Human Mitochondrial Genome Editing Toolkit Expands – Genetic Engineering & Biotechnology News


Scientists at the Center for Genome Engineering at the Institute for Basic Science (IBS) in Daejeon, South Korea, have developed a programmable tool that can perform adenine (A)-to-guanine (G) base conversions in the human mitochondrial genome.

The base editors used in the new mitochondrial gene-editing platform are called TALEDs (transcription activator-like effector-linked deaminases), and have three components: a custom-designed TALE (transcription activator-like effector) that targets a DNA sequence, an engineered deoxyadenosine deaminase (TadA8e) derived from the Escherichia coli protein TadA that converts A to G, and a catalytically impaired, full-length, or split cytidine deaminase (DddA) that unwinds DNA and makes it more accessible to TadA8e.

Sung-Ik Cho, PhD, first author of the new study, said, “We created a novel gene-editing platform, TALED, that can achieve A-to-G conversion. Our new base editor dramatically expanded the scope of mitochondrial genome editing. This can make a big contribution, not only to making a disease model but also to developing a treatment.”

The details of the new mtDNA editing platform were published in the journal Cell, in an article titled, “Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases.”

“This is a timely and interesting study expanding the genome editing toolbox with TALE-associated deaminases for A-to-G precision mutations. The proof-of-concept in human mitochondrial DNA is encouraging with efficiency numbers enabling the genesis of disease models. While there is a need to enhance specificity for translational and clinical applications, this is a practical expansion to the genome editing toolbox for an under-resourced set of applications in mitochondrial DNA, with potential for mammalian cells and beyond,” said Rodolphe Barrangou, PhD, distinguished professor of food, bioprocessing, and nutrition science at North Carolina State University and the chief editor of The CRISPR Journal.

TALEDs work in mitochondria by first deaminating adenine to inosine, then converting inosine to guanine by DNA repair or replication. [Institute for Basic Science]

The ability to alter the genomic code has progressively increased with the discoveries of restriction enzymes in 1968, polymerase chain reaction in 1985, zinc finger nucleases in 1996, TAL-effector DNA interactions in 2009, CRISPR-Cas9 gene editing in 2012, and base editing in 2016. These discoveries have made it possible to treat previously incurable genetic diseases by editing disease-causing mutations in the human genome. However, live-cell genome editing tools have largely focused on the nuclear genome.

Mutations in the maternally inherited, circular mitochondrial DNA (mtDNA) are responsible for several serious diseases. There are 90 known disease-causing point mutations in mtDNA that affect at least 1 in 5,000 individuals.

“There are some extremely nasty hereditary diseases arising due to defects in mtDNA. For example, Leber hereditary optic neuropathy (LHON), which causes sudden blindness in both eyes, is caused by a simple single point mutation in mtDNA,” said Jin-Soo Kim, PhD, director of the Center for Genome Engineering at IBS and senior author.

In addition, MELAS (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes) gradually attacks the patient’s brain. Other studies suggest abnormalities in mtDNA may be responsible for degenerative diseases such as Alzheimer’s disease and muscular dystrophy.

Existing genome editing tools cannot be used to edit mtDNA primarily due to limitations in methods of delivery. For example, guide RNAs used in the CRISPR-Cas platform are unable to enter mitochondria.

“Another problem is that there is a dearth of animal models of these mitochondrial diseases. This is because it is currently not possible to engineer mitochondrial mutations necessary to create animal models,” said Kim. “Lack of animal models makes it very difficult to develop and test therapeutics for these diseases.”

With the discovery of DddA-derived cytosine base editors (DdCBEs) in 2020 by a team led by David Liu, PhD, professor at the Broad Institute of Harvard and MIT, and Joseph Mougous, PhD, professor at the University of Washington, it was possible to conduct cytosine (C)-to-thymine (T) conversions in the mtDNA without creating double-stranded DNA breaks. However, DdCBEs were largely limited to C-to-T conversions in the TC motif and could correct only about 10% pathogenic mitochondrial point mutations.

Liu, who was not involved in the current study, told GEN, “Jin-Soo and his group beautifully show how integration of our mitochondrial cytosine base editor and the deaminase enzyme we evolved for our nuclear adenine base editors can achieve an important goal in the field—adenine base editing in mitochondrial DNA. Even though mutations in mitochondrial DNA cause many serious genetic diseases, mitochondria have previously resisted precision gene editing due to difficulty of delivering CRISPR guide RNAs into mitochondria.”

Liu added, “In 2020, we reported the first CRISPR-free base editors (DdCBEs) that overcome this limitation by using proteins, instead of CRISPR protein-RNA complexes, to programmatically target DNA in mitochondria. Using this DdCBE architecture, together with a deoxyadenosine deaminase that we evolved in our laboratory to support high-efficiency adenine base editing, Jin-Soo’s laboratory succeeded in developing the first mitochondrial adenine base editors. Since adenine base editing can, in principle, correct many of the mutations in mitochondrial DNA that cause genetic diseases, his group’s work is a key advance towards the precise correction of pathogenic mitochondrial mutations.”

Surprisingly, TadA8e can change A to G in the double-stranded human mitochondrial genome when the E. coli genome consists of single-stranded DNA.

Kim said, “No one has thought of using TadA8e to perform base editing in mitochondria before, since it is supposed to be specific to only single-stranded DNA. It was this outside-of-the-box approach that has really helped us to invent TALED.”

The team hypothesized that the split DddA allows TadA8a access to double stranded DNA by transiently unwinding it, which allows TadA8e to quickly make the necessary edits. In addition to tweaking the components of TALED, the researchers also developed a technology that is capable of both A-to-G and C-to-T base editing simultaneously, as well as A-to-G base editing only.

The authors reported that the custom designed TALEDs efficiently converted A to G in human cells at 17 target sites in different mitochondrial genes, with editing frequencies of up to 49%.

The team applied the new TALEDs by creating a single cell-derived clone containing desired mtDNA edits. Moreover, TALEDs showed no cytotoxicity and maintained the integrity of mtDNA. TALEDs generated no undesirable off-target edits in nuclear DNA and very few off-target effects in mtDNA.

In future studies, the researchers intend to improve TALEDs by increasing editing efficiency and specificity, to pave the way toward application of the technology in patients. The team is also developing TALEDs suitable for A-to-G base editing in chloroplast DNA.