# Mitochondrial genome microhomology-mediated editing by donor DNA delivery into mitochondria in human cells

Vadim Maximov, Nikita Shebanov, Natalia Nikitchina, Rachel Rapoport, Yehoshua Maor, Ivan Tarassov, Ophry Pines, Nina Entelis

► To cite this version:

Vadim Maximov, Nikita Shebanov, Natalia Nikitchina, Rachel Rapoport, Yehoshua Maor, et al. Mitochondrial genome microhomology-mediated editing by donor DNA delivery into mitochondria in human cells. BioRxiv, 2025, 10.1101/2025.11.02.686110. [https://doi.org/10.1101/2025.11.02.686110](https://doi.org/10.1101/2025.11.02.686110)

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Distributed under a Creative Commons Attribution 4.0 International License
1 Mitochondrial genome microhomology-mediated editing by donor DNA delivery into

2 mitochondria in human cells

3 Vadim V. Maximov¹#, Nikita Shebanov², Natalia Nikitchina², Rachel Rapoport³, Yehoshua

4 Maor¹, Ivan Tarassov²*, Ophry Pines³, Nina Entelis²

5 ¹ – Department of Molecular Biology, Phytor Ltd, Jerusalem, Israel

6 ² – UMR7156 Molecular Genetics, Genomics, Microbiology, CNRS/University of Strasbourg,

7 Strasbourg, France

8 ³ – Department of Microbiology and Molecular Genetics, IMRIC, The Hebrew University of

9 Jerusalem, Israel

10 The presented research was conducted in Jerusalem, Israel and Strasbourg, France

11 * – Correspondence should be sent to V.V.M. (vadim.phytor@gmail.com) and I.T.

12 (i.tarassov@unistra.fr)

13 # – V.V.M is currently a Senior Scientist at MitoCareX Bio Ltd, Ness Ziona, Israel. MitoCareX

14 Bio Ltd has not been involved in the presented research by any mean and this company is only
15 the current affiliation of V.V.M.

16 V.V.M. – Department of Molecular Biology, Phytor Ltd, 19 Ya'akov El'azar St., Jerusalem,
17 Israel, telephone: +972-(0)2-671-1911, fax: +972-1532-6711911, vadim.phytor@gmail.com

18 I.T. – UMR7156 Molecular Genetics, Genomics, Microbiology, CNRS/University of Strasbourg,
19 4, Allée Konrad Roentgen Strasbourg 67000, France, telephone: +33 3 68 85 14 81, email:

20 i.tarassov@unistra.fr

21 Short Title: Mitochondrial genome microhomology-mediated editing.
## 22 Summary

23 Mutations in the mitochondrial DNA (mtDNA) are associated with severe human diseases, lacking 24 efficient therapies. Direct correction of mtDNA mutations may offer a cure for such diseases. We 25 propose a novel strategy based on double-stranded DNA (dsDNA) oligonucleotide delivery into 26 mitochondria and intrinsic microhomology-mediated end joining (MMEJ) for mtDNA editing.

27 This strategy enables introduction of multiple predefined nucleotide changes in mtDNA. For this, 28 the presence of MMEJ activity in the human mitochondrial lysates was confirmed. 49 bp DNA 29 oligonucleotide duplexes, fused to an RNA hairpin previously identified as a mitochondrial import 30 signal, were delivered into the mitochondria of cultured human cells. Delivery of these donor 31 dsDNA molecules, homologous to an *ND4* site of mtDNA and bearing designed nucleotide 32 changes, led to a low but statistically significant introduction of the designed nucleotide changes 33 into mtDNA. Donor dsDNA delivery combined with the CRISPR/mito-AsCas12a system also 34 resulted in a statistically significant number of an expected concomitant change of five nucleotides 35 distributed across a 16-nucleotide *ND4* site of the mitochondrial genome. The proposed strategy 36 may become an efficient mtDNA editing tool suitable for the correction of near-homoplasmic 37 mutations such as Leber's Hereditary Optic Neuropathy (LHON)-associated mutations in the *ND4* 38 gene of mtDNA.

39

40

41

42
43 **Introduction**

44 Mutations in mitochondrial DNA (mtDNA) are associated with a spectrum of health conditions
45 including cancer, aging, and Parkinson's disease¹,², in addition to their roles in primary
46 mitochondrial diseases³,⁴. Leber's hereditary optic neuropathy (LHON), which causes central
47 vision loss at a young age, is the most common optic neuropathy and an important example of a
48 mitochondrial disease⁵. The most frequent mutation 11778G>A, which is associated with LHON,
49 occurs in the mitochondrial *NADH dehydrogenase subunit 4 (ND4)* gene⁶,⁷ (Figure 1A) which is
50 a component of the mitochondrial respiratory chain⁸. This mutation leads to R340H substitution
51 in the ND4 protein⁶ (Figure 1A). Functional studies of disease-associated mtDNA mutations, as
52 well as the study of the mammalian mitochondrial genome in general, were impeded by the lack
53 of efficient mammalian mitochondrial genome editing tools. Such tools could be also applied for
54 gene therapies against diseases caused by mutations in the mitochondrial DNA.

55 Cytosine and adenosine deaminases-based tools have enabled the introduction of point transition
56 nucleotide substitutions in mammalian mtDNA. Mitochondrially targeted rat APOBEC1, which
57 can act as an RNA and DNA cytosine deaminase, was initially applied to introduce random
58 cytidine to thymidine transition mutations in the Drosophila mitochondrial genome⁹. The first
59 prominent success in mammalian mitochondrial genome editing was achieved by applying the
60 double-stranded DNA deaminase toxin A (DddA)-derived cytosine base editor (CBE) (DdCBE)¹⁰⁻
61 ¹⁹. Mitochondrial adenosine base editors (ABEs) for efficient A-to-G editing, which are known as
62 TALE-linked deaminases (TALEDs), have been also developed. TALEDs are fusions of TALE
63 domains with a modified *E. coli* deoxyadenosine deaminase TadA—TadA8e¹⁸,²⁰,²¹. Despite these
64 impressive achievements, deaminase-based techniques have a fundamental limitation—they can
65 introduce only point transition nucleotide changes (purine-to-purine or pyrimidine-to-pyrimidine).
All other modifications of the mammalian mitochondrial genome—transversion nucleotide changes (purine-to-pyrimidine or pyrimidine-to-purine), deletions, insertions, inversions, etc.— remain a challenge and require development of novel mitochondrial genome editing strategies.

Mammalian mitochondria lack the classical non-homologous end joining (NHEJ)²², and linearized mtDNA is typically degraded by components of the replication machinery²³,²⁴. Remarkably, microhomology-mediated end joining (MMEJ), as well as homologous recombination activities, are detectable in mammalian mitochondrial extracts²²,²⁵. These activities could be potentially exploited for genome editing in mammalian mitochondria upon donor DNA delivery.

Delivery of linear DNA into isolated mammalian, including human, mitochondria has been reported²⁶,²⁷. However, DNA delivery into mitochondria of cultured mammalian cells has been a challenge. Amongst possible approaches are: (1) mitochondrial targeting peptides²⁸, and (2) RNA mitochondrial import signals (RMISs)²⁹,³⁰. RMISs were discovered using SELEX³¹ and represent hairpin RNA structures that serve as a signal to target small non-coding RNA molecules in human mitochondria³²,³³. Chimeric RNA/DNA molecules, which contain RMIS and DNA sequences, can be commercially synthesized. RMIS, which is based on the D-arm of yeast tRNA *LysCUU*', has been reported to successfully deliver not only RNA, but also short (20 nt) single-stranded DNA (ssDNA) oligonucleotides²⁹,³⁰. Nonetheless, it was unclear whether this RMIS can be applied for delivery of longer DNA duplexes. Here we report, for the first time, delivery of 49 nt DNA duplexes into mitochondria of cultured human cells, applying RMIS. Moreover, we demonstrate the possibility of human mitochondrial genome editing due to delivery of ss- and double-stranded DNA (dsDNA) oligonucleotides alone or in combination with CRISPR/mito-AsCas12a into mitochondria of human cells. Editing was carried out in the clinically relevant site of the
mitochondrial *ND4* gene encompassing nucleotide G11778, whose mutations are associated with
the LHON disease (Figure 1A).

## Results

### Microhomology-Mediated End Joining (MMEJ) in human mitochondrial extracts

To confirm the presence of MMEJ activity in human mitochondria²², we tested DNA end-joining activity in human mitochondrial and nuclear extracts. For this, we synthesized two oligonucleotide duplexes (Figure 1A, Table S1), bearing the same 22nt microhomology arm indicated as “HS” in Figures 1A and 1C. These duplexes were incubated with mitochondrial and nuclear extracts from HEK 293T cells. Recombination between microhomology arms of these sequences, resulting in joining their ends, indicates MMEJ activity. The recombination product is detectable by PCR (Figure 1C, MMEJ band) with primers P5 and P7 (Table S2). We detected MMEJ but not NHEJ activity, which is expected to give longer amplicons, in human mitochondrial extracts (Figure 1C, lanes 1 and 2), while both NHEJ and MMEJ activities could be detected in the human nuclear extract (Figure 1C, lane 3). This suggests that the detected mitochondrial MMEJ activity is not a result of nuclear contamination. The microhomology arm was purposely designed to be identical to the wild-type human mitochondrial genomic site, which is affected in LHON by the most common LHON mutation 11778G>A⁶,⁷ (Figure 1A), to test whether MMEJ recombination can occur at this site.

To further validate MMEJ activity in the mitochondrial lysate, we applied another test system consisting of three DNA fragments (Figure 1B, 1D and Table S1). Two DNA duplexes, MMEJ-L-HS1 and MMEJ-R-HS2, harbored different sequences HS-1 and HS-2 respectively. The third one, MMEJ-Linker, contained both HS-1 and HS-2 microhomology arms separated by *BamHI* site. Therefore, if two MMEJ-mediated recombination events successfully occur, this will produce
a DNA fragment bearing the BamHI recognition site “GGATCC”, which replaces the wild-type site “GTCGCA” in the mitochondrial genome between HS-1 and HS-2 (Figures 1B, 1D). The resulting recombinant DNA was detected by PCR, and BamHI cleavage of the amplicon generated DNA fragments of the expected sizes (Figure 1D). These results suggest that the intrinsic mitochondrial MMEJ activity can be utilized for introduction of novel DNA sequences into the human mitochondrial genome.

## Delivery of dsDNA oligonucleotides into mitochondria in human cells

To validate the utility of mitochondrial MMEJ activity for mitochondrial genome editing in the living human cells, we next needed to target the DNA molecules into mitochondria. Previously, RNA mitochondrial import signals (RMIS) have been successfully used for delivery of artificial RNA and short (20 nt) ssDNA molecules into human mitochondria<sup>29-31,34,35</sup>. Here, we aimed to test whether an RNA hairpin, corresponding to the D-arm of yeast tRNA<sub>CUU</sub><sup>Lys</sup>, can be used to deliver longer (49 nt) dsDNA molecules, which are suitable for human mitochondrial genome editing by MMEJ. We designed chimeric oligonucleotides, which are referred to as RMIS-Dir and RMIS-Rev (Figure 2A). The 5'-RNA parts of these oligonucleotides are composed of 16 ribonucleotides, corresponding to the D-arm of yeast tRNA<sub>CUU</sub><sup>Lys</sup> (in blue) where two dihydrouridine residues are substituted by uridine residues. The 3'-parts of the chimeric oligonucleotides represent 49 nt DNA sequence, corresponding to MMEJ-Linker (Figure 2A). These DNA sequences contain homologous arms, HS-1 and HS-2 (Figure 2A), which are required for recombination and integration in the human mitochondrial *ND4* gene. The five-nucleotide sequence at the center, together with the last nucleotide of the left flanking sequence, creates the six-nucleotide BamHI restriction site, which should replace the most common “LHON” mutation
site in the mitochondrial genome. Thus, these oligonucleotides are designed to test whether the *ND4* region of human mitochondrial genome can be edited upon their delivery into human mitochondria. The expected nucleotide substitutions would lead to the changes in the ND4 protein: Ser339Stop, Arg340Ile, and Ile341Leu (Figure 1B). Therefore, these changes in the mitochondrial genome are expected to yield a truncated *ND4* protein and impair mitochondrial respiratory function.

First, we asked whether dsDNA oligonucleotides with the RMIS on both strands (RMIS-Dir:RMIS-Rev, Figure 2C) or on only one strand (RMIS-Dir:Rev, Figure 2D) can be targeted into mitochondria in HEK 293T cells. For this, we modified the protocol previously developed to assess the mitochondrial import of small non-coding RNAs³¹,³²,³⁶. We transfected HEK 293T cells with the chimeric molecules and analyzed nucleic acids isolated from purified mitochondria, mitoplasts (obtained by swelling of mitochondria), and mitochondria lysed by detergent (Figure 2 B). To eliminate cytosolic nucleic acids, half of each fraction was treated with a mix of nucleases to ensure complete degradation of DNA duplexes outside the mitochondria (see Materials and Methods). Northern blot hybridization with probes to DNA sequences used for cell transfection (Dir and Rev), mitochondrial tRNA<sup>Thr</sup> and cytosolic 5.8S rRNA demonstrated that nucleic acids located within the mitochondrial matrix were protected from the nuclease cleavage (Figure 2C, 2D, probe to mt tRNA<sup>Thr</sup>) while being sensitive to the nuclease treatment upon the mitochondrial membrane disruption. The 5.8S rRNA, localized in cytosolic ribosomes, was mostly sensitive to the nuclease treatment (Figure 2C, 2D). In one experiment, mitochondrial tRNA<sup>Thr</sup> was degraded in mitochondria but not in mitoplasts (Figure 2C lanes 3 and 5), suggesting that mitochondria were accidently disrupted during purification. The chimeric duplexes (RMIS-Dir:RMIS-Rev and RMIS-
Dir:Rev) were partially protected from the nuclease treatment in the mitoplast fractions (Figure 2C, 2D) and completely degraded by the nucleases upon lysis of mitochondria (Figure 2C, 2D).

Noteworthily, in cells transfected with Duplex 1 (Figure 2C), both strands of the duplex, RMIS-Rev and RMIS-Dir, were detected in the mitoplasts upon the nuclease treatment which indicate their delivery into the mitochondrial matrix. In cells transfected with Duplex 2 (Figure 2D, left panel), we clearly demonstrated the mitochondrial import of the chimeric RNA-DNA strand RMIS-Dir. Remarkably, the other strand of the duplex, Rev, not harboring the RMIS signal, was also protected from the nuclease treatment in the mitochondria and mitoplast fractions (Figure 2D, right panel), indicating delivery of this strand into mitochondrial matrix. These data demonstrate that a dsDNA duplex, with only one strand fused to an RNA hairpin structure, can penetrate mitochondrial membranes, thus expanding our knowledge of the nucleic acids mitochondrial import and its possible therapeutic applications.

## Mitochondrial genome editing in human cells by donor DNA delivery

Our next objective was to test whether mitochondrial import of donor DNA fragments can induce a change in human mtDNA sequence. For this, HEK 293T cells were transfected with a mix of ssDNA oligonucleotides RMIS-Dir and RMIS-Rev or with various pre-formed duplexes (Figure 3A) capable of being targeted into mitochondria. Six days post-transfection, mitochondria were isolated, and the targeted mtDNA region (an ND4 gene site) was PCR-amplified and subjected to deep sequencing. Editing efficiency was defined as the number of reads containing the edited mitochondrial sequence per one million reads (Counts Per Million = CPM) that unambiguously aligned to the mitochondrial sequence (MT:11710-11855). Since multiple mitochondrial DNA insertions known as Nuclear mitochondrial Sequences (NumtS) are found in the human nuclear genome³⁷, we applied the BLAT tool of the UCSC Genome Browser to search for sequences,
which are similar to the sequenced amplicon, in human nuclear genome³⁸. The most similar human
nuclear genomic sequences (Table S3) were then aligned to the corresponding human
mitochondrial genomic sequence (Figure S1A, see also Materials and Methods section for details).
Ultimately, only those reads that clearly matched the mitochondrial but not the nuclear genome,
were analyzed and used for the editing efficiency estimation (Figure 3B).

We detected a low but statistically significant amount of the expected four-nucleotide change (see
Figure 1C) only in samples from transfected cells (Figure 3B), indicating on a successful
microhomology-mediated editing (further referred to as MME) of the targeted gene. Notably,
RMIS-bearing DNA duplexes were significantly more efficient for human mitochondrial genome
editing than the combination of single-stranded RMIS-Dir and RMIS-Rev oligonucleotides (see
Discussion section). Thus, we succeeded to introduce the predesigned four-nucleotide change in
the human mitochondrial genome in cultured human cells. It is important to mention that a four-
nucleotide modification of the mitochondrial genome is currently not achievable by using other
techniques, such as deaminase-based mitochondrial genome editing methods.

Next, trying to improve the efficiency of mitochondrial genome editing after donor DNA delivery,
we decided to test whether mitochondrial DNA cleavage at the editing site can improve the MME
efficiency. We used a recently constructed cell line, T-REx-293-Su9-AsCas12a, carrying a
tetracycline-inducible gene of the mitochondria-targeted AsCas12a effector nuclease³⁹. For
editing, we selected a site within the mitochondrial ND4 gene bearing the protospacer adjacent
motif (PAM) TTTN required for recognition by the mito-AsCas12a effector nuclease (Figure 4A).
For this experiment, new donor chimeric RNA/DNA oligonucleotides (RMIS-Dir-New and
RMIS-Rev-New) were designed, each containing two different Homology Sequences regions
(new HS-1 and new HS-2) flanking the site of specific cleavage by mito-AsCas12a/crRNA and
the editing site (Figure 4A). A five-nucleotide change, which we expected to introduce by MME, is located in the region corresponding to crRNA, therefore, the donor DNA oligonucleotides and the edited mtDNA sequence should not be recognized and cleaved by the mito-AsCas12a/crRNA complex. Moreover, all the nucleotide changes were introduced in the third position of the codon, leading to synonymous codons and thus not inducing any alteration in the ND4 amino acid sequence.

T-REx-293-Su9-AsCas12a cells were transfected with donor ssDNA or dsDNA together with the crRNA, in combination with induction of the mito-AsCas12a with tetracycline (Figure 4B, 4C). To prevent degradation of cleaved mtDNA molecules, some samples were also treated with an siRNA targeting mitochondrial genome maintenance exonuclease 1 (MGME1), as described previously³⁹. MGME1 plays a key role in the degradation of linearized mtDNA, thus, its downregulation was expected to stabilize the ends of cleaved mitochondrial DNA molecules²³ and facilitate their recombination and ligation.

After cultivation of transfected cells, mtDNA was isolated and the region of interest was PCR-amplified with the introduction of unique molecular identifiers (UMIs)⁴⁰. After the library preparation and deep sequencing, the editing efficiency was determined for different transfection conditions, including control experiment without donor DNA (Figure 4C). Differences between the mitochondrial sequence and similar NumtS outside the recombination region and the primers annealing sites (Figure S1B, Table S4) allow unambiguous alignment of reads to the mitochondrial sequence. Unexpectedly, we obtained extremely low levels of editing. To ensure the significance of the data, we compared the numbers of reads containing the expected five-nucleotide change and those with four- or three-nucleotide changes (Figure 1C and Figure S2). No four- or three-nucleotide changes were detected, indicating that the directed change of five nucleotides was not
a random event. Moreover, no editing events were found in control cells without donor DNA delivery, suggesting that the editing requires donor DNA. The five-nucleotide change was also not found in cells transfected with ss-donor DNA without *MGME1* downregulation, which can indicate on the ssDNA degradation in mitochondria by MGME1.

The most significant level of mtDNA editing was obtained in cells transfected with ds-donor DNA and crRNA, both with and without *MGME1* silencing. Thus, as in the previous experiment, RMIS-bearing DNA duplexes were more efficient for human mitochondrial genome editing than the single-stranded chimeric oligonucleotides. Extremely low efficiency of editing in the last experiment may be explained by specific features of the T-REx-293-Su9-AsCas12a cell line, characterized by reduced respiration level³⁹, which can influence the efficiency of cell transfection and the entire mitochondrial metabolism, including activity of enzymes involved in the microhomology mediated recombination.

Taken together, our results indicate that human mtDNA sequence can be modified by donor dsDNA delivery into mitochondria. This proof-of-concept study opens further possibilities to improve the efficiency of mtDNA editing and develop applications of this novel mtDNA editing approach.

## Discussion

### *Challenge of mtDNA editing*

The objective of the study was to apply the mitochondrial machinery of microhomology-mediated end joining (MMEJ) to human mtDNA editing, i.e., site-directed mutagenesis of the mitochondrial genome, a challenge that has not yet been fully resolved.

The 16.5 kb human mitochondrial genome encodes 13 transmembrane subunits of the respiratory chain complexes, 22 tRNAs, and two ribosomal RNAs required for the mitochondrial translation.
Therefore, any mutation in this extremely compact genome can lead to drastic changes in protein structure and/or expression, thereby causing respiratory chain dysfunction and decreased ATP production. Such defects particularly affect nervous and muscle tissue, but can also be associated with autoimmune diseases, cancer, diabetes, and many other disorders⁴¹. Despite substantial progress in understanding the molecular mechanisms of mitochondrial diseases, there is still a lack of treatments for patients affected by these disorders. The only notable exception is a recently approved in the European Union coenzyme Q analogue, idebenone, which has a positive effect in 50% early-stage LHON patients⁴².

Recent advances in direct editing of DNA sequences offer the possibility of converting one base pair into another, thus directly correcting certain pathogenic mutations⁴³. The recently reported DdCBE-based approach of mammalian mitochondrial DNA editing utilized cytosine deamination and allowed efficient conversion of C:G into T:A pairs in tissues of living mice¹⁰⁻¹². Similarly, conversion of A:T into G:C pairs has been achieved by applying mitochondria-targeted adenosine deaminase TALEDs¹⁸,²⁰,²¹. Besides a significant level of off-target and bystander edits¹³⁻¹⁵, as well as context-dependence¹⁰, these editing approaches can introduce only transition nucleotide changes (purine-to-purine or pyrimidine-to-pyrimidine). The strategy we propose in the current study, which uses RMIS-based DNA oligonucleotide delivery and the intrinsic MMEJ enzymatic machinery for mammalian mitochondrial genome editing, is capable of introducing virtually any type of nucleotide changes including multiple mutations in the region of interest. For this, DNA oligonucleotides bearing regions of homology with mtDNA and containing desired mutations should be delivered into mitochondria of human cells.
268 **DNA import into mammalian mitochondria**

269 Here, we demonstrated for the first time that RNA mitochondrial import signals (RMIS),
270 previously discovered in our laboratory, can be used to target relatively long (~ 50 bp) dsDNA
271 molecules into human mitochondria. We are aware that the experimental estimation of DNA, as
272 well as RNA, mitochondrial import can be biased³³. For this reason, we treated isolated
273 mitochondria and mitoplasts with a mixture of nucleases, optimized to remove possible DNA
274 contaminants from the mitochondrial surface. We also performed control experiments by lysing a
275 portion of mitochondria before nuclease treatment; the detection of DNA molecules in
276 mitochondria and mitoplasts but not in the lysates confirmed their localization in the mitochondrial
277 matrix (Figure 2). Interestingly, when we transfected cells with a DNA duplex carrying RMIS on
278 a single strand, the entire duplex was detected in mitoplasts, indicating that the DNA strands were
279 not separated during translocation across mitochondrial membranes (Figure 2D). This is consistent
280 with our previous data, which showed that a tRNA could be imported into yeast mitochondria as
281 a folded molecule⁴⁴, and with previous studies demonstrating the import of dsDNA in plant and
282 human mitochondria²⁶,²⁷,⁴⁵.

283 The molecular mechanism of nucleic acid translocation across mitochondrial membranes is not
284 fully understood. Polynucleotide phosphorylase (PNPase), located in the intermembrane space of
285 mitochondria, may be involved in the recognition of stem-loop RNA structures (RMIS)⁴⁶⁻⁴⁸. Outer
286 membrane translocase (TOM) and voltage-gated anion channel (VDAC) may facilitate
287 translocation across the outer mitochondrial membrane (reviewed in³³). It should be noted that
288 VDAC oligomers have been reported to form pores for the release of mtDNA fragments⁴⁹, and the
289 structure and role of Porin1 hexamers have recently been studied in yeast⁵⁰. We can thus
290 hypothesize that pores formed by VDAC hexamers may serve not only for the export of mtDNA
291 fragments, but also for the uptake of dsDNA fragments from the cytosol.

292 **Mitochondrial genome editing by donor DNA delivery**

293 We demonstrated here that donor DNA delivery into human mitochondria results in a low but
294 statistically significant introducing of designed nucleotide changes into intact mitochondrial loci
295 (Figure 3). This donor DNA-induced editing without the preliminary cleavage of mtDNA can be
296 explained in two ways: first, by occasional random mtDNA cleavage by reactive oxygen species
297 (ROS)⁵¹ or by stalling of the replication fork⁵². These events, if occurred in the region of homology
298 with donor DNA, can induce mtDNA reparation by MMEJ mechanism resulting in the designed
299 nucleotide changes which have been introduced into the donor DNA oligonucleotides. The second
300 possibility consists in the proposed MME model. The model of the MME is shown in Figure 5.
301 We hypothesize that RNA hairpin structures can be removed from the donor DNA molecules by
302 mitochondrial nucleases EndoG, which digests both DNA⁵³,⁵⁴ and RNA⁵⁴ ds-substrates with
303 preference for C:G tracks, and ExoG, which can cleave at RNA-DNA junctions⁵⁵. Noteworthily,
304 the RMIS sequence has a CG track in the ds-site (Figures 2A and 4A). Afterwards, like in MMEJ
305 mechanism, PARP1 recruits MRE11, BRCA1 and CtlP. These proteins, performing the 5'-3' ends
306 recession and production of 3' overhangs, have been identified within human mitochondria²².
307 Strand invasion, microhomology alignment and extension by DNA synthesis then can be
308 facilitated by RAD51 and POLQ, which have both nuclear and mitochondrial localizations⁵⁶⁻⁵⁹.
309 Non-homologous tails are removed by FLAP endonuclease FEN1, followed by DNA synthesis
310 and end-joining by DNA ligase III⁵²,⁵⁷. This model is supported by our data, which demonstrated
311 that DNA duplexes were more efficient for human mitochondrial genome editing than the single-
312 stranded chimeric oligonucleotides (Figure 3). This can be explained not only by the rapid
313 degradation of single-stranded DNA, but also by the fact that the ends of donor double-stranded
314 DNA molecules can mimic double-strand breaks in mtDNA, thereby recruiting PARP1 and the
315 entire MMEJ machinery.

316 Both possible mechanisms are characterized by very low efficiency of introducing nucleotide
317 changes (2×10⁻⁴–4×10⁻⁴ or less). Noteworthy, these values are close to the reported efficiency of
318 introducing changes into the intact mammalian nuclear genome of somatic cells by homologous
319 recombination (5×10⁻⁴)⁶⁰. One possible way to improve the efficiency of our approach is to
320 introduce site-specific cleavage into mitochondrial DNA. Double-strand breaks in editing
321 mitochondrial sites are expected to facilitate integration of delivered oligonucleotides into these
322 sites, enhancing efficiency of mammalian mitochondrial genome editing. Quick degradation of
323 linearized unedited mitochondrial genomic DNA²³,²⁴ should further improve efficiency of our
324 approach. Up to date, four types of DNA endonucleases have been successfully delivered into
325 mitochondria and used for highly efficient site-specific mitochondrial DNA cleavage: (1)
326 restriction enzymes (REs)⁶¹⁻⁶³, (2) zinc-finger nucleases (ZFNs)⁶⁴⁻⁶⁷, (3) transcription activator-
327 like effector nucleases (TALENs)⁶⁸⁻⁷⁰, and (4) the meganuclease ARCUS⁷¹,⁷². The primary
328 purpose of all these techniques is a heteroplasmy shift, which is supposed to improve the ratio of
329 wild-type to mutant mitochondrial genomic DNAs. Obviously, the heteroplasmy shift approach
330 cannot be applied to nearly-homoplasmic mitochondrial diseases such as LHON. Nonetheless, a
331 combination of our RMIS-based DNA oligonucleotide delivery approach with mitochondria-
332 targeted site-specific endonucleases promises to be applicable for homoplasmic mitochondrial
333 diseases as well. Importantly, this combined approach has the potential to introduce not only point
334 changes into mammalian mitochondrial genomic DNA, but also large deletions and insertions.
Recently, the applications of CRISPR technology for specific mitochondrial genome cleavage have been reported³⁵,³⁹. We, therefore, attempted to combine CRISPR/mito-AsCas12a technology with donor DNA delivery to improve mitochondrial genome editing efficiency (Figure 4). A statistically significant number of the expected five-nucleotide change was detected in transfected cells; however, global editing was unexpectedly less efficient. This could be explained by lower respiration capacity of the cell line and/or also by a very low efficiency of mtDNA specific cleavage by the CRISPR/mito-AsCas12a system³⁹. Notably, in these experiments, the engineered nucleotide changes were distributed over a larger region of the mitochondrial genome (16 nucleotides) (Figure 4A), which could also significantly reduce the editing efficiency. We believe that optimizing the mitochondrial CRISPR/mito-AsCas12a system together with donor DNA design can create an efficient mtDNA editing tool.

In summary, we report a novel strategy of mtDNA editing in living human cells via RMIS-based DNA oligonucleotide delivery. This approach was used for the predefined change of four or five nucleotides in the sequence of the human mitochondrial *ND4* gene. MME can introduce all possible changes into the mammalian mitochondrial genome, opening opportunities for mtDNA editing in basic and medical research.

## Materials and Methods

### Cell Culture

HEK293T cells (ATCC, Manassas, VA) were maintained in DMEM-high glucose medium (cat # 01-052-1A, Biological Industries) complemented with 4 mM L-Glutamine, 10% fetal bovine serum (FBS), and antibiotics (Penicillin G – 100 U/ml; Streptomycin Sulfate –100µg/ml; Nystatin – 12.5 U/ml). 1 mM pyruvate and 50 µg/ml uridine were added to the medium in order to culture HEK293T cells after mitochondrial genome editing.
358 Generation of the T-REx-293-Su9-AsCas12a cells with tetracycline-inducible expression of Su9-AsCas12a and culturing conditions for these cells are reported by us elsewhere³⁹. When required, 359 Su9-AsCas12a expression was induced by adding 100 ng/mL of tetracycline to culturing medium. 360

361 **Transfections**

362 All transfection with HEK293T cells were conducted with the lipofectamine transfection reagent 363 (cat # 18324012, ThermoFisher Scientific) according to the manufacturer instructions. Briefly, 364 HEK293T cells were seeded in DMEM-high glucose medium complemented with 4 mM L-365 Glutamine, 10% fetal bovine serum (FBS) without any antibiotics a day before transfections. The 366 HEK293T cells were transfected at confluence 80-90%. Transfection mixtures of the 367 lipofectamine reagent with oligonucleotides were prepared according to the manufacturer 368 instructions in DMEM-high glucose medium without serum and antibiotics. 30 µl of the 369 lipofectamine reagent and 500 pmol of an ss-oligonucleotide or 250 pmol of an oligonucleotide 370 duplex were used per one transfection in a T25 flask. 6 µl of the lipofectamine reagent and 100 371 pmol of an ss-oligonucleotide or 50 pmol of an oligonucleotide duplex were used per one 372 transfection in a well of a 12 well plate. The medium on HEK293T cells was changed DMEM- 373 high glucose medium without serum and antibiotics and the transfection mixtures were added to 374 the cells. The medium on the transfected HEK293T cells was changed back DMEM-high glucose 375 medium complemented with 4 mM L-Glutamine, 10% fetal bovine serum (FBS) without 376 antibiotics in 4 hours after transfection. The next day after transfections the medium on the cells 377 was changed to the same medium with antibiotics. 378

378 Transfections of T-REx-293-Su9-AsCas12a cells were conducted as described in the subsection 379 "Mitochondrial Genome Editing Assay with Donor DNA Delivery and CRISPR/Cas12a". 380
380 **Mitochondria Purification**

381 Mitochondria purification for the MMEJ assay was conducted according a shorten version of a
382 published protocol⁷³ with modifications. Briefly, HEK293T cells were collected by trypsinization
383 from 3 T75 flasks and washed with PBS. Then they were suspended in 2 ml of an ice-cold breakage
384 buffer Bcells-1 (225 mM mannitol, 75 mM sucrose, 0.1 mM EGTA, and 30 mM Tris-HCl pH 7.4)
385 and disrupted by one out of two methods. The cells were homogenized either with 400 strokes at
386 4,000rpm applying Dounce-type homogenizer with Teflon pestle, or by passing the cells 25-30
387 times through a 25-gauge needle attached to a 2 ml syringe. The homogenate was centrifuged 3
388 times at 600g for 5 minutes at +4°C in order to remove nuclei, unbroken cells, and cell debris. The
389 mitochondria containing supernatant was transferred to a new Eppendorf tube after each
390 centrifugation. 1/20 of the nuclei-containing pellet after the first centrifugation was lysed in a
391 mitochondrial lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgCl2, 10% glycerol,
392 2 mM EGTA, 2 mM EDTA, 0.2% Triton X-100, and 1 mM DTT) complemented with Half
393 Protease and Phosphatase Inhibitor Cocktail (cat # 78441, ThermoFisher Scientific) by suspending
394 the pellet in 100µl of the ice-cold mitochondrial lysis buffer and leaving the suspension on ice for
395 30 minutes and centrifuging it at 13,200g for 5 minutes at +4°C. Protein concentration in the
396 supernatant (nuclear extract) was assessed by the Bradford method and then it was aliquoted by
397 10 µl, snap frozen in liquid nitrogen, and kept at -80°C. Mitochondria was precipitated from the
398 mitochondria-containing supernatant by centrifugation at 7,000g for 10 minutes at +4°C. The
399 mitochondrial pellet was suspended in 2 ml of ice-cold buffer Bcells-2 (225 mM mannitol, 75 mM
400 sucrose, and 30 mM Tris-HCl pH 7.4) and centrifuged again at 7,000g for 10 minutes at +4°C.
401 The mitochondrial pellet was again suspended in 2 ml of ice-cold buffer Bcells-2 and centrifuged at
402 10,000g for 10 minutes at +4°C. The resulting mitochondrial pellet was suspended in 100µl of the
403 ice-cold mitochondrial lysis buffer with Half Protease and Phosphatase Inhibitor Cocktail. The
404 lysis was conducted and protein concentration was determined as it is described for the nuclear
405 extract. The resulting mitochondrial extract was aliquoted by 10 µl, snap frozen in liquid nitrogen,
406 and kept at -80°C.

407 Mitochondria purification from HEK293T cells for the oligonucleotide delivery assay and deep
408 sequencing was conducted as it was published elsewhere³²,³⁶. Briefly, HEK293T cells were
409 detached and collected from one T75 flask by incubating them in 1 mM EDTA in PBS. Then these
410 cells were washed in PBS and suspended in 1 ml of ice-cold buffer A (0.6 M sorbitol, 10 mM
411 HEPES-KOH, pH 7.5, 1 mM EDTA) with 0.1% BSA. The cells were disrupted by passing them
412 30 times through a 25-gauge needle attached to an 1 ml syringe. The homogenate was diluted in 2
413 times by adding 1 ml of ice-cold buffer A with 0.1% BSA. The disrupted cells then were
414 centrifuged 4 times at 600g for 5 minutes at +4°C in order to remove nuclei, unbroken cells, and
415 cell debris. The mitochondria containing supernatant was transferred into a new Eppendorf tube
416 each time. The mitochondria were precipitated at 15,000g for 20 minutes at +4°C. The
417 mitochondrial pellet was then treated as described in subsections "Oligonucleotide Delivery
418 Assay" and "Mitochondrial Genome Editing Assay".

419 Primer and Molecular Size Ladder Labeling and Radioactive PCR

420 20 pmol of P7 primer (Table S1) was labeled in 20 µl of a labeling reaction with 20 units of
421 polynucleotide kinase T4 (PNK T4) (cat # M0201, NEB) and 13.3 pmol of ATP-[γ-³²P] (6,000
422 Ci/mmol, cat # NEG035C001MC, PerkinElmer) in the PNK T4 buffer. 0.2 pmol of the
423 radioactively labeled P7 primer were used per 10 µl of a PCR reaction along with 5 pmol of each
424 – nonradioactive P5 and nonradioactive P7 primers (Table S1). The radioactive PCR was
425 conducted with Taq polymerase at the final concentration 0.1 U/µl and at the final dNTP
concentration 0.2 pmol/µl each in a commercial Taq polymerase buffer (cat # B9004S, NEB). The
PCR conditions are further specified in the subsection “MMEJ Assay”.

Oligonucleotides MMEJ-Dir (99nt), MMEJ-Rev (91nt), MMJE-Linker-Dir (66nt), MMEJ-L-Dir
(54nt), and MMEJ-L-Rev (46nt) (Table S1) were labeled at the same conditions as the P7 primer
and used as a molecular size ladder in the MMEJ assay.

MMEJ Assay

The MMEJ assay was conducted as it was described by researchers from Sathees C. Raghavan's
group<sup>22,74</sup> with modifications. Briefly, duplexes MMEJ-Ovr-L, MMEJ-Ovr-R, MMEJ-L, MMEJ-
Linker, and MMEJ-R were obtained by annealing of sense and antisense oligonucleotides: MMEJ-
Ovr-L-Dir + MMEJ-Ovr-L-Rev, MMEJ-Ovr-R-Dir + MMEJ-Ovr-R-Rev, MMEJ-L-Dir + MMEJ-
L-Rev, MMEJ-Linker-Dir + MMEJ-Linker-Rev, and MMEJ-R-Dir + MMEJ-R-Rev,
correspondingly. The oligonucleotide sequences can be found in Table S2. Overhangs on each side
of every duplex (Figure 1 A, B) are included in order to reduce PCR artifacts. The MMEJ assay
buffer composition was 50 mM Tris-HCl pH 7.6, 20 mM MgCl<sub>2</sub>, 10% PEG-3350, 1 mM ATP, 1
mM DTT. All MMEJ reactions were conducted in the volume = 20 µl. 5 µg of mitochondrial or
nuclear extract were added to one MMEJ reaction. In the MMEJ assays with MMEJ-Ovr-L and
MMEJ-Ovr-R duplexes each duplex was added in the final concentration 4 nM. In the MMEJ
assays with MMEJ-L, MMEJ-Linker, and MMEJ-R duplexes, MMEJ-L and MMEJ-R duplexes
were added in final concentrations 4 nM each, while MMEJ-Linker duplex was added in the final
concentration 8 nM. The MMEJ reactions were conducted for 4 hours at 37°C and stopped by heat
inactivation at 65°C for 20 minutes. Each MMEJ reaction was diluted in 2 times with water and 2
µl of the diluted MMEJ reaction were taken for radioactive PCR with P5 and P7 primers in the
volume of 10 µl. The radioactive PCR was conducted as it is described in the subsection “Primer
Labeling and Radioactive PCR". Precise PCR conditions for the MMEJ assays with MMEJ-Ovr-L and MMEJ-Ovr-R duplexes were: 94°C – 3min; cycle - 94°C – 30sec, 60°C – 1 min, and 68°C – 1 min (16 cycles); 68°C – 5 min. Precise PCR conditions for the MMEJ assays with MMEJ-L, MMEJ-Linker, and MMEJ-R duplexes were: 94°C – 3min; cycle - 94°C – 30sec, 60°C – 1 min, and 68°C – 1 min (26 cycles); 68°C – 5 min.

In the case of MMEJ assays with MMEJ-Ovr-L and MMEJ-Ovr-R duplexes, the recombination product is detectable by the radioactive PCR with P5 and P7 primers (Figure 1A and C, upper panel at the left). 5 µl of each radioactive PCR reaction were immediately loaded and resolved on a 10% denaturing polyacrylamide gel (PAAG) with 7M urea. The gels were dried and exposed to a phosphorimager screen, which was then visualized on Typhoon phosphorimager (FLA 7000).

In the case of MMEJ assays with MMEJ-L, MMEJ-Linker, and MMEJ-R duplexes, we asked whether the MMEJ activity can be used to join DNA ends through a linker containing an enzyme restriction site. Introduction of a novel DNA sequence (e.g. BamHI site) is an indication of MMEJ. Design of oligonucleotide duplexes in this case was different (Figure 1B and D, upper panel at the right). One duplex (MMEJ-L) has a 22nt microhomology arm, identical to the sequence located in the human mitochondrial genome to the left of the six-nucleotide sequence "GTCGCA" and designated "HS-1" (Figure 1B and D, upper panel at the right). The bold underlined G in this sequence is G11778 and is in fact the most commonly mutated nucleotide in LHON⁶,⁷. The other duplex has a 22nt microhomology arm, which is designated "HS-2", identical to the sequence located in the human mitochondrial genome (to the right of the above six-nucleotide sequence in Figure 1B and D, upper panel at the right). These duplexes (MMEJ-L and MMEJ-R) cannot recombine with each other but only with the linker MMEJ-Linker. Therefore, the radioactive PCR with primers P5 and P7 can detect a product of two MMJE-mediated recombination events with
the BamHI site in the middle (Figure 1B and D, upper panel at the right). In order to detect this product of double recombination, the radioactive PCR reactions were diluted in 2 times with water and 2 µl of each diluted radioactive PCR reaction were digested with 20 U of BamHI (cat # R0136S, NEB) in the BamHI buffer in 12 µl of the final volume for 6 hours at 37°C. The control undigested samples were incubated for the same time at the same conditions but without BamHI.

Then the samples were loaded and resolved on a 10% denaturing PAAG with 7M urea. The gels were dried and exposed to a phosphorimager screen, which was visualized on Typhoon phosphorimager (FLA 7000).

## Oligonucleotide Delivery Assay

Oligonucleotides, which are listed in Table S5, were used in the oligonucleotide delivery and mitochondrial genome editing assays. RMIS-Dir and RMIS-Rev hybrid oligonucleotides contain at their 5'-end a known mitochondrial RNA import signal – modified D-arm of yeast tRNA LysCUU – rGrCrGrCrArArUrCrGrGrUrArGrCrGrC<sup>29,30</sup>. Duplex 1, duplex 2, duplex 3, and duplex 4 were obtained by annealing of oligonucleotides RMIS-Dir + RMIS-Rev, RMIS-Dir + Rev, Dir + RMIS-Rev, and Dir + Rev correspondingly. HEK293T cells were transfected with oligonucleotides and duplexes in T25 flasks as it is described in the subsection "Cell Culture and Transfections". Immediately after transfections, the medium on the cells was changed to the medium with pyruvate and uridine. In 24 hours after transfections the transfected HEK293T cells were reseeded from T25 flasks to T75 flasks (one T25 flask to one T75 flask). In 72 hours after the transfections mitochondria were purified from the transfected cells as it is described in the subsection "Mitochondria Purification". The purified mitochondria were divided in 6 equal parts and subjected to 10-minute incubation in 100 µl of one of the following buffers – the breakage buffer A (0.6 M sorbitol, 10 mM HEPES-KOH, pH 7.5, 1 mM EDTA), the swelling buffer B (10 mM
HEPES-KOH, pH 7.5, 1 mM EDTA), and the lysing buffer C (10 mM HEPES-KOH, pH 7.5, 1 mM EDTA, 0.5% n-dodecyl-β-maltoside) with and without a mix of nucleases. Compositions of the buffers were already published elsewhere³²,³⁶. The mix of nucleases, which was added to each sample, consisted of 5 µl of the RNase A/T1 mix (2 mg/ml, 5,000 U/ml), 0.5 µl of RNase I<sub>f</sub> (50,000 units/ml), 5 µl of DNase I (2,000 units/ml), 5 µl of Exo I (20,000 units/ml), and 0.25 µl of Exo III (100,000 units/ml). Immediately after the treatment, 1.07 ml of TRI reagent (cat # 93289, Sigma-Aldrich) with adjusted pH was added to each sample. The TRI reagent pH was adjusted by adding 70 µl of 3M KOH to each milliliter of the TRI reagent in order to ensure that the TRI reagent pH is higher than 8.0. This pH allows to purify the total nucleic acids (RNA + DNA). The total nucleic acids were then purified according to the manufacturer protocol for RNA purification. 60 µg of glycogen were added to the aqueous phase of each sample during the purification in order to ensure more complete oligonucleotide recovery. The purified total nucleic acids were subjected to the small RNA/DNA Northern blot as it is described in the subsection "Small RNA/DNA Northern Blot" in order to assess oligonucleotide delivery into mitochondria.

### Mitochondrial Genome Editing Assay with Donor DNA Delivery only

HEK293T cells in wells of a 12 well plate were transfected with oligonucleotides and duplexes, which were described in the subsection "Oligonucleotide Delivery Assay", as it is described in the subsection "Cell Culture and Transfections". Immediately after transfections, the medium on the cells was changed to the medium with pyruvate and uridine. In 24 hours after transfections, a half of the transfected HEK293T cells from each well of the 12 well plate was seeded in a 35 mm cell culture dish. In 82 hours after the transfections, the transfected HEK293T cells from each 35 mm dish were seeded in one T75 and one T25 flasks. In 144 hours after the transfections the transfected HEK293T cells in T25 flasks were frozen while mitochondria were purified from T75 flasks as it
is described in the subsection “Mitochondria Purification”. The purified mitochondria were lysed
in 200 µl a lysis buffer (10 mM Tris-HCl pH 8.8, 1 mM EDTA, 0.5% Triton X-100) by heating
the samples at 95°C for 5 minutes. 2 µl of each lysate were used to amplify the edited site of the
human mitochondrial genome with primers Mit-ND4-DS-Dir and Mit-ND4-DS-Rev (Table S1)
applying Q5 high-fidelity DNA polymerase (Cat. # M0491, New England Biolabs, USA). The
resulting PCR products were submitted for library preparation and deep sequencing at the
Genomics Applications Laboratory (Core Research Facility, Faculty of Medicine, The Hebrew
University of Jerusalem, Jerusalem).

## Mitochondrial Genome Editing Assay with Donor DNA Delivery and CRISPR/Cas12a

For *MGME1* knockout (when required), T-REx-293-Su9-AsCas12a cells were transfected in suspension with 40 nM DsiRNA targeting *MGME1* (hs.Ri.MGME1.13.8, IDT) (Table S5) using Lipofectamine RNAiMAX (Invitrogen) in Opti-MEM (Gibco, Fisher Scientific). After 6 hours, the transfection medium was replaced with complete EMEM. Three days later, transfection was repeated under standard adherent cell conditions.

After additional 48 hours, cells were transfected in suspension with crRNA (320 ng/ml) targeting
the *ND4* gene of human mtDNA and donor DNA (RMIS-Rev-New or RMIS-Dir-New:RMIS-Rev-
New at 2 µg/ml) using Lipofectamine 2000 (Invitrogen) in Opti-MEM. After 6 hours, the medium
was replaced with EMEM supplemented with 100 ng/ml tetracycline to induce Su9-AsCas12a
expression.

Seven days later, cells were detached, lysed, and total DNA was extracted using the QIAamp DNA
Mini Kit (Qiagen). 100 ng of each purified mitochondrial DNA sample were used to amplify the
edited site of the human mitochondrial genome with introduction of unique molecular identifiers
(UMIs) according to a published protocol⁴⁰ with some modifications. Briefly, reactions of UMIs
541 introduction were conducted with the NGS-ND4-Dir primer (Table S2). Primer concentration was 542 0.02 µM. Total reaction volume was 10 µl. Q5 Hot Start High-Fidelity 2X Master Mix (Cat. # 543 M0494, New England Biolabs) was used for this reaction. The reaction conditions were: 98°C – 2 min, 59°C – 15 min, 65°C – 15 min, 72°C – 7 min. The reactions were purified from primers with 544 were purified with GeneRead Size Selection Kit (Cat. # 180514, QiaGen, Germany) according to 545 the manufacturer's instruction. Two rounds of purification were applied. The edited site with 546 introduced UMIs was further amplified from purified DNA with primers Ext Primer and NGS- 547 ND4-Rev applying the Q5 Hot Start High-Fidelity 2X Master Mix (Cat. # M0494, New England 548 Biolabs). PCR conditions were: 95°C – 3min; cycle - 95°C – 30sec, 59°C – 40sec, and 68°C – 549 550 40sec (16 cycles); 68°C – 7 min.

551 The obtained amplicons were submitted for library preparation and deep sequencing at the 552 Genomics Applications Laboratory (Core Research Facility, Faculty of Medicine, The Hebrew 553 University of Jerusalem, Jerusalem).

554 Small RNA/DNA Northern Blot

555 Total nucleic acid samples were resolved on denaturing 10% PAAG with 7 M urea. The transfer 556 was conducted in x0.5 TBE at 1 mA/cm² on zeta-probe membrane (cat # 1620159, BioRad). After 557 UV-crosslinking the membranes were hybridized with the following probes: Dir and Rev (Table 558 S5), Anti-tRNA-Thr (specific to mitochondrial Thr tRNA), and Anti-5-8S RNA (specific to 559 cytosolic 5.8S rRNA) (Table S6).

560 Deep Sequencing

561 The deep sequencing of PCR products was conducted at the Genomics Applications Laboratory 562 (Core Research Facility, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem). 563 Libraries were prepared according to the 16S metagenomic sequencing library preparation
protocol<sup>75</sup>. In brief, the PCR products were purified with AMPure XP beads and indexes, which are listed in Tables S7 and S8, were introduced by 8 cycles of index PCR. 150nt single end sequencing was conducted on the Illumina machine “NextSeq 500” (cat # SY-415-1001, Illumina) using the Nextseq 500/550 mid output kit v2.5 (150 cycles). The deep sequencing data are available in the Sequence Read Archive (SRA) database: https://www.ncbi.nlm.nih.gov/sra/PRJNA1332044.

The bioinformatic analyses of both sets of the deep sequencing data were conducted by Dr. Yuval Nevo (Info-CORE, Bioinformatics Unit of the I-CORE at the Hebrew University of Jerusalem, Jerusalem) as outlined below.

### Bioinformatic Analysis of Mitochondrial Genome Editing Assay with Donor DNA Delivery only

Briefly, sequence quality was inspected with FastQC software. Cutadapt software<sup>76</sup> was employed in order to trim low-quality and adapter sequences. This software was further applied for filtering sequences shorter than 75nt and/or lacking the forward primer sequence. Remaining low-quality reads were removed with the fastq_quality_filter software. The refined reads were further aligned to the human genome version GRCh38 employing Bowtie 2 software<sup>77</sup> with default parameters. Uniquely aligned reads with a single best alignment score, which span the expected amplicon mitochondrial genome positions (MT:11710-11855), were then inspected for changes in the editing site. Reads with the string “T0C0G1A” in their MD tag were designated as reads with edited sequence (“Mut”). Those of the remaining reads, which had an alignment score higher than -15, were designated as reads with wild type sequence (“WT”).

The Mut reads and eight the most similar human nuclear genomic sequences (Supplementary Table 6) were then aligned to the corresponding human mitochondrial genomic sequence applying the
Bowtie 2 software with permissive parameters (version 2.3.4.3, command: bowtie2 -f -L 12 --local --mp 2 --rdg 3,1 --rfg 3,1 --all -x MT -U genomic_homeologues.fa). The latter provided an unambiguous verification that most of the Mut reads are indeed better aligned to the human mitochondrial genomic sequence than to any human nuclear genomic sequence. This alignment also verified that most of the Mut reads have the predesigned change in the nucleotide sequence.

## Bioinformatic Analysis of Mitochondrial Genome Editing Assay with Donor DNA Delivery and CRISPR/Cas12a

Analogously to the previous analysis, the cutadapt software was applied to trim low quality, adapter, and poly-G sequences. The same software was utilized to remove reads, which became shorter than 95nt after trimming of low quality sequences. Fastq质量_filter software was used for removal of remaining low-quality reads. The cleaned reads were aligned to the human genome version GRCh38 employing Bowtie 2 software with the -a parameter. Uniquely aligned reads with a single best alignment score, which span the expected amplicon mitochondrial genome positions (MT:11790-11803) in the expected orientation, were de-duplicated in a such way that each de-duplicated read has either a unique UMI or a unique human mitochondrial genome-aligned sequence. The unique aligned and de-duplicated reads were checked for the presence of any of the possible combinations of the 5 intended nucleotide substitutions. The unique aligned and de-duplicated reads with all the 5 intended nucleotide substitutions were considered as edited. The remaining reads were considered as wild type reads.

## Statistics

Statistical significance of differences in the mitochondrial genome editing efficiency was assessed by the two-sided permutation test with plus-one correction and adjustment via Benjamini-Hochberg.
609 Hochberg false discovery rate. One simulation with 100,000 permutations was conducted for each pair of samples.
610

611 **Data availability statement**

612 The deep sequencing data are available in the Sequence Read Archive (SRA) database: https://www.ncbi.nlm.nih.gov/sra/PRJNA1332044. The raw gel autography and Northern blot data are available from the corresponding authors.
613 614

615 Acknowledgements

616 We thank Dr. Yuval Nevo from Info-CORE, the I-CORE Bioinformatics Unit of the Hebrew 617 University of Jerusalem for Bioinformatics data analysis.

618 We thank Anne-Marie Heckel (UMR 7156 GMGM, Strasbourg) for technical help in cells 619 manipulating.

620 N.E., N.S., N.N. and I.T. were supported by the Interdisciplinary Thematic Institute IMCBio+, as 621 part of the ITI 2021-2028 program of the University of Strasbourg, CNRS and Inserm, supported 622 by IdEx Unistra (ANR-10-IDEX-0002), EUR (IMCBio ANR-17-EUR-0016) and SFRI 623 (STRAT'US project, ANR-20-SFRI-0012) within the framework of France 2030 National 624 Program. N.N. was supported by Region Grand-Est (France). O.P. is supported by the German- 625 Israeli Foundation for Scientific Research and Development (GIF; Grant No. 1561) and the 626 German-Israeli Project Cooperation (DIP; Grant No. 17516).

627 Declaration of interests

628 The authors declare no competing interests.
## Keywords

Mitochondria, mitochondrial genome editing, mitochondrial DNA delivery, mitochondrial

CRISPR, microhomology-mediated mtDNA editing.

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897 **List of figure legends**

898 **Figure 1. MMEJ Assay.** (A) Sequences of the wild-type and LHON-associated mutant *ND4* gene alleles and ND4 proteins (upper panel) aligned to MMEJ-L-HS and MMEJ-R-HS duplexes (lower panel). Nucleotides and amino acid residues, which differ between wild-type and mutant alleles and proteins, are highlighted in purple. Sequence within the HS rectangle corresponds to the microhomology recombination site HS. (B) DNA duplexes applied for *BamHI* site insertion by MMJE (lower panel) and changes introduced in the *ND4* gene and ND4 protein during the mitochondrial genome editing by donor DNA delivery alone. The edited *ND4* gene nucleotides and corresponding amino acids are highlighted in bold gray. Sequences in the HS-1 and HS-2 rectangles correspond to the microhomology recombination sites HS-1 and HS-2. (C) End-joining activity in mitochondrial and nuclear extracts tested with DNA duplexes MMEJ-L-HS and MMEJ-R-HS (Supplementary Table 2) as schematically represented at the top. The MMEJ product was detected by radioactive PCR applying P5 and ³²p-labeled P7 primers (highlighted in red) and separated by 10% urea PAGE. The HS sequence is shown on the panel (A). Bands above the MMEJ band correspond to the NHEJ products due to NHEJ activity from the nuclear extract (lane 3). (D)
End joining activity tested in mitochondrial and nuclear extracts in presence of three DNA duplexes: MMEJ-L-HS1, MMEJ-Linker, and MMEJ-R-HS2, as schematically shown on the upper panel. The blue section in the middle of MMEJ-Linker represents the BamHI site. HS-1 and HS-2 sequences are shown on panel (B). Nucleotide G11778, which is commonly mutated in LHON, is underlined and enlarged. Nucleotides, which are different from those in the natural mitochondrial genome sequence and create the BamHI site, are in red and underlined. Autoradiography of amplicons, corresponding to MMEJ PCR product and its fragment (MMEJ BamHI Digestion Band) and separated by 10% urea PAGE, is shown. DNA Ladder represents a mix of ³²p-labelled oligonucleotides, their size is indicated at the right.

**Figure 2. Evaluation of mitochondrial import of dsDNA fragments.** (A) Chimeric RNA-DNA oligonucleotides RMIS-Dir, RMIS-Rev, as well as Dir and Rev, designed for mitochondrial delivery and editing experiments are shown in alignment to the MMEJ-Linker duplex. Nucleotides which differ from the wild-type *ND4* sequence are highlighted in gray. The *BamHI* site is shown by the small rectangle. Nucleotides, which differ from the wild-type sequence, are highlighted in bold. The blue hairpin structures represent RMIS. (B) Schematic representation of submitochondrial fractionation (modified from ³⁴). (C, D) *In vivo* import Assays. Mitochondria were purified from the HEK 293T cells transfected with Duplex 1 RMIS-Dir:RMIS-Rev (C) or with Duplex 2 RMIS-Dir:Rev (D). Total nucleic acids (RNA + DNA) were purified and separated by 10% urea-PAGE for Northern blot hybridization. Two representative blots are shown for each experiment (left and right panels), probed for oligonucleotides Dir or Rev (upper left and right panels, correspondingly) as indicated on the left of each panel. To detect possible cytosolic contamination, the probe for 5.8S ribosomal RNA was used (middle panels); the mitochondrial tRNA<sup>Thr</sup> signal (lower panel) served as a loading control and as an indicator of mitochondrial and
mitoplast integrity. Mitochondria, mitoplasts and lysed mitochondria are schematically indicated above the panels, as in (B). Markers of size and hybridization controls, oligonucleotides RMIS-Dir and Dir, were loaded on the right extremity of each gel, as indicated on the top.

**Figure 3. mtDNA editing by mitochondrially imported donor DNA in 293T cells.** (A) Schematic representation of ss-oligonucleotides and duplexes used in the corresponding transfections of HEK 293T cells. Duplex 1 – RMIS-Dir:RMIS-Rev; Duplex 2 – RMIS-Dir:Rev; Duplex 3 – Dir:RMIS-Rev. (B) Mitochondrial genome editing efficiency in counts of reads with the edited mitochondrial sequence per one million (Counts Per Million = CPM) of reads unambiguously aligned to the mitochondrial sequence (MT:11713-11853) for each transfection is shown on the graph. Statistical significance was assessed by the two-sided permutation test with the plus-one correction and adjustment via Benjamini-Hochberg False Discovery Rate. N.S. – no statistical significance (p > 0.3). * – moderate statistical significance (p < 0.05; # 1 vs # 2 – p-adjust = 0.0036; # 1 vs # 3 – pzejust = 0.043; # 1 vs # 4 – pzejust = 0.0069). ** – strong statistical significance (pzejust < 0.001).

**Figure 4. mtDNA editing by donor DNA delivery in mitochondria of T-REx-293-Su9-AsCas12a cells.** (A) crRNA, wild-type and edited *ND4* alleles, and their translated protein sequences, RMIS-Dir-New and RMIS-Rev-New are shown on the picture. New HS-1 and New HS-2 indicate the microhomology arms. The guide part of crRNA and corresponding sites of wild-type and edited *ND4* genes are highlighted in purple. (B) Schematic representation of a timeline of cells transfections and treatments. (C) Left panel: means of counts of reads with five-, four-, or three edited nucleotides per one million (Counts Per Million = CPM) of reads unambiguously aligned to the mitochondrial sequence (MT:11643-11870) for each transfection, are shown on the
957 3D graph. Each transfection was conducted in biological triplicate. Right panel: schematic
958 explanation of the transfections and treatments.

959 **Figure 5. Hypothetic mechanism of microhomology-mediated mtDNA editing (MME).** The
960 process may start with RMIS removal (likely mediated by EndoG and ExoG nucleases),
961 followed by end resection, which could be executed by mitochondrial MMEJ machinery, and 3'-
962 overhangs formation. The next steps may involve strand invasion and FLAP removal by FEN1,
963 followed by DNA synthesis and ligation by DNA ligase III. Newly synthesized DNA is
964 represented by the red dashed line.
<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<table><thead><tr><th>Lane #</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th></tr></thead><tbody><tr><td>Mitochondrial Extract 1</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Mitochondrial Extract 2</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Nuclear Extract</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td></tr><tr><td>MMEJ-Ovr-L Duplex</td><td>+</td><td>+</td><td>+</td><td>-</td><td>-</td></tr><tr><td>MMEJ-Ovr-R Duplex</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td></tr></tbody></table>

<figure><img src="image_5.png" /></figure>

<table><thead><tr><th>Lane #</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th><th>9</th><th>10</th></tr></thead><tbody><tr><td>Mitochondrial Extract 1</td><td>+</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Mitochondrial Extract 2</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td><td>-</td><td>-</td></tr><tr><td>Nuclear Extract</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>MMEJ-L Duplex</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>-</td></tr><tr><td>MMEJ-R Duplex</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>-</td></tr><tr><td>MMEJ-Linker Duplex</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>-</td></tr><tr><td>BamHI</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td></tr></tbody></table>

<figure><img src="image_6.png" /><figcaption>MMEJ Band</figcaption></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>Figure 5</figcaption></figure>

970

971