Base Editing the Cell’s Powerhouse: Bacterial Toxin Could Help Cure Mitochondrial Diseases

By Debarati Banik, Ph.D.

The CRISPR-Cas9 technology revolutionized the field of genetic engineering and bolstered the arsenal of molecular biologists, by offering a simple yet site-specific gene-editing technique. However, the editing was limited to nuclear DNA only. Scientists have now identified a bacterial toxin that can bypass this roadblock and edit mitochondrial DNA (mtDNA).

Although sparse in number, mtDNA is mainly responsible for maintaining the energy-producing features of mitochondria. Several maternally inherited genetic diseases may originate from mtDNA mutations. A few examples include myoclonic epilepsy and ragged-red fiber disease (MERRF), Leigh syndrome (LS), Leber hereditary optic neuropathy (LHON), etc., which affects the nervous system and muscles of the human body. The ability to edit mtDNA will crack down on these diseases, but CRISPR-Cas9 has not been able to rectify it. To understand why one must look closely into the toolbox.

CRISPR–Cas9 and Other Gene Editors

The CRISPR–Cas9 technology involves a strand of RNA that guides the Cas9 enzyme precisely to edit a desired site of DNA. The Cas9 enzyme cuts the double-stranded DNA (dsDNA) to a single strand, exposing the desired region for further site-specific editing. The guide RNA (gRNA), which renders the specificity, works well in nuclear DNA editing. However, in the case of mitochondria, the double-layered membrane poses a challenge as it prevents the entry of CRISPR machinery. This made scientists look for an alternative enzyme that could edit a dsDNA without the assistance from the gRNA.

Previously, mtDNA has been edited by RNA-free programmable nucleases, such as transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs). However, CRISPR, TALENs, and ZFNs create double-stranded breaks (DSBs) in the genome and rely on the host’s machinery for repair. An advanced technology called base editing, on the other hand, is much safer since it edits a single base in a gene without DSBs.

DddA – Bacterial Toxin that Base Edits dsDNA

In 2018, a team of microbiologists led by Joseph Mougous at the University of Washington identified a bacterial cytidine deaminase from Burkholderia cenocepacia. DddA, as they were called, contained sequences, suggesting they are substrates of intracellular protein delivery systems, such as the type VI secretion system (T6SS). Further research established DddA as an antibacterial toxin delivered by the T6SS. It harbors the toxicity conferring “DddAtox domain,” which specifically targets dsDNA and catalyzes the deamination of cytosine, converting it into uracil (C to U). The activity was highly specific for dsDNA and failed to exert the same editing effect on dsRNA or ssDNA.

The activity was also structure-dependent since a specific amino acid substitution could lead to a loss of function. After the first C to U conversion, in the next round of replication, uracil would require to be paired with adenine, the ultimate result being cytosine turning into thymine within the DNA sequence. This enzyme, which they called ‘double-stranded DNA deaminase toxin A’ or DddA, however, did not have any sequence specificity. Therefore, it could act non-specifically on any host DNA, causing a global mutagenic event and making it unsuitable for use at its native state.

Constructing the First Mitochondrial Gene Editor

In collaboration with David Liu’s team from the Broad Institute, they devised a ‘tamed version’ of the enzyme. They split the DddA into N-terminal and C-terminal halves, which individually were maintained in their non-toxic and inactive states. They could be activated only when brought together with their target DNA. Spacing region length, target cytosine position, and split orientation were the factors that dictated the editing efficacy.

A programmable DNA-binding protein, i.e., TALE array proteins, containing a bipartite nuclear localization signal for nuclear DNA and MTS-linked TALE proteins for mtDNA, were fused to the DddA halves, which executed the function of adding specificity of translocation. It was also found that fusing one or two molecules of uracil glycosylase inhibitor to the enzyme halves could enhance the editing efficacy by 8 fold while reducing the occurrence of indels (non-specific insertion or deletion of bases in the genome).

The final engineered form of the enzyme that catalyzed a C to T conversion is a fusion of split-DddA halves, transcription activator-like effector array proteins for translocation specificity, and a uracil glycosylase inhibitor (UGI) molecule. The resultant protein was called ‘RNA-free DddA-derived cytosine base editors’ (DdCBEs). Unlike TALENs and ZFNs, which remove mtDNA copies, these CRISPR-free DdCBEs enable the precise manipulation of mtDNA.

Besides demonstrating a high target specificity, the authors also modeled the enzyme’s utility in the process of respiration. Both the pathways of oxidative phosphorylation and basal and uncoupled respiration rates were decreased in the active DdCBEs treated cells, similar to the effect of a complex 1 inhibition.

Future Prospects

Although tested in at least two cell lines, the base editor needs further validation in multiple cell-types. Besides, it displayed an off-target effect, which was also a drawback of the CRISPR-Cas9 system. To be clinically approved, one must make sure that the by-stander genes are minimally affected, the path to which is still under construction. However, scientists are optimistic that this technology can prove to be an asset to the existing arsenal against mitochondrial diseases, such as mitochondrial replacement or degradation of mutated mtDNA through the delivery of genetic cargo into the mitochondria.

Editor: Rajaneesh K. Gopinath, Ph.D.

Related Article: CRISPR-Edited T cells Evaluated in Combating Lung Cancer

References

1. https://www.nature.com/articles/d41586-020-02054-5?error=cookies_not_supported&code=734b435b-ed79-4296-a865-559710abc806
2. https://www.nature.com/articles/s41586-020-2477-4
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6225103/
4. https://www.nature.com/news/reproductive-medicine-the-power-of-three-1.15253

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