A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing

Abstract

Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques1,2. Because previously described cytidine deaminases operate on single-stranded nucleic acids3, their use in base editing requires the unwinding of double-stranded DNA (dsDNA)—for example by a CRISPR–Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria4. As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases9,10.Here we describe an interbacterial toxin, which we name DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.

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Fig. 1: DddA is a double-stranded DNA cytidine deaminase that mediates T6SS-dependent interbacterial antagonism.
Fig. 2: Non-toxic split-DddAtox halves reconstitute activity when co-localized on DNA in HEK293T cells.
Fig. 3: TALE–split DddAtox fusions for mitochondrial base editing in HEK293T cells.
Fig. 4: DdCBE editing at five mtDNA genes in HEK293T cells.
Fig. 5: Mitochondrial genome-wide off-target DNA editing by DdCBEs.

Data availability

Coordinates and structure factors for DddA have been deposited in the PDB under accession code 6U08. High-throughput sequencing and whole-mitochondria sequencing data have been deposited in the NCBI Sequence Read Archive under accession code PRJNA603010. Amino acids sequences of all base editors in this study are provided in the Supplementary Information, sequences 1–4.

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Acknowledgements

We thank C. Wilson, L. Ludwig, T. Wang, K. Zhao, W. H. Yeh, S. Miller, A. Sousa, H. Hayden and A. Vo for materials and technical advice. R. Harris provided APOBEC3A and L. Eberl provided B. cen H111. This work was supported by the Merkin Institute of Transformative Technologies in Healthcare; US National Institutes of Health (NIH) grants R01AI080609, U01AI142756, RM1HG009490, R35GM122455, R35GM118062 and P30DK089507; US Defense Threat Reduction Agency (DTRA) grant 1-13-1-0014; and the University of Washington Cystic Fibrosis Foundation Research Development Program (RDP) SINGH15R0. B.Y.M. was supported by a Singapore A*STAR National Science Scholarship (NSS) fellowship; M.H.d.M. was supported by Cystic Fibrosis Foundation Fellowship DEMORAA18F0; A.V.K. was supported by a National Science Centre (NCN) Poland mobility grant UMO-2019/32/T/NZ1/00459; and A.R. was supported by NIH T32 GM095450 and a National Science Foundation graduate research fellowship. V.K.M., J.D.M. and D.R.L. are supported by the Howard Hughes Medical Institute.

Author information

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Authors

Contributions

B.Y.M., M.H.d.M., S.B.P., V.K.M., J.D.M. and D.R.L. designed the study; M.H.d.M., J.Z. and D.E.B. designed, performed and analysed experiments to characterize DddA; B.Y.M. designed, performed and analysed nuclear and mitochondrial editing experiments; B.Y.M., M.H.d.M., A.R. and M.C.R. performed sequence analyses; F.H. performed microscopy; A.V.K. designed, performed and analysed mitochondrial biology experiments; B.Y.M., M.H.d.M., S.B.P., A.V.K., V.K.M., J.D.M. and D.R.L. wrote the manuscript.

Corresponding authors

Correspondence to Joseph D. Mougous or David R. Liu.

Ethics declarations

Competing interests

The Broad Institute and the University of Washington have filed provisional patent applications on base-editing systems described in this study, listing B.Y.M., M.H.d.M., S.B.P., J.D.M. and D.R.L. as inventors. D.R.L. is a consultant and co-founder of Prime Medicine, Beam Therapeutics, Pairwise Plants and Editas Medicine, companies that use genome editing. V.K.M. is a consultant to 5am Ventures and Janssen Pharmaceuticals.

Additional information

Peer review information Nature thanks Rahul Kohli, Fyodor Urnov and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Analysis of the bactericidal activity of DddA and its activity against dsDNA and RNA substrates.

a, Genomic context of dddA (purple) and dddIA (blue) in B. cenocepacia H111. b, Viability of B. cenocepacia ΔdddA ΔdddIA (recipient) over time during competition with B. cenocepacia donor strains carrying wild-type dddAtox or dddAtoxE1347A. Values and error bars represent the mean ± s.d. of three technical replicates. The experiment was repeated three times with similar results. c, α-VSV-g western blot analysis of total cell lysates of E. coli expressing the indicated deaminases tagged with VSV-G epitope. RNAP-β was used as a loading control. Results are representative of n = 2 independent biological replicates. d, In vitro DNA cytidine deamination assays using double-stranded 36-nt DNA substrates containing AC, TC, CC, and GC with a FAM fluorophore on the forward (A) or reverse (B) strand. Deamination activity results in a cleaved product (P). Images are representative of n = 2 independent biological replicates. e, f, Poisoned primer extension assay to detect deamination of cytidine in single-stranded (e) or double-stranded (f) RNA substrates. Images are representative of n = 2 independent biological replicates. A mix of RNA substrates containing the sequences GUCG or GUUG at the indicated ratios were incubated with purified DddAtox and reverse transcriptase. Primer extension was performed in reactions with ddGTP to terminate primer extension at cytidines. Cytidine deamination yields the 31-mer product.

Extended Data Fig. 2 DddAtox deaminates cytidines in bacteria with strong sequence context preference.

a, Number of SNPs from the indicated nucleotide classifications observed in E. coli Δudg following intoxication with DddAtox or DddAtox(E1347A). b, c, The position of SNPs on the chromosome of E. coli Δudg isolates intoxicated with DddAtox (b) or DddAtox(E1347A) (c). SNPs above the line indicate C-to-T transitions on the plus strand; SNPs below indicate C-to-T transitions on the minus strand. Other mutations are represented on the plus strand. Sequencing coverage was 203–265-fold. d, Deamination assay on DddAtox with double-stranded DNA substrates containing a single C with different nucleotides (A, T, C or G) at the position immediately 5′ of the C (red) (S, substrate; P, product). Images are representative of n = 3 independent biological replicates.

Extended Data Fig. 3 Base-editing efficiencies and indel frequencies of all DddAtox splits in HEK293T cells.

ah, Each split was assayed in the aureus-N and aureus-C orientation (see Fig. 2b) across spacing region lengths of 12-bp (a), 17-bp (b), 23-bp (c), 28-bp (d), 33-bp (e), 39-bp (f), 44-bp (g) and 60-bp (h). Cells were collected 3 days post-transfection for DNA sequencing. Colours reflect the mean of n = 2 independent biological replicates.

Extended Data Fig. 4 TALE–split DddAtox proteins mediate efficient base editing in nuclear DNA of U2OS cells.

a, Left–G1333-DddAtox-N and Right–G1333-DddAtox-C bind DNA sequences within CCR5. Target cytosines are shown in purple and TALE binding sites are shown in blue. Two copies of UGI proteins (2×-UGI) were fused to the N- or C terminus through a 2- or 16-amino acid linker. Editing efficiencies and indel frequencies for the possible combinations of UGI positions and linker lengths are shown. In the absence of UGI protein, only C9-to-T9 edit was observed. b, Architecture of nuclear-targeting CCR5-DdCBE (see Fig. 3c for optimized DdCBE architecture targeting mtDNA). Target cytosines are shown in purple. c, Editing efficiencies and indel frequencies of cells treated with CCR5-DdCBE and ND6-DdCBE 3-days-post transfection are shown. Dead-DdCBEs containing the inactive DddAtox(E1347A) mutant were used as negative controls. d, Outcomes among edited alleles in which the specified target C is mutated are shown for the indicated base editor. Values and error bars in a, c and d reflect the mean ± s.d. of n = 3 independent biological replicates.

Extended Data Fig. 5 Unoptimized mitoTALE–split DddAtox fusions mediate modest editing of mitochondrial ND6 in HEK293T cells.

a, Architectures of non-UGI containing ND6-mitoTALE–DddAtox fusion pair. DddAtox was split at G1333 or G1397, with each half fused to either the left TALE or the right TALE. TALEs bind to mtDNA sequences (blue) that flank a 15-bp spacing region in mitochondrial ND6. Target cytosines are shown in purple. The last TALE repeat (*) did not match the reference genome9 (see Supplementary Table 4). b, mtDNA editing efficiencies of mitoTALE–DddAtox pairs in the listed split orientations. The dashed line is drawn at 0.1%. Values and error bars reflect the mean ± s.d. of n = 3 independent biological replicates.

Extended Data Fig. 6 DdCBE editing in HEK293T cells persist over multiple divisions while maintaining cell viability and mitochondrial DNA integrity.

ae, Editing efficiencies for optimized ND6-DdCBE (a), MTND5P1-DdCBE (denoted ND5.1-DdCBE) (b), MTND5P2-DdCBE (denoted ND5.2-DdCBE) (c), ATP8-DdCBE (d) and BE2max and BE4max (e) are shown for each time point. C•G-to-T•A conversions at protein-coding genes that generate missense mutations (green) of the putative amino acid (red) are shown. f, Western blots of ND6-, ND5.1-, ND5.2- and ATP8-DdCBE at various time points. The right halves were Flag-tagged and the left halves were HA-tagged. Day 3 images are representative of n = 3 independent biological replicates; n = 1 for day 6 and day 12 images (see Supplementary Data 3 for uncropped images and fluorescent tagging of each half). Nuclear β-actin was used as a loading control. g, Cell viability was measured by recording the luminescence at the indicated time points. Luminescence values were normalized to the untreated control. h, DNA gel of PCR-amplified mtDNA captured as two amplicons (red). Images are representative of n = 3 independent biological replicates (see Supplementary Data 4 for uncropped images). i, mtDNA levels of DdCBE-edited cells were measured by qPCR relative to untreated cells. Values and error bars in ae, g and i reflect the mean ± s.d. of n = 3 independent biological replicates. For ae, asterisks indicate significant editing based on a comparison between indicated time points. *P < 0.05 and **P < 0.01 by Student’s two-tailed paired t-test. Individual P values are listed in Supplementary Table 7.

Extended Data Fig. 7 Stalling mtDNA replication impairs mitochondrial base editing in human cells.

a, Schematic of experimental design. Addition of doxycycline (Dox) induces the stable expression of a dominant-negative mutant of DNA polymerase-gamma containing a D1135A substitution (POLGdn) in a HEK293-derived cell line31. Total cell lysate was collected at indicated time points for western blotting of POLGdn in n = 3 independent biological replicates. b, mtDNA levels of uninduced (no Dox) and induced (+Dox) cells treated with indicated DdCBE 48 h post-transfection. mtDNA levels were measured by qPCR and normalized to uninduced cells without DdCBE treatment. c, Editing efficiencies of indicated DdCBE in uninduced and induced cells 48 h post-transfection. All values and error bars in b and c reflect the mean ± s.d. of n = 3 independent biological replicates.

Extended Data Fig. 8 Effect of DdCBE editing on mitochondrial function and mtDNA homeostasis.

a, mtDNA levels of ND4-edited cells measured by qPCR relative to cells treated with dead ND4-DdCBE. b, mtRNA levels of ND4-edited cells measured by reverse transcription-qPCR relative to cells treated with dead ND4-DdCBE. cf, Confirmation of editing by Sanger sequencing and OCR of cells treated with ND5.1-DdCBE (c), ND5.2-DdCBE (d), MTND5P3-DdCBE (denoted ND5.3-DdCBE) (e) and ND1-DdCBE (f). Untreated cells were used as controls. All cells were collected 6 days post-transfection. For all Sanger sequencing plots, n = 3 independent biological replicates. All values and error bars shown in a, b and OCR plots in cf reflect the mean ± s.e.m. of n = 3 independent biological replicates. For a and b, Student’s unpaired two-tailed t-test was applied. NS, not significant (P > 0.05).

Extended Data Fig. 9 Off-target editing activity of DdCBEs in nuclear DNA of HEK293T cells.

ac, The on-target editing site in mtDNA and the corresponding nuclear DNA sequence with the greatest homology are shown for ND6-DdCBE (a), ND5.1-DdCBE (b) and ND4-DdCBE (c). TALE binding sites begin at N0 and are shown in blue. Target cytosines are in purple. Nucleotide mismatches between the mtDNA and nuclear pseudogene are in red. Editing efficiencies are measured by targeted amplicon sequencing 3 days post-transfection (a, b) or six days post-transfection (c) (see Methods for primer sequences). Each amplicon was sequenced at >44,000× coverage. All values and error bars reflect the mean ± s.d. of n = 3 independent biological replicates. Student’s unpaired two-tailed t-test was applied. *P < 0.05; NS, not significant (P > 0.05).

Extended Data Fig. 10 TALE arrays need to bind to mtDNA sequences positioned in close proximity to reassemble catalytically active DddAtox for off-target editing.

a, The identities and relative binding positions of each mismatched (MM) TALE–DddAtox half is shown. MM-1 and MM-2 contain a TALE-bound DddAtox half and a TALE-free DddAtox half. MM-3 and MM-4 contain DddAtox halves fused to TALE repeat arrays that bind to distant regions in mtDNA. ND6-Right TALE contains a permissive N-terminal domain (see Supplementary Table 4). b, The average percentage of genome-wide C•G-to-T•A off-target editing in mtDNA by indicated DdCBE and MM pairs are shown. The dashed line represents the percentage of endogenous C•G-to-T•A conversions in mtDNA as measured in the untreated control. Values and error bars reflect the mean ± s.e.m. of n = 3 independent biological replicates.

Extended Data Fig. 11 Predicted effects of off-target SNVs on mitochondrial DNA sequence and protein function.

a, Classification of off-target SNVs into noncoding or coding mutations. Mutations occurring in protein-coding regions of mtDNA were further categorized into synonymous, missense or nonsense mutations. b, For nonsynonymous SNVs, SIFT was used to predict the effect of these mutations on protein function. High- or low-confidence calls (indicated in parentheses) were made according to the standard parameters of the prediction software. c, Editing efficiencies of selected off-target TC bases in the indicated sequence contexts are shown. HEK293T cells were treated with the indicated DdCBE and collected 3 days post-transfection for DNA sequencing. Values and error bars reflect the mean ± s.d. of n = 3 independent biological replicates.

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion, Supplementary Data Figures 1–4, Supplementary Tables 1, 4, 10-13 and Supplementary Sequences 1-4.

Reporting Summary

Schematic and sequences of guide RNAs for split-DddA

Supplementary Table 2 tox–Cas9 screen. dSpCas9 guide RNAs (spG7 and spG6) are paired with with SaKKH guide RNAs (saG1 to saG4) to generate spacing regions with lengths between 12 and 60 bp.

Base percentages at each position of the

Supplementary Table 3 EMX1 locus for DddAtox–Cas9 splits with no guide RNAs and monomers with their respective gRNAs. For G1333/G1397 DddAtox–dSpCas9 or G1333/G1397 DddAtox–SaKKH-Cas9(D10A) halves, these halves are directed to a site within EMX1 by a guide RNA spG4 (magenta) or saG4 (blue), respectively. The reciprocal DddAtox half of each fusion was absent. The 60-bp spacing region in highlighted in yellow. All shorter spacing regions are nested within the 60-bp spacing. Base percentages are obtained 3 days post-transfection and representative of n=2 independent biological replicates.

Indel frequencies of DdCBEs in their optimized orientations

Supplementary Table 5 . Shown are the percent of indels in HEK293T cells for each DdCBE in its optimized split orientation (see Fig. 4a-g for on-target editing efficiencies of optimized DdCBEs). Cells treated with ND5.1-, ND5.2-, and ATP8-DdCBE were harvested 3 days post-transfection; cells treated with ND1-, ND2, ND4- and ND5.3-DdCBE were harvested 6 days post-transfection.

Base percentages at each position of the H-strand of mtDNA for indicated DdCBE in its optimized split orientation

Supplementary Table 6 . TALE-binding sites are highlighted in blue. On-target C•G bases are highlighted in yellow. Base percentages are measured 3 days post-transfection of HEK293T cells.

-values from comparison of editing efficiencies from time course experiments in HEK293T cells and U2OS cells

Supplementary Table 7 P-values from comparison of editing efficiencies from time course experiments in HEK293T cells and U2OS cells. For a given DdCBE, the P-value for editing efficiencies of target cytidine across two cumulative timepoints is shown. P-values were calculated using the Student's two-tailed paired t-test. Entries are highlighted in red if the P-value indicated a significant difference (P<0.05).

Unique off-target SNVs mediated by DdCBEs

Supplementary Table 8 . SNVs called by VarScan 2 were considered high-confidence if the percentage frequency of a given SNV is >0.1% in one or more replicates. For samples treated with DdCBEs, dead DdCBEs, and TALE-free G1397 DddAtox, the combined number of unique off-target SNVs from all three independent biological replicates that are absent in the untreated control are shown. For the untreated control, heteroplasmic mutations were excluded. SNV positions for each DdCBE treatment were from the NC_012920 reference genome. On-target SNVs are highlighted in red.

Overlapping off-target SNVs

Supplementary Table 9 . Shown are the list of overlapping off-target SNVs between ND6- and ND5.1-DdCBE, ND6-, ND5.1- and ATP8-DdCBE and ND6-, ND5.1-, ND5.2- and ATP8-DdCBE. The average frequency of each SNV is shown.

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Mok, B.Y., de Moraes, M.H., Zeng, J. et al. A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature 583, 631–637 (2020). https://doi.org/10.1038/s41586-020-2477-4

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