02.09.

2020

Project Name: TARRTMA

(TARdigrade Radiation Tolerance Mechanism and its Application)

Written by: Metehan Kara

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 ABSTRACT
Tardigrades are one of the most tolerant organisms to radiation damage. They can resist
many abnormal environments- e.g., low/high temperature, high/low pressure, space vacuum, high
radiation, desiccation-. To figure out these tolerance mechanisms can be essential in unexpected
situations or to help in order to humans' civilisation development beyond the Earth. After
understanding these mechanisms, it is necessary to integrate into real life in order to advance
humans' civilisation by using genetic engineering technique, especially the CRISPR method

AIM
The main purpose of this project is to understand the mechanism underlying the radiation
tolerance of tardigrades and to integrate this mechanism into real life by utilize genome editing
system particularly CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)
method. To understand this mechanism is very crucial for 7 points for humans:
1)Exploring Deep Space: If humankind 2) To raise organism: If the humans want
wants to explore deep space, they must to live on other planets, for this purpose,
overcome to the radiation damage because organisms take an important place in order
we already know that even though explorer to make an unliveable planet liveable.
may not travel so far from Earth-so they Therefore, these organisms must be
cannot take radiation so harshly-, the resistant to radiation while growing up,
radiation effect is a serious health problem, since in high probability, amount of
especially with the long term because radiation is very different from Earth, so in
amount of radiation exposure radiation order to produce oxygen or any material
accumulates during their time in space. that needs to live in other planets, Living
Therefore, this accumulation can damage organisms are essential.
the explorer body particularly their DNA.
(Furukawa et al.,2020)

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3) To understand DNA repair all organism containing plants.
mechanism: It is already known that Furthermore, most skin cancers are caused
radiation-especially X-ray and UV beams- by UV radiation damaging the DNA in skin
can damage the DNA. Radiation can induce cells. Therefore, if the ozone layer gets
DNA double-strand breaks (DBS) that can thinner or removes completely, all
lead to tumorigenesis or cell death. In this organisms can face the fatal outcomes.
way, the organism has developed an Thus, whole organisms should be had
efficient DNA repair mechanism to repair tolerant to the side of fatal radiation effects
DSB damage during living in high radiation against undesired situations.
exposure. That is why, in this project, the 5) To provide some perspective to the
model organism can be tardigrade because Cancer research: In today's world, cancer
the past research told us that tardigrade is taken a considerable place. The similarity
tolerated the radiation about 3000-4200 Gy- between cancer treat and tardigrade's
note that human can survive only 3-5 radiation tolerance mechanism - also other
Gy-.Therefore somehow tardigrades can cryptobiosis organisms- is to prevent
suppress the radiation effect in their DNA damage to DNA. In this way, the
some through repair mechanism. (Furukawa understanding of tardigrade's radiation
et al.,2020) For these reasons, this project is tolerance mechanism can be given to us a
essential to understand underlying the DNA perspective about the cancer treatment.
repair mechanism. [ CITATION KIn19 \l 1055 ]
4) In the case of losing Ozone layer: There 6) To protect all organisms from the
are three kinds of UV radiation. UV-C nuclear power station's possible radiation
(<280 nm), UV-B (280-315 nm), UV-A leakage: Radiation leakage from nuclear
(315-400 nm). UV-C, which is highly power stations can be considered as a low
energetic wavelength, is removed by chance, and even though the accident in
stratospheric ozone layer and is not reached nuclear power plants is unlikely, we have
to plants. UV radiation induces damage to experienced examples of this in the past
such as Chernobyl or Fukushima Daiichi
nuclear power plants. Since there would be radiation. Otherwise, in an unexpected
still such accidents in the future, all living situation, all life on earth may come to
organisms on earth must be resistant to extinction. [CITATION Sur20 \l 1055 ]

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7) To protect all organisms from possible
nuclear war: Although the possibility of a
nuclear war is unlikely, such as in the case
of a radiation leak from a nuclear power
plant, it should be noted that such a
possibility still exists. Therefore, in case of
Environment of Space Radiation
One of the biggest challenging of
the humankind's desire to explore deep
space is that it has to spend plenty of time in
deep space. Therefore, ionizing radiation
(IR) is one of the most fundamental topics
to consider. Even though in low earth orbits,
there are three kinds of radiation sources:
Galactic cosmic rays (GCRs), which range
broadly from protons to Fe ions, Solar
particle events (SPEs), and Electrons and
Protons trapped in the Van Allen Belts
(TPs). Primary GCRs consist of protons and
high energy heavy ion (HZE) charged
particles. The GCRs fluxes depend heavily
on the ISS altitude and their different shield
an undesired situation, all living organisms
in the world must necessarily have radiation
resistance. Otherwise, we can experience
the extinction of a collective life caused by
the nuclear bomb like in Hiroshima and
Nagasaki.
to use. The fluxes of primary TPs increase
substantially as the altitude of the ISS
increases. The space radiation environment
differs in and beyond LEO (Low-Earth
orbits), including the surface of the Moon,
Mars, deep space, and their comparisons.
Space radiation doses alter drastically due
to the varying intensity and peak amplitude
of SEP (solar energetic particles) events in
and near the Moon and Mars environments,
where a protective magnetic field is almost
entirely absent. Moreover, on the surface of
Mars, the UV radiation is remarkably higher
than that on Earth and exceeds the safety
limit for terrestrial life.[ CITATION Sur20 \l
1055 ]
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Tardigrade

Tardigrades, also known as water to maintain their hydroskeleton function.

bears, are small aquatic animals that inhabit These small animals need water to be
marine fresh water or limno-terrestrial active- they also have adapted to extreme
environments.[ CITATION Did19 \l 1055 ]. conditions such as desiccation, freezing,
Moreover, Tardigrades belong to the high pressure, heavy metals, and even
smallest invertebrates with a body length outer-space conditions. This highly resistant
that may reach 1 mm in the largest species, state is called cryptobiosis. This state gives
but usually is in the range of 0.25-0.5 mm. tardigrades the ability to survive conditions
They are essential aquatic animals, that are far more extreme than those
requiring to be surrounded by water in order imposed by their natural habitats. [ CITATION
Mar19 \l 1055 ]
.
A) B)

Figure1. (A): Illustration of Tardigrade in its natural habitat. (B): Illustration of “active” and

“Tun state”

During desiccation state, tardigrades conditions. In "Tun" state, tardigrades'

become "Tun" state. While tun state, physical features change to become
tardigrade can be more tolerant the extreme undistinguishable from dust, but even in this

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case, with only 10-12% water content they
can maintain their body's functional duties.
The evolutionary background to the extreme
radiation tolerance in desiccation-tolerant
animals is still largely unknown. However,
it is hypothesized to be side effect of
mechanism connected to desiccation
tolerance.[ CITATION EJC10 \l 1055 ] Also,
interestingly, some hydrated tardigrade
tolerance to UV radiations is much higher
than desiccation state. Higher tolerance to
extreme stress by hydrated tardigrades
1: DNA Repair Mechanism
As mentioned above, the X-ray and
UV beams can damage to the DNA by
causing DSB (double-strand breaks). DSBs
are the most severe DNA lesion, which can
induce to develop a tumour and/or cell
death. Therefore, organisms have various
DNA damage repair pathways to ensure
genome stability. DSBs are mainly repaired
by nonhomologous end-joining (NHEJ) and
homologous recombination (HR) in
eukaryotes. Once DSB damages are
generated following exposure to IR
(ionizing radiation), the complex-include
KU70/KU80 or RAD50/RAD51- is
clustered to the DSB damage sites. Rad51
is very important protein in the DNA repair
system and also is highly conserved.
compared to desiccation state might be due
to their ability to repair damages, containing
DNA damages, rather than to biochemical
prevention of damages itself, because in
hydrated tardigrades an ongoing
metabolism is present. In order to explain
these tolerance mechanisms, especially
radiation tolerance, there are two
mechanisms hypothesized. One of them is
an efficient DNA repair mechanism, and
another mechanism is to have protector
protein/molecules to help DNA in order to
protect DNA from the radiation damage.
Moreover, its proteins from tardigrades
possess the characteristic properties of
recombinase, mainly DNA binding and
ATPase activity. An important research
showed that lacking Rad51 show
hypersensitivity to DNA damaging agents
and meiotic defects. [ CITATION Eli13 \l 1055 ]
KU70/KU80 can activate the NHEJ
pathway by causing to re-join DSB ends.
Since the exposure of DNA to IR creates
DSB ends in various forms, genetic stability
and some nucleotides may be lost as a result
of the merging of these DSB ends. Hence,
NHEJ can be regarded as an error-prone
repair system. On the other hand, HR can be
regarded as a more reliable mechanism
compared to NHEJ because HR can use the
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sister chromatid as reference in order to
accurate repair of damaged regions.
However, NHEJ is used more often
preferentially in eukaryotes, but as NHEJ
.
2: DNA protector protein/molecules
According to widely accepted ideas,
the radiation tolerance mechanism of
tardigrades is depicted as the DNA repair
mechanism and DNA-protective molecules
working together in harmony. The DNA-
protective molecules include heat/cold
shock proteins, antioxidant defence and
various bio-protectants, and these provide a
comprehensive picture of the molecular
machinery underlying tardigrade stress
tolerance. Trehalose accumulation can be
regarded as the one of the protector
molecules. Trehalose, the non-reducing
disaccharide, has been demonstrated to
accumulate to a very high concentration in
various anhydrobiotic animals. Trehalose
can protect membranes and macromolecules
from desiccation damage by replacing
water. However, trehalose accumulation
alone cannot explain cryptobiotic survival.
Therefore, to explain these survival
mechanisms emerge different molecules.
One of them is late embryogenesis abundant
proteins (LEA). The intrinsically disordered
LEA proteins act as a molecular shield
cannot repair complicated DNA damage
(e.g., DSB, SSB, and oxidative damage),
HR is often activated for the repair of this
DNA damage in a cell-independent manner
preventing protein aggregation during
desiccation and that LEAs further may
function as membrane protectants, ion
skins, hydrated buffers and antioxidants, so
LEA proteins may show various protective
abilities within the tardigrades. Another
protein is heat shock proteins (HSP). A
large number of putative HSP genes were
detected in all investigated tardigrade
species. HSPs act as molecular chaperons
with significant roles in preventing protein
aggregation, supporting refolding of
denatured proteins and degradation of
aberrant proteins. HSP expression can be
induced by many environmental stressors
apart from heat, cold, food depletion,
osmotic stress and toxicants. Like HSP,
cold shock domain (CSD) can be said as
stress linked post-transcriptional gene
regulation. CSDs involve in adaptation to
low temperatures. Another protective
system is antioxidant defence mechanisms
during cryptobiosis, and a well-developed
antioxidant system has been suggested as a
possible explanation for the highly
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increased radiation tolerance seen among
tardigrades. In the antioxidant defence
system, catalases and peroxisomes can be
involved. Catalases can prevent H 2 O2
accumulation by its removal in a catallactic
or peroxidatic manner. Peroxisomes
METHODS
With the development of genome
editing systems last 10-15 years, the
scientific community want to alter genome
sequences in organisms in order to treat
genetic disorders and generate organisms
that can withstand different conditions. For
this purpose, there arose different
techniques over the years. Until the 5-6
years ago, there exist 5-6 techniques that are
Zinc Finger Nucleases (ZFNs)
ZFNs are a fusion protein of Cys2-
His2 zinc finger proteins (ZFPs) and a non-
specific DNA restriction enzyme derived
from Fok1 endonucleases. They are
associated with transcriptional regulation
and protein-protein interactions. One of the
biggest challenges with ZFNs include
constitute a key source of cellular
ROS/RNS. However, they also possess
well-developed protective mechanisms that
counteract oxidative stress and maintain
redox balance. However, it remains unclear
if and how tardigrades can utilize
peroxisomes.[ CITATION Mar19 \l 1055 ]
DNA recombinant technology, ZFNs (Zinc
Finger Nucleases), TALENs (Transcription
Activator-Like Effector Nucleases) etc.,
However, 5 years ago, A new technique that
is named CRISPR (Clustered Regularly
Interspaced Short Palindromic Repeats) has
emerged. This technique can be regarded as
the most useable, cheapest, easiest
programmable technique until today.
design and engineering of the ZFP for high-
affinity binding of the desired sequence,
which can prove non-trivial. Moreover, not
all sequences are available for ZFP binding,
so site selection is limited. Another
significant challenge is off-target cutting.
ZFN design improvements addressing off-
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target concerns have included ZFNs that
work in pairs, with each pair recognizing
two sequences that flank the target cleavage
Transcription activator- like effector nucleases (TALENs)
After discovering ZFNs, a new class
of natural DNA-binding proteins was
discovered in the plant pathogenic bacteria
Xanthomas sp. The proteins, named
transcription activator-like effectors
(TALEs), include 33-35 amino acids repeats
that flank a central DNA binding region
(amino acids 12 and 13). This DNA binding
region called as the repeat variable di-
residues (RVDs) specifically binds the
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)
Prokaryotes possess defence
mechanisms against viral and plasmid
invader like eukaryotic organisms. One of
these defence mechanisms is an adaptive
immune system found in many bacteria and
archaea, that is called CRISPR along with
the CRISPR-associated proteins-Cas
proteins-. It is first discovered in
Escherichia coli. Then, it was discovered
that a set of genes adjacent to the CRISPR
locus-termed CRISPR -associated system,
or Cas. Further, scientists were reported that
site. One ZFN binds forward strand, and the
second binds the reverse strand.[ CITATION
Chr18 \l 1055 ]
DNA. Like ZFNs, TALENs are a fusion
protein consisted of a TALE and Fok1
nuclease. Unlike ZFNs, design and
engineering of TALENs is much simpler
and can be done in a shorter time. One of
the challenging of TALENs is packaging
and delivery of TALENs in some viral
vectors might be problematic due to the
high level of repetition in the TALENs
sequence.[ CITATION Chr18 \l 1055 ]
the non-repeating CRISPR spacers
contained sequences derived from foreign
chromosomal DNA, specifically DNA from
bacteriophages. Moreover, some bacteria
that carried a given viral DNA sequence in
the CRISPR locus were known to be
resistant to infection by that phage. In this
way, this can indicate that the CRISPR
system may be a type of adaptive immune
system in prokaryotes. [ CITATION Deb18 \l
1055 ] A primary difference of this system
from the protein-based binding to DNA of
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ZFNs and TALENs is the use of a short specific nuclease against a specific DNA
RNA sequence as the specificity- target sequence, requiring only the synthesis
determining element to drive the formation of a new piece of RNA. This dramatically
of a DSB at the targeted site. In addition, simplifies and greatly reduces the time
the use of CRISPR/Cas9 avoids the need for needed for gene editing design and
protein engineering to develop a site- implementation.

Figure 2. Demonstration of CRISPR locus

Even though there are three kinds of
the CRISPR system, the widely used system
is Type II. Type II CRISPR systems require
only one protein, cas9, to scan, bind and
cleave the target sequence. Before
explaining the working mechanics of the
CRISPR system, the genomic CRISPR
locus is comprised of three components: the
trans-activating CRISPR RNA (tracrRNA)
gene, the Cas gene, and the CRISPR repeat
and spacer sequences. These are transcribed
into tracrRNA, Cas9 protein, and pre-
crRNA (pre-CRISPR RNA). The tracrRNA
has a region that is complementary to the
repeat region of the CRISPR locus and
binds to the newly transcribed pre-crRNA
creating a double-stranded RNA which gets
cleaved by Rase III, and with this process,
pre-crRNA becomes crRNA. In the working
mechanism of the CRISPR system, there are
three stages of this system: Acquisition,
interference (immunity). In the acquisition
stage, searching the invading DNA for
sequences complementary to the crRNA
occurs through Cas9 binding to the
sequences in invading viral or plasmid,
genome termed Proto-spacer Adjacent
Motifs (PAMs). Different Cas9 proteins

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from different species of bacteria or archaea
recognize different PAM sites. In the
interference stage, once crRNA and
tracrRNA is bounded to the Cas9. Then,
Cas9 is activated and cleave invading
nucleic acid sequences (interference). Cas9
Figure 3. General mechanism of CRISPR system
In response to Cas9 induced double-
strand breaks, cells employ one of two
DNA repair pathways to repair the damage:
either non-homologous end joining (NHEJ)
or homologous- directed repair (HDR).
NHEJ can occur through canonical NHEJ
(C-NHEJ), which ligates or essentially
"glues" the broken ends back together.
Additionally, there is an alternative end-
cleaves the DNA 3 base pairs upstream of
the PAM, resulting in a blunt-end cleavage
of DNA. Cleaving the DNA is deleterious
to the invading plasmid or virus, caused
degradation and protection against these
invaders.
joining pathway (alt-NHEJ), in which one
strand of the DNA on either side of the
break is resected to repair the lesion. Both
of these repair methods are error-prone,
meaning that the lesion is repaired
unproperly, caused insertions or deletions.
In another pathway, if there is a nearby
DNA molecule with homology to the region
around the double-strand break, then the
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homologous DNA can be used as a template
to repair the break through the homology-
directed repair (HDR) pathway. This
pathway can be exploited to introduced
precise edits or large insertions or deletions
by introducing a donor template for repair.
In this way, by making a cut at a specific
locus and taking advantage of the cellular
DNA repair pathways, there is the potential
Figure 4. Demonstration of gene adding in CRISPR system
to generate targeted mutations and insert
sequences of interest. Since a PAM site is
required for Cas9 binding, the target must
be upstream of a 5'-NGG-3' site (in the case
of SpCas9-Streptococcus pyogenes-). Thus,
as long as the sequence of your target gene
is known, Cas9 can be targeted to almost
any site given the presence of nearby PAM
(5'-NGG-3').[ CITATION Deb18 \l 1055 ]
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 In addition to site-specific gene
editing, the DNA-binding properties of
CRISPR/Cas9 may prove useful in other
important applications. For instance,
CRISPR can be used to controlling gene
expression by developing a catalytically
dead Cas9 enzyme (dCas9) that retained its
capability to recognize and bind a target
DNA sequence. Instead of cleaving the
bound DNA, the dCas9 enzyme remained
bounded the target sequence, disrupting
RNA polymerase or transcription factor
binding. This system is called CRISPR
interference (CRISPRi), and it could repress
the expression of multiple genes
CONCLUSION
Genome editing can be regarded as a
dangerous tool for altering nature balance
for some people. Also, these people may see
this technology as trying to play the role of
God. For this reason, they may want to
forbid this technology, but this attempt may
be more dangerous than using this
technology because it is obvious that not
using this technology will be a big problem
besides the bad results that may arise when
it is used because it can be known that if the
scientists can utilize this system, they can
treat cancer or genetic disorders. Therefore,
the community should not ignore these
simultaneously without altering the genome.
On the other hand, CRISPR activation
(CRISPRa) can act the same mechanism,
but instead of suppressing the genomic
activity, it can activate the genomic activity
through stimulating the promoter. As in the
case with CRISPRi and CRISPRa, the Cas9
components utilized for its precision
targeting rather than for catalytic activity.
As a result, the outcome of this system is
gene editing with no double-stranded
breaks. Since many disorders are led to by
single point mutations, this implementation
of CRISPR can prove to be one of the
system's most powerful gene-editing tools.
[ CITATION Deb18 \l 1055 ]
benefits of this system. Also, if we think of
CRISPR as a weapon for humanity, and
diseases as a monster, we can neutralize
these monsters with this weapon, but we
can also kill ourselves with this weapon.
Here, all humanity should behave in a
wisely mind and make this decision very
properly. Also, this system can be likened to
computer programming too. Some people
can utilize the programming for hacking,
but many people use programming for
people benefits, so do we prohibit
programming just because some people
hack computers through programming?
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 To sum up, in this project, I want to
integrate Tardigrade's radiation tolerance
mechanism and CRISPR system together.
In this way, firstly, we may understand the
underlying mechanism of radiation
tolerance of Tardigrade and secondly, as
mentioned above, with CRISPR method,
Tardigrade's radiation tolerance mechanism
can be used to many points for human
benefits.

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