anti-HA mAb. The localization of the ZFN precursors (‘p’) and their mature (‘m’) form in total cell lysate (‘T’), cytosolic (‘C’) and a mitochondrial fraction treated with proteinase K under various conditions as indicated, was compared with the localization of marker proteins. The precursors of mitochondrial ZFNs, found in the mitochondrial fractions, were located outside the mitochondria, since they were accessible to protease digestion. In contrast, the mature form of ZFNs was protected and became accessible to proteolysis only after the mitochondria were lysed with Triton X-100. The following endogenous proteins were used as fractionation markers: (i) TFAM: the transcription factor that is localized in the mitochondrial matrix; (ii) Cox2; a subunit of the cytochrome oxidase complex localized in the mitochondrial inner membrane; (iii) Tom22: a subunit of mitochondrial translocase of outer membrane; and (iv) GAPDH: a protein localized in cytoplasm. (C) The F-NARPd-Fok and F-NARPd-Fok-L35-Fok ZFNs co-localize with mitochondria. The intra-cellular localization of ZFNs was additionally analysed by immunofluorescence in transiently transfected 143B cells. ZFNs were detected with antibodies against the HA epitope-tag followed by secondary antibodies conjugated to FITC (1 and 4; green) Mitochondria were stained with MitoTracker CMX Red (2 and 5; red). Both ZFNs exhibited mitochondrial-staining pattern that in represented by yellow staining on digitally overlaid pictures (3 and 6).
Figure 5.
Figure 5.
Mitochondrial heteroplasmy in cells expressing mutation-specific ZFNs. The m.8993T>G mutation-specific or control ZFNs were co-expressed with eGFP from a single vector in heteroplasmic cybrid cells containing ∼85% of mtDNA molecules with the NARP m.8993T>G mutation (dashed line). Two days post-transfection, cells were FACS sorted using eGFP as a marker and the degree of heteroplasmy was assessed in the sorted cells. Mock indicates cells transfected with vector alone. Symbols: *P < 0.05; **P < 0.01 in two-tailed t-test, unequal variance. (A) Schematic illustration of the experimental approaches taken. (B) Differences in the proportion of the wt and mutant mtDNA in cells transfected with F-NARPd-Fok or F-NARPd-Fok-L35-Fok specific for the m.8993T>G. The result is an average of three independent experiments. The difference between mock and F-NARPd-Fok-L35-Fok transfected cells is highly significant (P = 0.015), while the difference between mock and the F-NARPd-Fok monomer is not significant (P = 0.2).(C) Total (sum of mutant and wt) mtDNA copy number in the cells transfected with the m.8993T>G mutation-specific ZFNs expressed in arbitrary units (a.u.). The results are normalized to mock-transfected cells. (D) Differences in the proportion of the wt and mutant mtDNA in cells transfected with F-NCR-Fok or F-NCR-Fok-L35-Fok targeting a site in the mitochondrial non-coding region. The result is an average of three independent experiments. No statistically significant differences between the constructs have been found. (E) Total mtDNA copy number in the cells transfected with the mitochondrial NCR-specific ZFNs expressed in arbitrary units (a.u.). The results are normalized to mock-transfected cells.
Figure 6.
Figure 6.
Stable shift in mitochondrial heteroplasmy in cells expressing mutation-specific ZFNs. The m.8993T>G mutation-specific ZFNs, F-NARPd-Fok or F-NARPd-Fok-L35-Fok, were co-expressed with eGFP from a single vector in heteroplasmic cybrid cells containing ∼85% of mtDNA molecules with the NARP m.8993T>G mutation (dashed line). Two days after the transfection cells were FACS sorted using eGFP as a marker, then the sorted cells were grown on non-selective medium for a further 28 days. The percentage of mutant mtDNA was assessed again at 2 and 30 days. Mock indicates cells transfected with vector alone. Symbols: *P < 0.05; **P < 0.01 in two-tailed t-test, unequal variance. (A) Schematic illustration of the experimental approaches taken. (B) Differences in the proportion of the wt and mutant mtDNA in bulk population of transfected cells at 2 and 30 days. The difference between mock and F-NARPd-Fok-L35-Fok transfected cells after 30 days is highly significant (P = 0.007), whereas the difference between mock and the F-NARPd-Fok monomer is not significant (P = 0.54). (C) Total mtDNA copy number in the transfected cells measured at 2 and 30 days expressed in arbitrary units (a.u.). The results are normalized to mock transfected cells. (D) Differences in the proportion of the wt and mutant mtDNA in individual clones, randomly picked at 30 days post-transfection. The red horizontal bar indicates an average value for each construct. The difference between mock and F-NARPd-Fok-L35-Fok transfected cells after 30 days is highly significant (P = 0.003), the difference between mock and the F-NARPd-Fok monomer is of much lower significance (P = 0.4).

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