mitoTev‐TALE: a monomeric DNA editing enzyme to reduce mutant mitochondrial DNA levels
2018; Springer Nature; Volume: 10; Issue: 9 Linguagem: Inglês
10.15252/emmm.201708084
ISSN1757-4684
AutoresCláudia V. Pereira, Sandra R. Bacman, Tania Arguello, Ugne Zekonyte, Siôn L. Williams, David R. Edgell, Carlos T. Moraes,
Tópico(s)CRISPR and Genetic Engineering
ResumoReport16 July 2018Open Access Source DataTransparent process mitoTev-TALE: a monomeric DNA editing enzyme to reduce mutant mitochondrial DNA levels Claudia V Pereira Claudia V Pereira Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA Search for more papers by this author Sandra R Bacman Sandra R Bacman Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA Search for more papers by this author Tania Arguello Tania Arguello Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA Search for more papers by this author Ugne Zekonyte Ugne Zekonyte Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA Search for more papers by this author Sion L Williams Sion L Williams Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA Search for more papers by this author David R Edgell David R Edgell Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada Search for more papers by this author Carlos T Moraes Corresponding Author Carlos T Moraes [email protected] orcid.org/0000-0002-8077-7092 Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA Search for more papers by this author Claudia V Pereira Claudia V Pereira Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA Search for more papers by this author Sandra R Bacman Sandra R Bacman Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA Search for more papers by this author Tania Arguello Tania Arguello Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA Search for more papers by this author Ugne Zekonyte Ugne Zekonyte Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA Search for more papers by this author Sion L Williams Sion L Williams Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA Search for more papers by this author David R Edgell David R Edgell Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada Search for more papers by this author Carlos T Moraes Corresponding Author Carlos T Moraes [email protected] orcid.org/0000-0002-8077-7092 Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA Search for more papers by this author Author Information Claudia V Pereira1, Sandra R Bacman1, Tania Arguello1, Ugne Zekonyte1, Sion L Williams1, David R Edgell2 and Carlos T Moraes *,1 1Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA 2Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada *Corresponding author. Tel: +1 305 243 5858; E-mail: [email protected] EMBO Mol Med (2018)10:e8084https://doi.org/10.15252/emmm.201708084 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Pathogenic mitochondrial DNA (mtDNA) mutations often co-exist with wild-type molecules (mtDNA heteroplasmy). Phenotypes manifest when the percentage of mutant mtDNA is high (70–90%). Previously, our laboratory showed that mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs) can eliminate mutant mtDNA from heteroplasmic cells. However, mitoTALENs are dimeric and relatively large, making it difficult to package their coding genes into viral vectors, limiting their clinical application. The smaller monomeric GIY-YIG homing nuclease from T4 phage (I-TevI) provides a potential alternative. We tested whether molecular hybrids (mitoTev-TALEs) could specifically bind and cleave mtDNA of patient-derived cybrids harboring different levels of the m.8344A>G mtDNA point mutation, associated with myoclonic epilepsy with ragged-red fibers (MERRF). We tested two mitoTev-TALE designs, one of which robustly shifted the mtDNA ratio toward the wild type. When this mitoTev-TALE was tested in a clone with high levels of the MERRF mutation (91% mutant), the shift in heteroplasmy resulted in an improvement of oxidative phosphorylation function. mitoTev-TALE provides an effective architecture for mtDNA editing that could facilitate therapeutic delivery of mtDNA editing enzymes to affected tissues. Synopsis This work describes the development of a mitochondrial-targeted DNA editing enzyme that can specifically cleave the MERRF m.8344A>G mtDNA mutation. The novel feature of this enzyme is that it is monomeric, in contrast to mitoTALEN and mitoZFN, which are heterodimeric. The homing endonuclease I-TevI was fused to the N-terminus of a TALE motif that binds specifically to the mtDNA MERRF m.8344A>G site. A mitochondrial targeting sequence and a FLAG tag were also added to the N-terminus. When MERRF cells harboring heteroplasmic mutant mtDNA were transfected with mitoTev-TALE there was a reduction in mutant mtDNA by approximately 20%. The monomeric nature of this reagent should facilitate packaging into AAV vectors. Introduction Mitochondria contain their own double-stranded circular genome composed of 16,569 base pairs (bp). There are approximately one thousand copies of mitochondrial DNA (mtDNA) inside a cell. The mtDNA contains 37 genes, encoding 22 transfer RNAs (tRNAs), two ribosomal RNAs (12S and 16S), and 13 subunits of the oxidative phosphorylation system (OXPHOS; Anderson et al, 1981; Wallace, 2007). Mutations in the mtDNA have been associated with several clinical syndromes, which are in most cases maternally inherited and have a variable onset (Giles et al, 1980; Wallace, 2007; Christie et al, 2015). More than 270 point mutations and large-scale rearrangements in human mtDNA have been associated with diverse clinical phenotypes, including muscle weakness, cardiomyopathy, stroke-like episodes, optic neuropathies, and neurodegenerative disorders, among others (DiMauro & Moraes, 1993; Schon et al, 2012; Stewart & Chinnery, 2015). MtDNA mutations are commonly heteroplasmic, whereby wild-type and mutant genomes co-exist (Wallace & Fan, 2010; Wallace & Chalkia, 2013; Stewart & Chinnery, 2015) and the threshold levels for disease onset are usually between 70 and 90% mutant mtDNA (DiMauro & Moraes, 1993; Davis & Sue, 2011). Therefore, a small shift in mtDNA heteroplasmy should produce a significant improvement in patient's phenotype (Smith & Lightowlers, 2011). Because mitochondria lack an efficient double-strand break (DSB) repair system, mitochondria-specific endonucleases can lead to a quick degradation of the mutant mtDNA followed by a repopulation with wild-type (WT) mtDNA. This concept has been demonstrated by the use of different mitochondrial-targeted endonucleases including mitochondrial restriction endonucleases (REs), mitochondrial zinc-finger nucleases (mitoZFNs), and mitochondrial TAL effector nucleases (mitoTALENs) (Srivastava & Moraes, 2001; Tanaka et al, 2002; Minczuk et al, 2006, 2008; Bacman et al, 2007, 2010, 2013; Alexeyev et al, 2008; Gammage et al, 2014; Hashimoto et al, 2015; Reddy et al, 2015). Although these approaches could effectively shift heteroplasmy in cultured cells, they have limitations for gene therapy use. In the case of the REs, their usage is limited to rare mutations that create naturally occurring restriction sites; mitochondrial ZFNs and mitoTALENs, even though capable of targeting virtually any mitochondrial DNA sequence, work as dimers. TALENs are particularly large, which imposes restrictions in their packaging into viral vectors. The standard ZFN and TALEN architecture utilizes the dimeric FokI Type IIS endonuclease domain (Bitinaite et al, 1998). Thus, both require two DNA recognition sites flanking a central spacer region. Our group has previously described a dimeric mitoTALEN specific for the m.8344A>G MERRF mutation (Hashimoto et al, 2015). Here, we describe a strategy to overcome the architectural constraints imposed by mitoTALENs against the same target. Based on the GIY-YIG homing nuclease I-TevI, we assembled a monomeric I-TevI-TALE nuclease (Kleinstiver et al, 2012, 2014; Beurdeley et al, 2013) targeting m.8344A>G, by taking advantage of the previously tested mitoTALEN-DNA binding domain. This novel design is a smaller alternative for mitochondrial genome editing, which is more readily applicable to in vivo delivery using adeno-associated virus (AAV) vectors. Results Designing mitoTev-TALEs against the m.8344A>G tRNALys mtDNA point mutation We sought to design a mutant mtDNA-specific monomeric mitochondrial-targeted endonuclease. The homing nuclease I-TevI catalytic domain recognizes a short DNA sequence 5′CNNNG3′, thereby being relatively non-specific. However, I-TevI was reported to be effective in editing nuclear DNA as a monomeric fusion with a more specific TALE DNA binding domain (Beurdeley et al, 2013; Kleinstiver et al, 2014). Based on our previous TALE DNA binding domain targeting the MERRF m.8344A>G mtDNA (Hashimoto et al, 2015), we engineered two monomeric mitoTev-TALE nucleases, which differed in size by the TALE DNA binding domain repeated variable di-residues (RVD), either 8.5 or 11.5 RVDs. We also added a mitochondrial localization sequence (COX8/Su9) preceding the I-TevI catalytic domain, which was fused to the TALE DNA binding domain, through a flexible linker (depicted in Fig 1A). The fluorescent marker eGFP was placed downstream of the TALE domain, translated independently from the same transcript as the mitoTev-TALE due to a T2A picornavirus ribosome stuttering sequence (Fig EV1A), which also serves as an epitope tag for the nuclease (Bacman et al, 2013). The mitoTev-TALE was positioned to bind the anti-sense strand, where m.8344A>G contains a C (at position 3 of the binding domain, depicted in red in Fig 1B), which can be discriminated from the T present in the WT strand (Hashimoto et al, 2015). The I-TevI domain was positioned 12 bp apart from the TALE obligated T at position zero (T0, depicted in green in Fig 1B), which was the closest I-TevI required canonical sequence (5′CNNNG3′), in this case 5′CACTG3′ for binding and catalytic activity (Fig 1B). Figure 1. Development of a mitoTev-TALE targeting the m.8344A>G mitochondrial DNA mutation A. The structure of a mitoTev-TALE. It consists in a mitochondrial localization sequence (MLS) and a TALE DNA binding domain which is fused to a monomeric endonuclease (I-TevI), through a flexible linker (linker). B. Diagram of mtDNA illustrating the binding site for two monomeric mitoTev-TALEs (8.5 and 11.5 RVDs) designed to target the m.8344A>G in the tRNALys gene. The two mitoTev-TALEs differ in the number of RVDs. The A:T base pair at position T0 is shown in green and the G:C discriminatory base pair in red. The I-TevI endonuclease domain targets a 5′CNNNG3′ sequence, in this case 5′CACTG3′ shown in green which is spaced by 12 bp from the TALE DNA binding site. C. HEK293T cells were transfected for 48 h with each mitoTev-TALE (11.5 and 8.5 RVDs), and total cell extracts were used for Western blots with anti-T2A antibody. Molecular weights of the two different mitoTev-TALEs are ˜85 kDa and ˜70 kDa, respectively. D. Immunocytochemistry using the same antibody as in (C) along with MitoTracker Red was performed in COS7 cells, 24 h after transfection. Nuclei were counterstained with DAPI (blue). 11.5 RVD and 8.5 RVD mitoTev-TALEs both co-localized with mitochondria, as seen in the merge image. Scale bar: 10 μm. Source data are available online for this figure. Source Data for Figure 1 [emmm201708084-sup-0003-SdataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Cell sorting of cybrids transfected with the plasmid coding for the MERRF mitoTev-TALE A. Plasmid expressing the MERRF mitoTev-TALE, showing the location of the eGFP gene. B. FACS gating of cells transfected (24 h) and sorted by GFP fluorescence. Cells sorted as positive for the eGFP marker were termed "GFP++", and cells without detectable fluorescence were termed "GFP−+". C. Fluorescence microscopy showing the presence of some GFP-positive cells in the GFP−+ population (4 and 24 h after sorting). D. Determination of the total mtDNA levels 1 and 15 days after transfection in clone 7 from the first set of sortings. The graph shows the ratio of (COXI/ACTB) PrimeTime probes. Data are mean ± SEM of n = 9–10, 1 day; n = 3–4, 15 days. Statistical analysis was performed using two-tailed Student's t-test between two groups (UNT. vs GFP−+ or UNT. vs GFP++, **P < 0.005; ****P < 0.0001). Download figure Download PowerPoint Transient transfection in HEK293T and COS7 cells showed expression of full-length proteins (Fig 1C). As previously reported, the monomers showed more than one band with the T2A antibody, with the higher molecular weight band likely corresponding to a small fraction of the newly synthesized monomer with an uncleaved mitochondrial localization signal (Hashimoto et al, 2015). Both the 11.5 RVD and 8.5 RVD mitoTev-TALEs co-localized with the MitoTracker Red mitochondrial marker (Fig 1D). The 11.5 RVD mitoTev-TALE effectively shifted mtDNA heteroplasmy The capacity of the monomeric mitoTev-TALE to shift mtDNA heteroplasmy was first tested in patient-derived transmitochondrial cybrids harboring approximately 40–45% of the MERRF mutation (MERRF Low Mut cells), previously characterized in our laboratory (Hashimoto et al, 2015). The cells were transfected with either plasmids coding the dimeric mitoTALEN (Hashimoto et al, 2015) or the monomeric mitoTev-TALEs. Cells were sorted for the respective fluorescent markers, by mCherry plus eGFP (in the case of the dimeric mitoTALEN), or by eGFP only (Fig EV1). Sorted cell populations expressing both green and red after the mitoTALEN transfection were termed "Yellow", and cell populations sorted as negative for the fluorescent markers were termed "Black". The cells showing high green fluorescence after the mitoTev-TALE transfection were termed "GFP++", whereas non-fluorescent sorted cells were termed "GFP−+" (Fig EV1B). This nomenclature was chosen because of the consistent observation of low numbers of GFP-positive cells in the latter population, which increased overtime (Fig EV1C). Using PCR/RFLP, we found a shift in heteroplasmy after transfection with all three tested constructs. Although the shift was robust with the 11.5 RVD mitoTev-TALE, the shorter 8.5 RVD mitoTev-TALE produced a smaller shift (Fig 2A and B). Because of these initial results, we continued to use exclusively the 11.5 RVD mitoTev-TALE, hereafter referred to as "MERRF mitoTev-TALE". Our data showed a robust shift of 25% toward the WT mtDNA in the "GFP++" cells after transfection with the MERRF mitoTev-TALE, which was similar to the shift produced by the mitoTALEN (30%, Fig 2A and B). The "GFP−+" showed a small reduction in mutant mtDNA (8%) when compared to the untransfected cells. In addition, sorted cells were cultured for up to 15 days post-transfection and mtDNA heteroplasmy was re-analyzed (Fig 2C). The "GFP++" cells maintained the lower mutant load 15 days after sorting (Fig 2D). Figure 2. Monomeric mitoTev-TALEs reduce mutant mtDNA loads in low mutant cybrids harboring the m.8344A>G mtDNA point mutation A. MtDNA heteroplasmy analyzed by PCR/RFLP, 24 h after mitoTev-TALE and mitoTALEN transfection. The RFLP analysis shows increased %WT mtDNA in sorted cells when compared to the untransfected. mitoTALEN monomers positive for both eGFP and mCherry were isolated as "Yellow". The "Black" cells represent the mitoTALEN sorted population of cells negative for eGFP and mCherry, and the "GFP−+" population represents mitoTev-TALE sorted cells with low levels of eGFP fluorescence. GFP++ were cells positive for GFP after mitoTev-TALEs transfections. B. Quantification of the % of WT load in cybrid sorted populations after transfection. C. Representative RFLP analysis showing the change in heteroplasmy at 1 and 15 days, after sorting. D. Quantification of the heteroplasmic shift over time. E. Determination of the levels of mtDNA (ND1/ACTB) by qPCR of mutant cybrids transfected with MERRF mitoTev-TALE. F. mtDNA levels in wild-type sorted cells transfected with the MERRF mitoTev-TALE. Data are expressed as percentage of the untransfected cells (%UNT). Data information: Data are expressed as mean ± SEM of n = 5–12 independent experiments (B), n = 3 independent experiments (D), n = 3–6 independent experiments (E), and n = 6 independent experiments (F). Statistical analyses were performed by the use of unpaired two-tailed Student's t-test, *P < 0.05, **P < 0.005, ***P < 0.001. For exact P-values and n number, see Appendix Table S1. Source data are available online for this figure. Source Data for Figure 2 [emmm201708084-sup-0004-SdataFig2.pdf] Download figure Download PowerPoint Heteroplasmic cybrids transfected with the MERRF mitoTev-TALE showed expression level-dependent reduction in mtDNA levels Quantitative PCR analysis was performed in DNA samples from the sorted cybrids collected at two different time-points, 1 and 15 days after transfection. The data showed a decrease in the total mtDNA levels in the GFP++ cells, 1 day after transfection (Figs 2E and EV1D) but not in the GFP−+ cells. The lower levels of total mtDNA persisted in the GFP++ cells up to 15 days in culture (Figs 2E and EV1D). However, this decrease was not observed in WT cells 1 day after transfection which indicates that off-site cleavage by the MERRF mitoTev-TALE is relatively low, under the tested conditions (Fig 2F). The associated mtDNA depletion in the MERRF Low Mut cybrids is likely related to the elimination of the mutant mtDNA (Fig 2F). Changes in mutant load and total mtDNA levels in a cell clone with high levels of MERRF mutant mtDNA In order to test the biological significance of the change in mtDNA heteroplasmy, we used a different cybrid clone with higher mutant load, with approximately 91% mutant mtDNA. This MERRF High Mut line (clone 20) was transfected with the MERRF mitoTev-TALE. Similar to what was described above, after 2–3 days, the cells were sorted for eGFP. A portion of the sorted cells was allowed to grow up to 27 days. A time-course experiment showed a shift from 8 to 23% WT mtDNA as early as 2–3 days after transfection (Fig 3A and B). The ratio of WT/Mut mtDNA was also analyzed at later time-points after sorting (15–20 days, 23–27 days) and increased to 43% WT mtDNA in the GFP++ population of cells, stabilizing over time (Fig 3B). In accordance with previous observations, the GFP−+ population of cells also showed a small shift in heteroplasmy toward the WT mtDNA which also increased significantly overtime (Fig 3B). The mtDNA levels were reduced in the GFP++ cells but not in the GFP−+ (Fig 3C). To better understand the correlation between MERRF mitoTev-TALE expression levels and mtDNA depletion, we conducted a narrower gating approach during FACS sorting. The high mutant cells were transfected for 48 h and gated/sorted by the intensity of GFP fluorescence. We aimed to separate the GFP−+ (low green) from the GFP++ (intermediate green) and GFP+++ (bright green)-positive cells (Fig 3D). We analyzed the total mtDNA levels in the different populations of sorted cells either at 2 days or at 15 days after sorting. As expected, the GFP−+ cells did not show a significant decrease in the total mtDNA levels, whereas the GFP++ had a partial reduction, and the GFP+++ population showed a higher mtDNA depletion (Fig 3E). WT cybrids were analyzed in a similar manner and also showed decreased levels of total mtDNA levels in the GFP++ and GFP+++ cells, 2 days after transfection. However, the decrease in the mtDNA levels in these populations of WT cells was transient (Fig 3F). Figure 3. The MERRF mitoTev-TALE effectively shifts heteroplasmy in MERRF high mutant cybrids A. RFLP analyses of high mutant cybrids transfected with the MERRF mitoTev-TALE. The sorted cells were analyzed at different times of growth by PCR/RFLP as previously described. B. Quantification of WT loads of 6–8 independent experiments/sortings, analyzed at different time-points. Data are expressed as mean ± SEM. C. Total mtDNA levels determined by qPCR in 6–7 separate sortings/experiments. Data are expressed as mean ± SEM. D. Flow cytometry diagram of gated sorted cells. The GFP−+ represent the cells with low levels of fluorescence, GFP++ showed intermediate levels of fluorescence, while GFP+++ had the highest levels. E. Quantification of mtDNA levels by PrimeTime qPCR probes. The mtDNA levels were analyzed in the different populations of sorted cells. Data are expressed as mean ± SEM of 3–4 separate experiments. F. mtDNA levels were also determined in the WT cybrids as described above. Data are expressed as mean ± SEM of 4–7 separate experiments. Data information: Statistical analyses were performed by a two-tailed Student's t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs UNT. For more details, see Appendix Table S1. Source data are available online for this figure. Source Data for Figure 3 [emmm201708084-sup-0005-SdataFig3.pdf] Download figure Download PowerPoint Cybrids with high mutant loads transfected with the MERRF mitoTev-TALE showed improved mitochondrial function To determine whether the treatment with mitoTev-TALE had phenotypic benefits to MERRF cells, we analyzed mitochondrial function in both GFP−+ and GFP++ cells. Cells transfected with the MERRF mitoTev-TALE (both GFP−+ and GFP++) significantly increased oxygen consumption rates (OCR), when compared to the untransfected control (Fig 4A and B). Because it takes several weeks after sorting to obtain enough cells for functional analyses, the increase in OCR in the GFP−+ population was not surprising as the small number of GFP-positive cells in this population likely grow faster than untransfected/defective cells. The results of several independent experiments showed a significant increase in all four bioenergetic parameters determined in the Seahorse apparatus (basal respiration, maximum respiratory capacity, spare capacity, and ATP-linked respiration) in both 27-day-grown GFP−+ and GFP++ cells when compared to the untransfected cells, which were grown in parallel (Fig 4B). The OCR in GFP++ cells was still lower than the WT cybrids (Fig 4B). Coupling efficiency was similar between the MERRF mitoTev-TALE-treated cells and the control WT cybrids, a bioenergetic parameter that was significantly decreased in the untransfected cells (Fig 4C). Figure 4. The MERRF mitoTev-TALE significantly improves mitochondrial oxidative phosphorylation function of high mutant cybrids A. Oxygen consumption rate (OCR) upon sequential injection of oligomycin (Oligo), FCCP, and rotenone (Rot) + antimycin (AA), in untransfected, sorted "GFP−+" and "GFP++". Data represent the mean ± SEM of 5–7 separate experiments. B. Quantification of 4–6 independent experiments comparing the basal respiration, maximal respiration, spare respiratory capacity, and ATP-linked respiration of the UNT vs "GFP−+", "GFP++", and WT cells. The cells were analyzed between 25 and 28 days after sorting. Data represent the mean ± SEM. C. Coupling efficiency, measured as the ratio of ATP-linked/basal respiration × 100. Data represent the mean ± SEM of 4–6 separate experiments. D. Representative membrane of a mitochondrial protein synthesis experiment. The results were normalized to the gel loading. Graph represents five separate experiments (with exception of wild-type samples, n = 2). Data represent the mean ± SEM. E. Western blot showing the levels of NDUFB8 and COXI in untransfected and sorted cells. The SDHA and tubulin levels were also analyzed. The panel also shows the quantification of these markers normalized by the stain-free loading of six independent sortings/experiments. Data represent the mean ± SEM. Data information: Statistical analysis was performed using two-tailed Student's t-test between each group pair (UNT vs GFP−+; UNT vs GFP++, and UNT vs WT), *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant. For exact P-values and n numbers, see Appendix Table S1. Source data are available online for this figure. Source Data for Figure 4 [emmm201708084-sup-0006-SdataFig4.pdf] Download figure Download PowerPoint The MERRF mutation in the tRNA lysine gene is known to cause impairment of mitochondrial translation by reducing tRNA lysine aminoacylation and mitochondrial protein synthesis (Enriquez et al, 1995; Yasukawa et al, 2000). To understand whether the increase in respiration in treated cells was accompanied by an increase in mitochondrial protein synthesis and OXPHOS protein levels, we analyzed mitochondrial translation. 35S-methionine labeling of cells in the presence of a cytoplasmic protein synthesis inhibitor showed a significant increase in the synthesis of mtDNA-encoded subunits in both GFP−+ and GFP++ populations (which were grown for 25–28 days after sorting to obtain enough cells; Fig 4D). In agreement, the levels of COXI (mtDNA-encoded complex IV subunit) and NDUFB8 (nuclear-encoded complex I subunit that is sensitive to unassembled complex I) showed increases in GFP−+ and GFP++ cells, although because of variability, some markers did not reach significance (Fig 4E). The nuclear-coded subunit of complex II, SDHA, did not change between the different cells, neither did tubulin (Fig 4E). Discussion The advantages of the mitoTev-TALE architecture The manipulation of mtDNA heteroplasmy has great potential as a therapeutic tool. Although CRISPR/Cas9 has been reported to cleave mtDNA, a number of concerns suggest that it may be challenging to use CRISPR/Cas9 for mtDNA editing due to difficulties in nucleic acid import (Gammage et al, 2018). On the other hand, a broad range of DNA editing enzymes has been tested in mitochondria (Srivastava & Moraes, 2001; Tanaka et al, 2002; Bayona-Bafaluy et al, 2005; Bacman et al, 2007, 2010, 2012; Alexeyev et al, 2008), the most flexible being mitoTALEN and mitoZFN (Minczuk et al, 2008; Bacman et al, 2013; Gammage et al, 2014; Hashimoto et al, 2015). However, both architectures are dimeric, posing challenges to viral delivery due to limitations in construct size. The current study presents a promising alternative, which is more amenable to viral packaging and in vivo delivery. Our laboratory has previously used mitoTALENs, which robustly target mtDNA and shift the heteroplasmy of cybrids harboring point mutations (m.14459G>A in MT-ND6, m.13513G>A in MT-ND5, and m.8344A>G in MT-TK) and the common deletion (m.8483_13459del4977; Bacman et al, 2013; Hashimoto et al, 2015). We have also explored reducing the DNA binding domain sequence in mitoTALEN monomers and demonstrated that monomers with 7.5–12.5 RVDs were able to discriminate single base differences (Hashimoto et al, 2015). Taking advantage of the success of the mitoTALEN approach, we now developed a different monomeric architecture that was initially developed for nuclear DNA editing (Kleinstiver et al, 2012; Beurdeley et al, 2013). We termed these enzymes "mitoTev-TALEs", which encompass the monomeric nuclease domain derived from the I-TevI homing endonuclease fused to a TALE DNA binding domain. This design can overcome the package limitations of bulky dimeric mitoTALENs, as the latter require approximately 7 kb per dimer, which are not well suited to viral vectors such as AAV that can only accommodate about 4.5–4.9 kb inserts. Of the two mitoTev-TALEs sizes tested, 8.5 RVDs and 11.5 RVDs, the longer one showed a more robust activity in changing heteroplasmy, and the size of this gene is approximately 2.5 kb (~ 3.2 kb with the CMV promoter). The fusion point of the I-TevI catalytic domain to the N-terminal TALE DNA binding domain influences the catalytic activity of the endonuclease, and the linker region between the two domains can also influence the specificity of the binding and the activity in the target DNA (Beurdeley et al, 2013; Kleinstiver et al, 2014). In this case, we did not change the fusion point and we kept the same I-TevI linker; thus, a possible explanation could be that the shorter version did not provide enough specificity for an appropriate binding to the target sequence and therefore decreased the overall efficiency of the heteroplasmy shift. Optimization of specificity can be further explored by a different TALE binding domain recognizing the opposite strand sequence fused to I-TevI mutants that recognize and cleave GNNNG (which is created by the MERRF m.8344A>G mutation). I-TevI mutants, such as the K26R, T95S, and Q158R mutations, recognize such sequence, instead of the canonical CNNNG (Roy et al, 2016). Balancing mtDNA mutant elimination and mtDNA depletion Our study showed that the MERRF mitoTev-TALE could reduce the mutant mtDNA load by 15–20% after 2–3 days. The shift obtained after one cycle of transfection had a major impact on mitochondrial function. The MERRF mitoTev-TALE produced a similar increase in the WT/Mut ratios when compared to the dimeric mitoTALEN previously described (Hashimoto et al, 2015), which strongly supports the efficacy and potential u
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