A new era of mitochondrial genome editing has begun

Date:
April 25, 2022
Source:
Institute for Basic Science
Summary:
A new era of mitochondrial genome editing has begun. Scientists
successfully achieve A to G base conversion, the final missing
piece of the puzzle in gene-editing technology.



FULL STORY ========================================================================== Researchers from the Center for Genome Engineering within the
Institute for Basic Science developed a new gene-editing platform called transcription activator-like effector-linked deaminases, or TALED. TALEDs
are base editors capable of performing A-to-G base conversion in
mitochondria. This discovery was a culmination of a decades-long journey
to cure human genetic diseases, and TALED can be considered to be the
final missing piece of the puzzle in gene- editing technology.


==========================================================================
From the identification of the first restriction enzyme in 1968,
the invention of polymerase chain reaction (PCR) in 1985, and the
demonstration of CRISPR- mediated genome editing in 2013, each new
breakthrough discovery in biotechnology further improved our ability
to manipulate DNA, the blueprint of life. In particular, the recent
development of the CRISPR-Cas system, or "genetic scissors," has
allowed for comprehensive genome editing of living cells. This opened
new possibilities for treating previously incurable genetic diseases by
editing the mutations out of our genome.

While gene editing was largely successful in the nuclear genome of
the cells, however, scientists have been unsuccessful in editing the mitochondria, which also have their own genome. Mitochondria, the
so-called "powerhouse of the cells," are tiny organelles in cells that
serve as energy-generating factories.

As it is an important organelle for energy metabolism, if the gene is
mutated, it causes serious genetic diseases related to energy metabolism.

Director KIM Jin-Soo of the Center for Genome Engineering explained,
"There are some extremely nasty hereditary diseases arising due to defects
in mitochondrial DNA. For example, Leber hereditary optic neuropathy
(LHON), which causes sudden blindness in both eyes, is caused by a
simple single point mutation in mitochondrial DNA." Another mitochondrial gene-related disease includes mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), which slowly destroys the
patient's brain. Some studies even suggest abnormalities in mitochondrial
DNA may also be responsible for degenerative diseases such as Alzheimer's disease and muscular dystrophy.

The mitochondrial genome is inherited from the maternal line. There
are 90 known disease-causing point mutations in mitochondrial DNA,
which in total affects at least 1 in 5,000 individuals. Many existing
genome editing tools could not be used due to limitations in the method
of delivery to mitochondria.

For example, the CRISPR-Cas platform is not applicable for editing these mutations in mitochondria, because the guide RNA is unable to enter the organelle itself.

"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,"
Director Kim added. "Lack of animal models makes it very difficult to
develop and test therapeutics for these diseases." As such, reliable technology to edit mitochondrial DNA is one of the last frontiers of
genome engineering that must be explored in order to conquer all known
genetic diseases, and the world's most elite scientists have endeavored
for years to make it a reality.



==========================================================================
In 2020, researchers led by David R. LIU of the Broad Institute of Harvard
and MIT created a new base editor named DddA-derived cytosine base editors (DdCBEs) that can perform C-to-T conversion from DNA in mitochondria. This
was made possible by creating a new gene-editing technology called base editing, which converts a single nucleotide base into another without
breaking the DNA.

However, this technique also had its limitations. Not only is it
restricted to C-to-T conversion, but it is mostly limited to the TC
motif, making it effectively a TC-TT converter. This means that it can
correct only 9 out of 90 (= 10%) confirmed pathogenic mitochondrial point mutations. For the longest time, the A-to-G conversion of mitochondrial
DNA was thought to be impossible.

First author CHO Sung-Ik said, "We began to think of ways to overcome
these limitations. As a result, we were able to create a novel
gene-editing platform called 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." As of note, being able to
perform A-to-G conversions in human mtDNA alone could correct 39 (= 43%)
out of the 90 known pathogenic mutations.

The researchers created TALED by fusing three different components. The
first component is a transcription activator-like effector (TALE),
which is capable of targeting a DNA sequence. The second component is
TadA8e, an adenine deaminase for facilitating A-to-G conversion. The
third component, DddAtox, is a cytosine deaminase that makes the DNA
more accessible to TadA8e.

One interesting aspect of TALED is TadA8e's ability to perform A-to-G
editing in mitochondria, which possess double-stranded DNA (dsDNA). This
is a mysterious phenomenon, as TadA8e is a protein that is known to be
specific to only single-stranded DNA. Director 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 thinking outside of the box approach that has really helped us to
invent TALED." The researchers theorized that DddAtox allows dsDNA to
be accessible by transiently unwinding the double-strand. This fleeting
but temporary time window allows TadA8e, a super fast-acting enzyme, 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 group demonstrated this new technology by creating a single
cell-derived clone containing desired mtDNA edits. In addition, TALEDs
were found to be neither cytotoxic nor cause instability in mtDNA. Also,
there was no undesirable off-target editing in nuclear DNA and very
few off-target effects in mtDNA. The researchers now aim to further
improve the TALEDs by increasing the editing efficiency and specificity, eventually paving the way to correct disease-causing mtDNA mutations in embryos, fetuses, newborns, or adult patients. The group is also focusing
on developing TALEDs suitable for A-to- G base editing in chloroplast DNA, which encodes essential genes in photosynthesis in plants.


========================================================================== Story Source: Materials provided by Institute_for_Basic_Science. Note:
Content may be edited for style and length.


========================================================================== Journal Reference:
1. Sung-Ik Cho, Seonghyun Lee, Young Geun Mok, Kayeong Lim, Jaesuk
Lee, Ji
Min Lee, Eugene Chung, Jin-Soo Kim. Targeted A-to-G base editing
in human mitochondrial DNA with programmable deaminases. Cell,
2022; DOI: 10.1016/ j.cell.2022.03.039 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2022/04/220425121101.htm

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