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

Mok BY, de Moraes MH, Zeng J, Bosch DE, Kotrys AV, Raguram A, Hsu F, Radey MC, Peterson SB, Mootha VK, Mougous JD, Liu DR
Source: Nature
Publication Date: (2020)
Issue: 7817: 631-637
Research Area:
Molecular Biology
Cells used in publication:
U-2 OS
Species: human
Tissue Origin: bone
Fibroblast, lung (DHFL), human CF
Species: human
Tissue Origin: lung
4D-Nucleofector® X-Unit

U2OS cell plasmid nucleofection
We combined 500 ng of Left DdCBE monomer and 500 ng of Right DdCBE monomer in a volume that did not exceed 2 µl. This combined plasmid mixture was nucleofected in a final volume of 22 µl per sample in a 16-well Nucleocuvette strip (Lonza). U2OS cells were nucleofected using the SE Cell Line 4D-Nucleofector X Kit (Lonza) with 30,000–50,000 cells per sample (program DN-100), according to the manufacturer’sprotocol.

Human primary fibroblast nucleofection
Human primary fibroblasts were nucleofected as previously described51.In brief, 500 ng of in vitro-transcribed Left-DdCBE mRNA and 500 ng of in vitro-transcribed Right-DdCBE mRNA were combined in a volume that did not exceed 2 µl. This combined mRNA mixture was nucleofected in a final volume of 22 µl per sample in a 16-well Nucleocuvette strip (Lonza). Human primary fibroblasts (GM04541, Coriell) were nucleofected using the P2 Primary Cell 4D-Nucleofector kit (Lonza) with 2.5 × 105 cells per sample (program DS-150), according to the manufacturer’s protocol. The medium was changed after 24 h of nucleofection and cultured for 5 days before collection for high-throughput sequencing.


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
mitochondria. As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases. 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.