▎ WuXi AppTec content team editor
A paper published online in the journal Cell introduced an important breakthrough: for the first time, a research team from South Korea achieved the conversion of A bases to G bases in mitochondrial DNA, filling a crucial puzzle for gene editing technology and bringing the hope of curing a variety of mitochondrial genetic diseases.
From the first discovery of restriction enzymes in the 1960s, to the invention of PCR technology in 1985, to the gene editing of CRISPR technology for living organisms in the past decade, in just half a century, human beings have achieved a huge leap in the ability to manipulate DNA in one breakthrough after another. Among them, the emergence of CRISPR allows scientists to efficiently edit pathogenic mutations in the genome and provide new treatment solutions for many genetic diseases.
However, there is another dark cloud that has long hung over the field of gene editing: the editing of mitochondrial DNA.
As important organelles involved in energy metabolism, mitochondria also contain small amounts of DNA from the mother line. If this part of the gene is mutated, it may lead to a variety of genetic diseases related to metabolism.
For example, Leber hereditary optic neuropathy (LHON), a dangerous disease caused by point mutations in mitochondrial DNA, can lead to blindness in patients. Mitochondrial DNA mutations can also lead to certain mitochondrial encephalomyopathy, in which the patient's brain suffers damage and symptoms such as epilepsy and psychobehavioral abnormalities may occur. On average, one in every 5,000 people suffer from genetic diseases caused by mutations in the upper mitochondrial dots.
Although gene-editing tools have exploded in the last decade, they have always struggled to work in the face of mitochondrial genetic diseases. For example, the CRISPR-Cas system, which is most prevalent today, cannot be used to treat mitochondrial diseases because guide RNA cannot cross the mitochondrial membrane.
Image credit: 123RF
The editing of mitochondrial DNA can be said to be the last untouched land in the field of gene editing, and this problem has become an obstacle that must be crossed to cure many genetic diseases.
In 2020, a revolutionary breakthrough arrives. Professor Ruqian Liu's team at the Broad Institute has developed a gene editing tool called DdCBE that can directly modify the bases on double-stranded DNA to achieve the conversion from C bases to T bases, which is also the first time scientists have edited mitochondrial DNA in human cells.
However, as a pioneering achievement in mitochondrial DNA editing, DaCBE also has shortcomings: it can only efficiently perform TC-TT sequence conversion, and only 9 of the 90 known pathogenic mitochondrial mutation sites can be repaired.
Of these 90 mutation sites, as many as 39 can be repaired by the conversion of A-G bases. If we can find the means to achieve A-G conversion, then a variety of mitochondrial genetic diseases, including the above two diseases, are expected to usher in a cure.
In the latest study, published in Cell, the research team from the Gene Editing Center of the Korea Institute of Basic Sciences finally completed this "impossible task" by developing a new gene-editing platform named transcription activator-like effector-linked deaminases (TALET).
▲SCHEMATIC DIAGRAM OF TALED EDITING MITOCHONDRIAL DNA (Image source: Reference[1])
Cho Sung-Ik, lead author of the paper, said: "The new base editor we designed greatly expands the scope of mitochondrial DNA editing, which not only helps to build disease models, but also helps people develop new therapies." ”
What exactly is TALED? As we'll see next, TALED is made up of three major parts with different functions that work together to accomplish this gene editing task.
The first part can be seen as a guide to THETALED. As mentioned earlier, common gene-editing systems cannot enter mitochondria. To solve this problem, TALED, like the DaCBE developed by Liu's team, used transcriptional activator-like effectors (TALE). As a DNA-binding protein, the TALE protein is able to target specific DNA sequences that enter mitochondria under the guidance of mitochondrial targeting sequences (MTS) and bind to specific mitochondrial DNA sequences.
At this point, TALED is taken to the workplace. Here, it's THE TURN OF THE SECOND PART OF THE TALET TO APPEAR. The research team needed to find the right dehydrogenase to achieve the conversion of A-G bases here. To do this, they chose an adenine dehydrogenase called TadA8e, which is modified from the adenine dehydrogenase of E. coli.
This option is quite creative because TadA8e is thought to be a protein that works specifically on single-stranded DNA, but here, it requires base editing in the mitochondrial double-stranded DNA.
The paper's corresponding author, Professor KIM Jin-Soo, director of the Gene Editing Centre, said: "No one has thought about base editing in mitochondria with TadA8e because it is thought to work only on single-stranded DNA. It was this idea that went outside the traditional framework that helped us invent TALED. ”
What helps TadA8e do this is the last major part of TALED: the cytosine dehydrogenase DddAtox. DddAtox allows double-stranded DNA to be briefly unraveled, and TadA8e seizes this fleeting window of time to efficiently catalyze the conversion of A-G bases in the mitochondria of human cells, with an editing frequency of up to 49%.
▲TALED realizes the conversion of A-G bases (Image source: Reference[1])
Through the adjustment of THE TALED, the research team developed technologies that can realize both A-G and C-T base conversion, and only A-G conversion.
Of course, as a pioneering technology, TALED still has imperfections, such as the possibility of conversion of nucleotides adjacent to the target site when base editing is performed; in addition, it remains to be seen whether TALED will have off-target effects in mammalian cells.
But there is no doubt that the advent of TALED technology has given us another highly promising gene editing tool, blowing away the last dark cloud of gene editing. Looking forward to the future application scenarios of this technology, the research team hopes to improve the editing efficiency and specificity of TALED, and eventually be able to correct the pathogenic mitochondrial mutations in embryos, newborns and adult patients, respectively. We expect that those who are trapped by mitochondrial genetic diseases will finally usher in a moment of healing.
Resources:
[1] Sung-Ik Cho, Seonghyun Lee, Young Geun Mok, Kayeong Lim, Jaesuk Lee, Ji Min Lee, Eugene Chung, Jin-Soo Kim. (2022). Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases. Cell. https://doi.org/10.1016/j.cell.2022.03.039
[2] A new era of mitochondrial genome editing has begun. Retrieved Apr 25th, 2022 from https://www.eurekalert.org/news-releases/950486
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