Mitochondrial DNA editing in mice with DddA-TALE fusion deaminases

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Plasmid construction

We adapted our transcription activator-like effector nucleases (TALEN) assembly system to construct expression vectors for the split DddA halves, as well as final TALE–DddAtox constructs8. Beginning with the expression vector from the TALEN system, we replaced the fragments encoding the nuclear localization sequence and FokI obligatory heterodimeric halves with fragments encoding mitochondrial translocation sequences (MTS), DddA deaminase dimeric halves, and uracil glycosylase inhibitor (UGI). The MTS-, DddA-, and UGI-encoding sequences were synthesized by IDT. To construct expression plasmids, DNA fragments for Gibson assembly were amplified using Q5 DNA Polymerase (NEB), and subjected to PCR and gel purification. Purified gene fragments were assembled with a HiFi DNA assembly kit (NEB); assembled plasmids were chemically transformed into Escherichia coli DH5ɑ (Enzynomics), and their identity confirmed by Sanger sequencing. Thereby, we obtained eight expression plasmids, which include BsaI restriction enzyme sites between regions encoding the N-terminal domain and C-terminal half domain (NG) of TALE for Golden-gate assembly. For DdCBE plasmid assembly, each expression plasmid was mixed with six module vectors (each encoding a TALE array), BsaI (10 U), T4 DNA ligase (200 U), and reaction buffer in a single tube (Supplementary Fig. 1). Next, restriction–ligation reactions were performed in a thermocycler, with 20 cycles of 37 °C and 50 °C for 5 min each, followed by final incubations at 50 °C for 15 min and 80 °C for 5 min. Ligated plasmids were chemically transformed into E. coli DH5ɑ, and subjected to Sanger sequencing to confirm the identity of the constructs. Correct plasmids were midi-prepped (Qiagen) for cell transfection.

Mammalian cell culture and transfection

NIH3T3 (CRL-1658, American Type Culture Collection) cells were cultured and maintained at 37 °C with 5% CO2. Cells were grown in DMEM supplemented with 10% (v/v) bovine calf serum (Gibco) without any antibiotics. For lipofection, cells were seeded in 12-well cell culture plates (SPL, Seoul, Korea) at a density of 1.5 × 104 cells per well, 18–24 h before transfection. Lipofection using Lipofectamine 3000 (Invitrogen) was performed with 500 ng of each TALE half monomer plasmid to make up 1000 ng of total plasmid DNA. Cells were harvested at day 3 post transfection.

mRNA preparation

The mRNA templates were prepared by PCR using Q5 High-Fidelity DNA Polymerase (NEB) with the following primers (F: 5′-CATCAATGGGCGTGGATAG-3′, R: 5′-GACACCTACTCAGACAATGC-3′). DdCBE mRNAs were synthesized using an in vitro RNA transcription kit (mMESSAGE mMACHINE T7 Ultra kit, Ambion) and purified with a MEGAclear kit (Ambion).

Animals

Experiments involving mice were approved by the Institutional Animal Care and Use Committee of Institute for Basic Science. Super ovulated C57BL/6 J females were mated to C57BL/6 J males, and females from the ICR strain were used as foster mothers. Mice were maintained in a specific pathogen-free facility under a 12 h dark–light cycle, and constant temperature (20–26 °C) and humidity maintenance (40–60%).

Microinjection of mouse zygotes

Steps prior to microinjection, including superovulation and embryo collection, as well as microinjection itself, were performed as described previously16. For microinjection, a mixture containing left DdCBE mRNA (300 ng/μl) and right DdCBE mRNA (300 ng/μl) was diluted in DEPC-treated injection buffer (0.25 mM EDTA, 10 mM Tris, pH 7.4), and injected into the cytoplasm of zygotes using a Nikon ECLIPSE Ti micromanipulator and a FemtoJet 4i microinjector (Eppendorf). After injection, embryos were cultured in micro drops of KSOM + AA (Millipore) at 37 °C for 4 days in a humidified atmosphere containing 5% CO2. Two-cell-stage embryos were implanted into the oviducts of 0.5-d.p.c. pseudo-pregnant foster mothers.

Genotyping

Blastocyst stage embryos and tissues were incubated in lysis buffer (25 mM NaOH, 0.2 mM EDTA, pH 10) at 95 °C for 20 min, after which the pH was adjusted to 7.4 using HEPES (free acids, without pH adjustment) at a final concentration of 50 mM. Genomic DNA was extracted from pups for PCR genotyping using DNeasy Blood & Tissue Kits (Qiagen), and subjected to Sanger and targeted deep sequencing.

Mitochondrial DNA isolation for high-throughput sequencing

To isolate mitochondria from NIH3T3 cells in 12-well cell culture plates, the culture medium was aspirated, and 200 µl of Mitochondrial isolation buffer A (ScienCell) was added to each well. Cells were scraped with cell lifter, collected into microtubes, and homogenized with a disposable pestle designed for cell grinding. After 15 strokes, the homogenate was centrifuged at 1000 × g for 5 min at 4 °C. The supernatant was transferred to a clean microtube and centrifuged at 10,000 × g for 20 min at 4 °C. The pellet was resuspended in 20 µl of lysis solution (25 mM NaOH, 0.2 mM EDTA, pH 10), and incubated at 95 °C for 20 min. To lower the pH, we added 2 µl of 1 M HEPES (free acids, without pH adjustment) to the lysed mitochondrial solution. A total of 1 µl of lysate was used as a template for high-throughput sequencing.

Targeted deep sequencing

To create a high-throughput sequencing library, nested first PCR and second PCR were performed, and final index sequences were incorporated, using Q5 DNA Polymerase. The library was subjected to paired-end read sequencing using MiniSeq (Illumina). In all cases, the paired-end sequencing results were joined into a single fastqjoin file and analyzed via CRISPR RGEN Tools (http://www.rgenome.net/)17.

Data analysis and display

Microsoft Excel (2019) and Powerpoint (2019) was used for drawing figures, graphs, and tables. Genome alignment, primer design, and cloning design were performed with Geneious (version 2021.0.1) and Snapgene 5.2.3, using NC_005089 genome as a reference.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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