◎ Science and Technology Daily reporter Zhang Mengran
Researchers at the Genome Engineering Center of the Korea Institute of Basic Sciences (IBS) have developed a new gene-editing platform called transcriptional activator effect-associated deaminase (TALED). TALED is a base editor capable of A-to-G base conversion in mitochondria. The discovery is the culmination of a decades-long journey to cure human genetic diseases, and TALET is considered the last missing piece of the puzzle in gene-editing technology. The results were published in the latest issue of the journal Cell.
The magic and flaws of "genetic scissors"
From the discovery of the first restriction enzyme in 1968, the invention of polymerase chain reaction in 1985 to the demonstration of CRISPR-mediated genome editing in 2013, each new breakthrough in biotechnology has further improved the ability to manipulate DNA. In particular, the newly developed CRISPR-Cas system ("gene scissors") allows for comprehensive genome editing of living cells. This opens up new possibilities for treating previously incurable genetic diseases by editing mutations in the human genome.
Historical timeline of major discoveries in biotechnology. Source: Korea Institute of Basic Sciences
While gene editing has had great success in a cell's nuclear genome, scientists have not been successful in editing mitochondria that have their own genomes. Mitochondria, the so-called "power chambers of the cell," are tiny organelles in the cell that act as energy-producing factories. Since it is an important organelle for energy metabolism, if a gene mutates, it can lead to serious genetic diseases related to energy metabolism.
Kim Jin-so, director of the IBS Genome Engineering Center in South Korea, explains: "There are some very serious genetic diseases due to mitochondrial DNA defects. For example, Leber's hereditary optic neuropathy, which causes sudden blindness in both eyes, is caused by simple, single-point mutations in mitochondrial DNA. "Another mitochondrial gene-related disorder includes mitochondrial encephalomyopathy with lactic acidosis and stroke-like attacks, which slowly destroys a patient's brain. Some studies have even suggested that abnormalities in mitochondrial DNA may also be responsible for degenerative diseases such as Alzheimer's disease and muscular dystrophy.
Mitochondrial DNA can be edited
The mitochondrial genome is inherited from the mother line. There are 90 known pathogenic point mutations in mitochondrial DNA, affecting a total of 1 in at least 5,000 people. Due to limitations in delivery methods to mitochondria, many existing genome editing tools are not available. For example, the CRISPR-Cas platform is not suitable for editing these mutations in mitochondria because the guide RNA cannot enter the organelles themselves.
Image source: Visual China
"Another problem is the lack of animal models of these mitochondrial diseases. This is because it is currently not possible to design the mitochondrial mutations needed to create animal models. Kim added, "The lack of animal models makes it very difficult to develop and test treatments for these diseases." ”
Therefore, reliable technology for editing mitochondrial DNA is one of the frontiers of genome engineering, a frontier that must be explored in order to conquer all known genetic diseases, and the world's best scientists have been working for years to make it a reality.
In 2020, a research team led by harvard's Broad Institute and MIT's Ruqian Liu created a new base editor called the DddA-derived cytosine base editor, which performs C-to-T conversion from DNA in mitochondria. This is achieved by creating a new gene-editing technique called base editing, which converts a single nucleotide base into another without damaging DNA. However, this technique also has its limitations. It is not limited to C-to-T conversion, but is primarily limited to the TC base sequence, making it an efficient TC-TT converter. This means that it can only correct 9 of the 90 confirmed pathogenic mitochondrial point mutations, or 10 percent. The A-to-G conversion of mitochondrial DNA has long been thought to be impossible.
Zhao Xingyi, the first author of the study, said: "We began to think about ways to overcome these limitations. So we created a new gene editing platform called TALED that enables A-to-G conversion. Our new base editor greatly expands the scope of mitochondrial genome editing. This can contribute greatly not only to the development of disease models, but also to the development of therapeutic approaches. Notably, its ability to perform A-to-G conversion in human mtDNA corrects 39 of the 90 known pathogenic mutations, or about 43 percent. ”
The researchers created TALED by fusing three different ingredients. The first component is the transcriptional activator-like effector, which is capable of targeting DNA sequences. The second component is TadA8e, an adenine deaminase used to facilitate A-to-G conversion. The third component, DddAtox, is a cytosine deaminase that makes DNA more readily available to TadA8e.
A graphical summary showing how TALEDs work in mitochondria. First adenine is deaminoized to inosine, followed by inosine is converted into guanine through DNA repair or replication. Source: Korea Institute of Basic Sciences
An interesting aspect of TALED is TadA8e's ability to perform A-to-G edits in mitochondria with double-stranded DNA. This is a mysterious phenomenon because TadA8e is a protein known to be specific only for single-stranded DNA. Kim said: "No one had thought of using TadA8e for base editing in mitochondria before because it should only be specific for single-stranded DNA. It's this out-of-box approach to thinking that really helped us invent TALED. ”
Nobel Prize-level achievements
The researchers speculate that DddA tox allows access to double-stranded DNA by instantaneously untying the double strands. This fleeting, temporary window of time allows TadA8e to act as an ultrafast enzyme to quickly make the necessary edits. In addition to tweaking the components of THE TALED, the researchers developed a technique capable of simultaneous A-to-G and C-to-T base editing, as well as A-to-G base editing only.
The research team demonstrated the new technique by creating a single cell-derived clone containing the required mtDNA edits. They found that TALDs were neither cytotoxic nor did they cause mtDNA instability. In addition, there are no undesirable off-target edits in nuclear DNA, and there are few off-target effects in mtDNA. The researchers now aim to further improve TALETs by increasing editing efficiency and specificity, ultimately paving the way for correcting pathogenic mtDNA mutations in embryos, fetuses, newborns, or adult patients. The research team is also focused on developing A-to-G base editing in chloroplast DNA, which encodes essential genes in plant photosynthesis.
William Sue, a science communicator at the Institute of Basic Sciences, praised: "I believe the significance of this discovery is comparable to the invention of the blue LED that won the Nobel Prize in 2014." Just as blue LEDs are the last piece of the puzzle that will allow us to have energy-efficient white LED light sources, IT IS EXPECTED TOALED WILL USHER IN A NEW ERA OF GENOENGINEERING. ”
Source: Science and Technology Daily
Editor: Li Xiaohang (Intern)
Review: Julie
Final Judgement: Wang Yu