Strand-selective base editing of human mitochondrial DNA using mitoBEs

Insufficiency of TALE–TadA8e-V106W alone to facilitate the editing of mtDNA

TadA8e-V106W is an engineered deoxyadenosine deaminase that is commonly fused with Cas protein to perform adenosine-to-inosine (recognized as guanine) editing on nuclear DNA14. To investigate the potential for mitochondrial A-to-G editing, we fused the TadA8e-V106W protein with the appropriate TALE array and mitochondrial targeting sequence. We detected a very low level of editing at all three targeting sites, MT-ND1, MT-ND4 and MT-RNR2, with an editing rate of up to 0.39%, which is barely above the deep sequencing error (>0.10%) (Fig. 1a and Extended Data Fig. 1a,b). Because TadA is an essential tRNA-specific adenosine deaminase originating from Escherichia coli15, it is unsurprising that TadA8e-V106W enables mtDNA editing. Nevertheless, TadA8e-V106W alone was unable to induce efficient deamination because TALE cannot unravel the DNA double helix16,17, and this deaminase preferentially acts on ssDNA1,18, even after optimization through multiple rounds of mutations14. For base editing, the Cas9 and single-guide RNA complex unravels the DNA double helix at the target site to expose an ssDNA structure that serves as the substrate for ssDNA deaminase (for example, TadA8e-V106W or rAPOBEC1)1. We hypothesized that generating an ssDNA structure at the target loci might unleash the full power of TadA8e-V106W’s deaminase activity.

Fig. 1: Strand-biased editing of mtDNA using programmable nickase and deaminase.figure 1

a, Mitochondrial A-to-G editing efficiency of HEK293T cells treated with paired TALE–TadA8e-V106W at the MT-RNR2 site. b, Mitochondrial A-to-G editing efficiency of HEK293T cells treated with left TALE–MutH and right TALE–TadA8e-V106W at the MT-RNR2 site. c, Mitochondrial A-to-G editing efficiency of HEK293T cells treated with left TALE–MutH-D70A and right TALE–TadA8e-V106W at the MT-RNR2 site. d, Product distributions at the MT-RNR2 site in (b). e, A speculative model for improving the editing efficiency of mtDNA by combining nickase with deaminase (take TadA8e-V106W as an example in this model). TALE–nickase binds the target DNA and nicks the dsDNA. The nicked dsDNA may be prone to form ssDNA structures. TALE–TadA8e-V106W binds the target DNA and efficiently deaminates the adenine(s) on the resulting ssDNA. The resulting inosine(s) can be converted permanently to guanine(s) after DNA repair or DNA replication. f, Editing efficiency of mitoABEMutH with MutH and TadA8e-V106W at different distances from 5′-GATC-3′ at MT-ND4. g, A speculative model for mitoABEMutH. When the distance between TALE–MutH and 5′-GATC-3′ is 0–4 bp, the opposite strand is nicked, thus causing editing of the TALE–MutH recognition strand; when the distance between TALE–MutH and 5′-GATC-3′ is 5–9 bp, the TALE–MutH recognition strand is nicked, thus causing editing of the opposite strand. For ad, data are presented as mean ± s.d. of n = 3 independent biological replicates; for f, the mean values from n = 3 independent biological replicates are shown. The blue rectangular blocks represent the left TALE array binding sequences, and the purple rectangular blocks represent the right TALE array binding sequences. In ac, e and g, the blue rounded rectangle represents TadA8e-V106W, the purple rounded rectangle represents nickase and the gray rectangle represents TALE.

Efficient targeted A-to-G editing of mtDNA by a combination of TALE–TadA8e-V106W and TALE–nickase

To investigate whether deamination can occur on ssDNA, we used nickase to cleave only one strand. Our initial attempt involved using MutH, a sequence-specific (5′-GATC-3′) nickase encoded by E. coli that is involved in initiating mismatch repair to remove nucleotides misincorporated by DNA polymerase19. Wild-type MutH can nick unmethylated DNA strands in unmethylated or hemimethylated DNA20. In previous work, Gabsalilow et al.21 achieved strand-specific nicking of DNA by fusing TALE and MutH, and the strand-specific nick outcome depended on the distance between the TALE-array binding sequence and MutH recognition site. By fusing MutH with an appropriate TALE array, we introduced TALE–MutH and TALE–TadA8e-V106W in pairs to target the loci MT-ND1, MT-ND4 and MT-RNR2. We observed high levels of targeted A-to-G editing at all three loci, with a maximum efficiency of up to 77% (Fig. 1b and Extended Data Fig. 1c,d). Nickase activity was crucial for editing activity because the endonuclease-inactivated MutH mutant22, TALE–MutH-D70A, failed to enable any base editing when paired with its corresponding TALE–TadA8e-V106W (Fig. 1c and Extended Data Fig. 1e,f). These results indicated that TALE–nickase may generate ssDNA that exposed adenines, which serve as more suitable substrates for TadA8e-V106W. We thus created a new, to our knowledge, mitochondrial A-to-G base editing system, designated mitoABEMutH. As expected, the dominant editing outcome was A•T to G•C, with a product purity of over 95% at the MT-ND1 locus (Extended Data Fig. 1g) and close to 100% at both the MT-RNR2 and MT-ND4 loci (Fig. 1d and Extended Data Fig. 1g). We confirmed the durability of mtDNA editing results in HEK293T cells over 15 days at three target loci (Extended Data Fig. 1h–j).

On the basis of the above results, we proposed a theoretical model to explain how the TALE–nickase and TALE–deaminase combination enables the base editing of mtDNA. By nicking the target site, TALE–nickase generates transient ssDNA and consequently allows a TALE–deaminase (such as TALE–TadA8e-V106W) to deaminate adenine(s) on the ssDNA. After repair and replication of mtDNA, the deamination of adenine(s) probably remains in one strand, leading to strand-biased A-to-G conversion (Fig. 1e).

Strand-biased editing of mitoABEs

mitoABEMutH-enabled adenine editing occurred preferentially on the top strands of the three target loci (MT-ND1, MT-ND4 and MT-RNR2) (Fig. 1b and Extended Data Fig. 1c,d). Despite the presence of multiple adjacent Ts next to the edited A within the editing windows, the A on its opposite strand appeared unedited (Extended Data Fig. 1c,d) or only barely edited (Fig. 1b). These results suggest that the editing of mitoABEMutH may be strand specific. To verify this notion, we switched the TALE arrays for each pair of TALE–MutH and TALE–TadA8e-V106W and found that the strand-biased edits were reciprocally switched (Extended Data Fig. 2a,b). In MT-RNR2 site 1, MT-ND1 site 1 and MT-ND4 site 1, we placed the MutH nicking sequence (5′-GATC-3′) in the center of the editing window, 3 base pairs (bp) away from each TALE (Extended Data Fig. 2a). In this case, editing occurred mainly on the top DNA strand using left TALE–MutH and right TALE–TadA8e-V106W, whereas editing occurred mainly on the bottom strand when switching the positions of TALE–MutH and TALE–TadA8e-V106W (Extended Data Fig. 2a). In MT-RNR2 site 2 and MT-ND4 site 1, the MutH nicking sequence is 5 or 6 bp away from each end of the editing window (Extended Data Fig. 2b), and editing occurs mainly on the bottom DNA strand with left TALE–MutH and right TALE–TadA8e-V106W. The top DNA strand became the A-edited strand after switching the MutH and TadA8e-V106W TALEs (Extended Data Fig. 2b). On the basis of these results, we speculated that strand-biased editing is related to the strand selection of nicking, which is, in turn, related to the number of bases between TALE binding and the MutH recognition motif (5′-GATC-3′).

Next, we fixed the position of left TALE–TadA8e-V106W and shifted the position of right TALE–MutH from 0 to 10 bp away from the nicking sequence (5′-GATC-3′) successively by 1 bp (Fig. 1f). Editing occurred mainly on the bottom DNA strand at both MT-ND4 and MT-RNR2 site 1 when the distance between TALE binding and the MutH nick motif was within 0 to 4 bp, and editing occurred mainly on the top DNA strand when the distance between TALE binding and the MutH nick motif was between 5 and 9 bp (Fig. 1f and Extended Data Fig. 2c). In contrast, when we fixed TALE–MutH and changed the position of TALE–TadA8e-V106W, the A-edited strand did not change, the editing windows only widened gradually (Fig. 1f).

We conducted experiments to determine whether the orientation of MutH relative to TALE affected the strand-biased editing of mitoABEMutH. Because the MutH nick motif (5′-GATC-3′) is a palindromic sequence, we suspected the fusion of MutH with left TALE or right TALE would have the same effect. By gradually widening the editing window at the MT-ND4 site (Extended Data Fig. 2d), the orientation-switched TALE–MutH still showed the distance-dependent, edited-strand preference in editing outcome. Editing was successful between the 8- and 24-bp editing windows at this site (Extended Data Fig. 2d). Therefore, we presumed a working model for mitoABEMutH in which TALE–MutH nicks the strand opposite its binding strand when the distance between TALE binding and MutH nick motif is 0–4 bp, and it nicks the same strand when the distance is 5–9 bp, which is consistent with previous work21. Nicking generates ssDNAs, and all adenines on ssDNA are subjected to TadA8e-V106W-mediated deamination within the window. After repair and DNA replication, most deaminated A on the nonnicked strand was retained (Fig. 1g). These findings indicated that TadA8e-V106W editing outcome depends on which strand is nicked by TALE–nickase, and adenine deamination is predominantly preserved on the nonnicked strand. We also observed that the linker sequences between TALE and MutH had no effect on the strand-biased editing of mitoABEMutH in our experiments (Supplementary Fig. 1a–d).

Expanded targeting scope of mitoABEMutH by site-directed mutations

The combination of TALE–MutH and TALE–TadA8e-V106W enables targeted strand-biased editing of mtDNA. However, MutH requires a specific sequence for nicking (5′-↓GATC-3′), limiting the editing scope of mitoABEMutH. Based on structural information (PDB: 2AOQ)22, we attempted to expand the editing scope of mitoABEMutH by introducing point mutation(s) to MutH. We found that K48A, R184A and Y212S abolished the editing activity of mitoABEMutH at the MT-ND4 site (Fig. 2a,b). In MutH, F94 helps loop 67 (amino acid residues 184–190) to make sequence-specific interactions with 5′-GATC-3′, and F91 interacts with the cytosine in 5′-GATC-3′ (ref. 22) (Fig. 2a). The E91A or F94A variant maintained the editing activity of mitoABEMutH, and the combination of these two mutations enhanced editing efficiency at the MT-ND4 site (Fig. 2b). We designated this special type of MutH mutant harboring E91A and F94A MutH*. We then investigated whether MutH* could generate nicks at 5′-GATD-3′ (D stands for A, T or G) sites and become a new type of mtDNA editing tool, mitoABEMutH*. By targeting three loci, MT-ND5, MT-CO2 and MT-MTTR, which, respectively contain 5′-GATA-3′, 5′-GATG-3′ and 5′-GATT-3′ sequences on the top strand, we confirmed that mitoABEMutH* indeed worked as an effective editing tool that generated bottom-strand edits at all three sites (Fig. 2c–e). Importantly, none of these sites could be edited by mitoABEMutH because of the absence of the MutH motif (Extended Data Fig. 3a–c).

Fig. 2: Broadened editing to 5′-GAT-3′ by introducing mutations to MutH.figure 2

a, Crystal structure of key amino acids of MutH interacting with unmethylated 5′-GATC-3′ (PDB: 2AOQ). b, Editing efficiency of MutH mutants (including K48A, E91A, F94A, R184A, Y212S and double mutation of E91A and F94A) combined with TadA8e-V106W at the 5′-GATC-3′ position. ce, The editing efficiencies of target regions at the 5′-GATA-3′ (c), 5′-GATG-3′ (d) and 5′-GATT-3′ positions (e) with different mitoABEMutH* orientations and distances. In be, the mean values from n = 3 biologically independent replicates are shown. f,g, The designable targeting range of TALE–MutH (f) and TALE–MutH* (g) in human mitochondria. The numbers 0, 2, 4 and 6 indicate the frequency of MutH and MutH* recognition sequences within a 40-bp region. In b, c, d and e, the blue rounded rectangle represents TadA8e-V106W, the orange rounded rectangle represents the MutH mutant and the gray rectangle represents TALE. The borders of rounded rectangles of different colors (blue and green) represent TALE–MutH* designed to nick different strands. WT, wild type.

The MutH motif, 5′-GATC-3′, is a palindromic sequence; thus, TALE–MutH* (5′-GATD-3′) can only nick the top strand by the 5′-guanine in certain designs, and only adenine edits on the bottom strand are retained (Fig. 2c–e). On the other hand, TALE–MutH cannot nick 5′-GATD-3′, resulting in no edits (Extended Data Fig. 3a–c). In addition, MutH* yielded the best editing efficiency when placed 3 bp from 5′-GATD-3′ at the right end, probably due to its high nicking efficiency (Fig. 2c–e). These results confirmed the presumed working principle of mitoABEMutH, and TALE–MutH* indeed expands its editing scope. There are 23 recognition sites (5′-GATC-3′) of MutH in human mtDNA, but MutH* (5′-GATN-3′) has 485 sites. We speculate that TALE–TadA8e-V106W could function within a range of 20-bp upstream and downstream of the nick position. Based on this, we estimated the proportion of designable mitoABEs in the human mitochondrial genome. TALE–MutH has a designable targeting range of only ~6% of the mitochondrial genome, whereas TALE–MutH* has a range of ~71%, with an average of two 5′-GATN-3′ sites per 40 bp in the mitochondrial genome (Fig. 2f,g). Despite expanding the editing scope of mitoABEs greatly, there are still many sequences that cannot be edited by mitoABEs. Therefore, further expansion of the editing scope is necessary.

Screening for alternative nickases with no sequence-context constraints

To further broaden the editable scope of mitoABEs, we tested multiple enzymes with potential nickase activity. As some nucleases have separate active centers for cutting dsDNA, mutation(s) inactivating one active center might convert the nuclease to nickase. In particular, the cleavage and recognition domains of type IIS restriction endonuclease are separable23, which makes it an ideal candidate of nickase for the half-deactivation cleavage domains. For enzymes without crystal structures, we attempted to predict their cleavage domains for engineering purposes24 (Extended Data Fig. 4). We began by replacing the MutH component of mitoABEMutH with the naturally existing nickase Nt.BspD6I(C)25 and engineered nickases, such as FokI-FokI-D450A26, Nb.BsaI(C)-N441D/R442G27, Nt.BsaI(C)-R236D27, Nb.BsmBI(C)-R438D27, Nt.BsmAI(C)-R221D27, Nb.BsrDI(C)28, Nt.CviPII (5′-↓CCD-3′)29, BspQI(C)30, N.AlwI(C)31 and I-TevI (5′-CNNN↓G-3′)32 to verify whether any of these enzymes nicks DNA when fused with an appropriate TALE array. We removed the recognition domains of all the enzymes mentioned above, except for Nt.CviPII (5′-↓CCD-3′) and I-TevI (5′-CNNN↓G-3′) because these two nickases recognize more extensive sequences. Our objective was to identify nickases that solely rely on the TALE array for recognition and do not possess recognition motifs. Therefore, we included only those candidates that do not have recognition sequences or have extensive recognition sequences in our system.

By fusing the above nickases with left TALE, we tested their potential editing activities when teamed up with right TALE–TadA8e-V106W (Fig. 3a). The three editing sites MT-ND1, MT-ND5 site 2 and MT-ND4 were selected for testing. Among all TALE array-fused candidate nickases, the TALE–FokI-FokI-D450A, TALE–I-TevI and TALE–Nt.BspD6I(C) enabled editing on targeted sites when combined with TALE–TadA8e-V106W. TALE–Nt.BspD6I(C) yielded higher base editing activity at all three targeted sites (Fig. 3a). Nt.BspD6I is a nickase that can form a heterodimer with BspD6I (the small subunit, 20 kDa) and function as a restriction endonuclease called R.BspD6I25. Nt.BspD6I(C), which we used to fuse with TALE, is only the C-terminal cleavage domain (382–604 amino acids). In comparison with TALE–MutH, TALE–Nt.BspD6I(C) showed lower strand preference at the MT-ND4 site (Fig. 3a and Extended Data Fig. 1d), possibly due to its imprecise nick on dsDNA. We named this editing tool mitoABENt.BspD6I(C).

Fig. 3: Screening nickases without recognition sequence restriction for mitoBEs.figure 3

a, Nickases without sequence restriction or with extensive recognition sequences were screened for mitochondrial base editing at three sites. The mean values from n = 3 biologically independent replicates are shown. b, The editing efficiencies of different mitochondrial sites when left TALE–Nt.BspD6I(C) was combined with right TALE–TadA8e-V106W. c, The editing efficiencies of different mitochondrial sites when right TALE–Nt.BspD6I(C) combined with left TALE–TadA8e-V106W. For b and c, data are presented as mean values ±s.d. of n = 3 independent biological replicates. In ac, the blue rounded rectangle represents TadA8e-V106W, the red rounded rectangle represents Nt.BspD6I(C) and the gray rectangle represents TALE.

To further characterize the editing pattern of mitoABENt.BspD6I(C), we applied mitoABENt.BspD6I(C) to target more diverse mtDNA sequences. mitoABENt.BspD6I(C) reached up to ~40% editing efficiency at some of these sites (Fig. 3b). In addition, when the TALEs of TALE–Nt.BspD6I(C) and TALE–TadA8e-V106W were switched, the edited strand was switched reciprocally (Fig. 3b,c). The linker sequences between TALE and Nt.BspD6I(C) did not affect the editing features of mitoABENt.BspD6I(C) (Supplementary Fig. 1e–h). From all tested sites, we speculated that TALE–Nt.BspD6I(C) produced the nick on the same DNA strand recognized by itself, resulting in the editing of adenine(s) in the strand recognized by TALE–TadA8e-V106W.

Mitochondrial C-to-T editing via cytosine deaminase

With the success of mitoABEs, we speculated that such a strategy could be extended to other types of deaminases, including rAPOBEC1, which converts C to T on ssDNA1. By replacing TadA8e-V106W with rAPOBEC1-fused UGI33, we were able to achieve mitochondrial C-to-T editing using the combination of TALE–rAPOBEC1–2×UGI and TALE–MutH, with a maximum editing efficiency of ~30% (Fig. 4a–c). Similar to mitoABEMutH, mitochondrial C-to-T editing, designated mitoCBEMutH, also displayed strand preference, with the top strands edited for MT-ND4 and MT-RNR2 site 3 (Fig. 4a,b), and the bottom strand edited for MT-RNR2 site 1 (Fig. 4c). In contrast, editing of DdCBEs was not biased toward a specific strand at these three sites (Fig. 4d–f).

Fig. 4: Mitochondrial C-to-T base editing by nickase combined with rAPOBEC1 and UGI.figure 4

ac, The editing efficiency of mitoCBEs at MT-ND4 (a), MT-RNR2 site 3 (b) and MT-RNR2 site 1 (c). The green rounded rectangle represents rAPOBEC1, the brown rounded rectangle represents UGI, the purple rounded rectangle represents MutH and the gray rectangle represents TALE. df, Comparison of the editing profiles of mitoCBEs and DdCBEs at MT-ND4 (d), MT-RNR2 site 3 (e) and MT-RNR2 site 1 (f). For af, data are presented as mean values ±s.d. of n = 3 independent biological replicates.

Monomeric mitoBEs for base editing on mitochondrial genome

Although it is beneficial to have nickase and deaminase domains in two separate TALE arrays, it is tempting to test if they could still work when fused with the same TALE array. We constructed four versions of such mitoABEs: TALE–MutH–TadA8e-V106W, TALE–TadA8e-V106W–MutH, TALE–Nt.BspD6I(C)–TadA8e-V106W and TALE–TadA8e-V106W–Nt.BspD6I(C). Monomeric mitoABEs, mitoABEMutH and mitoABENt.BspD6I(C) enabled efficient A-to-G editing (Extended Data Fig. 5a,b). The monomeric versions of mitoABEMutH achieved higher editing efficiency at the MT-ND1 target site compared with dimeric mitoABEMutH (left MutH), whereas the dimeric mitoABENt.BspD6I(C) yielded higher editing efficiency. For the MT-ND4 site, both monomeric and dimeric mitoABEs showed comparable editing efficiency. Moreover, monomeric mitoABEs have a wider editing window compared with dimeric mitoABEs, with a consistent strand preference observed for both types within the editing windows (Extended Data Fig. 5a,b). The smaller size of monomeric mitoBEs makes them easier to deliver, especially when using AAVs (adeno-associated virus) as a vector. In addition, we also successfully constructed monomeric mitoCBEs (mitoCBEMutH and mitoCBENt.BspD6I(C)) and achieved efficient C-to-T editing at targeted sites (Extended Data Fig. 5c,d).

Editing specificity of mitoBEs

To evaluate the editing specificity of mitoBEs, we performed mtDNA sequencing analysis. HEK293T cells transfected with either mitoABEMutH- or mitoABENt.BspD6I(C)-expressing plasmids were subjected to mtDNA sequencing analysis, in which the untreated group (Fig. 5a) and nontargeting groups, including mitoABEMutH and mitoABENt.BspD6I(C) not associated with the TALE array (Fig. 5b,c), were used as a control. The mean sequencing coverage across the mitochondrial genome was approximately 1193× (Fig. 5l). mtDNA sequencing analysis detected no nonspecific editing in all experimental groups compared with controls (untreated and nontargeting); only on-target editing was observed (Fig. 5a–i). Of note, the fact that there was no difference between the nontargeting (Fig. 5b,c) and untreated groups (Fig. 5a) suggests that the free form of either TALE–deaminase or TALE–nickase does not cause any unwanted off-target effects. We also assessed the editing specificity of monomeric mitoABEs (monomeric mitoABEMutH and mitoABENt.BspD6I(C)) and found their specificity to be comparable with that of dimeric mitoABEs (Extended Data Fig. 6a–h). This suggests that both monomeric and dimeric mitoABEs display high specificity when editing the mitochondrial genome. Additionally, we compared the off-target editing of mitoCBEs to that of DdCBEs with the same TALE array and found that mitoCBEs induced lower off-target editing in the mitochondrial genome, particularly at the MT-ND4-targeted site (Fig. 5j,k and Supplementary Fig. 2a,b). These results demonstrated that mitoBEs represent a reliable set of mitochondrial editing tools with minimal off-target editing on mtDNA.

Fig. 5: Editing specificity of mitoBEs.figure 5

ak, The average frequency and mitochondrial genome position of each unique single nucleotide variant are shown for untreated HEK293T cells (a) and HEK293T cells treated with nontargeting mitoABEMutH (b), nontargeting mitoABENt.BspD6I(C) (c), MT-ND4-targeting mitoABEMutH (left TALE–MutH with right TALE–TadA8e-V106W) (d), MT-ND4-targeting mitoABEMutH (left TALE–TadA8e-V106W with right TALE–MutH) (e), MT-RNR2-targeting mitoABEMutH (left TALE–MutH with right TALE–TadA8e-V106W) (f), MT-RNR2-targeting mitoABEMutH (left TALE–TadA8e-V106W with right TALE–MutH) (g), MT-ND1-targeting mitoABENt.BspD6I(C) (left TALE–Nt.BspD6I(C) with right TALE–TadA8e-V106W) (h), MT-ND1-targeting mitoABENt.BspD6I(C) (left TALE–TadA8e-V106W with right TALE–Nt.BspD6I(C)) (i), MT-ND4-targeting mitoCBEMutH (left TALE–rAPOBEC1–2×UGI with right TALE–MutH) (j) and MT-RNR2-targeting mitoCBEMutH (left TALE–MutH with right TALE–rAPOBEC1–2×UGI) (k). l,m, The deep sequencing average coverage of the mitochondrial genome (l) and nuclear genome (m). n, The nuclear genome average frequency of each unique single nucleotide variant are shown for the EGFP group (control), nontargeting groups and targeting groups. For ak and n, all data are three or more biological replicates, the arrow points to the targeted editing site and the blue or red dots represent the editing efficiency of adenines or cytosines in the editing window. For l and m, all data are presented as mean values of n = 3 independent biological replicates. o, The copy number of mtDNA was detected by quantitative PCR. Data are presented as mean values ±s.d. of n = 3 independent biological replicates.

Mitochondrial gene editing tools such as DdCBEs are known to cause off-target effects in the nuclear genome13. To investigate whether mitoBEs also have off-target effects in the nucleus, we performed whole-genome sequencing (with an average coverage of ~58.4×) and compared the overall off-target editing in the targeting group (including mitoABEMutH and mitoABENt.BspD6I(C)) with that of the EGFP and nontargeting control groups. We found no significant difference between the targeted groups and the control groups (Fig. 5m,n). Furthermore, we analyzed the presence of TALE-dependent off-target effects using the whole-genome sequencing data and found no off-target editing within ±50 bp of the TALE-array binding sequence (including zero or one mismatch) in the nuclear genome (Supplementary Table 1). These findings suggest that mitoBEs exhibit low off-target effects in the nuclear genome. Due to the limitations of current off-target assay methods, more precise approaches will be necessary to assess off-target editing of the nuclear genome in the future.

To further evaluate the effect of mitoABEs on mitochondria, we measured the copy number and integrity of mtDNA. Using the above mtDNA sequencing data, we examined the indels of mtDNA. We found no difference between the targeted groups (Extended Data Fig. 7d–i) and the controls (Extended Data Fig. 7a–c). By real-time quantitative PCR and long-range PCR analysis, we further confirmed that the copy number and integrity of mtDNA in the targeted groups remained the same as those in the controls (Fig. 5o and Extended Data Fig. 7j). Collectively, mitoABEs showed high specificity in human cells.

Circular RNA-encoded mitoABEs enable strand-biased editing in multiple cell lines

Treatment of disease by direct delivery of RNA shows good potential. Because mitoABEs, unlike the CRISPR system, do not require RNA components to function, we tested mitochondrial editing using circular RNA (circRNA)34 to encode mitoABEs. circRNA-encoded mitoABEs conferred strand-biased editing in various human cell types, including H1299, MCF7, Huh7 and RPE1, indicating mitoABEs are versatile tools compatible with various delivery routes to achieve efficient and precise mtDNA base editing (Fig. 6a–c).

Fig. 6: circRNA-encoded mitoABEs successfully created disease models and corrected mutation in cells derived from individuals with LHON.figure 6

a, Overview of circRNA-encoded mitoABEMutH-transfected cells. b,c, circRNAs of two mitoABEMutH orientations were transfected into different cell lines to achieve strand-biased editing, left TALE–MutH with right TALE–TadA8e-V106W (b), left TALE–TadA8e-V106W with right TALE–MutH (c). Genomic DNA was collected 2 days posttransfection. d, Overview of circRNA-encoded mitoABENt.BspD6I(C)-transfected HEK293T cells and genomic DNA collected 2 days posttransfection. e, The editing efficiencies of circRNA-encoded mitoABENt.BspD6I(C) targeted the start codon of MT-ND4. f, The ATP levels of cells transfected with circRNA-encoded mitoABENt.BspD6I(C) targeted the start codon of MT-ND4. Student’s t test, P = 2.71 × 10−5. g, Oxygen consumption rate (OCR) in HEK293T cells treated with circRNA-encoded mitoABENt.BspD6I(C) targeted the start codon of MT-ND4 for 2 days. h, Overview of circRNA-encoded mitoABENt.BspD6I(C)-transfected LHON disease cells GM10742, with genomic DNA was collected 3 days posttransfection. i, The editing efficiency of mitoABENt.BspD6I(C) corrected the 11778G>A mutation of LHON disease cell GM10742. j, The ATP levels of cells transfected with circRNA-encoded mitoABENt.BspD6I(C) targeting the 11778G>A mutation of LHON disease cell GM10742. Student’s t test, P = 6.98 × 10−5. k, OCR of the LHON disease cell line GM10742 treated with circRNA-encoded mitoABENt.BspD6I(C) targeting the 11778G>A mutation for 2 days. l, Types of mitochondrial diseases (MITOMAP) and the proportion of diseases that can theoretically be treated by mitoBEs. For b, c, f, g, j and k, the data are presented as mean values ±s.d. of n ≥ 3 independent biological replicates. For e and i, the mean values from n = 3 biologically independent replicates are shown. For g,k, FCCP represents carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone.

Editing start codons of mitochondrial genes perturbed the function of the respiratory chain

Mitochondrial diseases are genetic disorders caused by mutations in either the nuclear DNA or mtDNA that are characterized by defects in oxidative phosphorylation35. Approximately 90% of mitochondrial genetic disorders caused by mtDNA mutations are due to single base mutations of the mitochondrial coding genes36. The leading cause of these genetic disorders is a decrease in ATP production due to the defective assembly of the mitochondrial respiratory complex37. Using circRNA-encoded mitoABEs to target the start codons of three genes in HEK293T cells, we tested our editing tools to generate phenotypes mimicking real mitochondrial diseases (Fig. 6d). We chose to t

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