Protectng human DNA against radiaton using genes from tardigrades

Bas Nieuwenhuis, Bowy la Riviere, Bram Kouwenhoven, Kristan Blom, Romano van
Genderen, Anna Daamen, Merel de Vries, Melitza Hesseling and Maarten van Tartwijk
Abstract:
The problem we will try to solve is the negatve efect
of UV light on our skin and DNA. In order to solve this
problem we will try to improve the repair mechanism
of our cells by using the repair mechanism of tardi-
grades and insertng parts of their DNA into ours. We
will frst test this method on mice before we try it on
human beings. These improvements will make us less
vulnerable to cancer and the negatve efects of UV
light.
Introducton:
The problem we are dealing with is ultraviolet light (UV
light) and its negatve efect on human DNA, which can
cause skin cancer. Our goal is to fnd a way to improve
our DNA repair mechanism so our DNA will be able to
withstand higher doses of UV light. We will take cer-
tain genes of tardigrades (organisms that can endure
extreme doses of radiaton) that encode for their DNA
repair mechanism and insert this DNA into the human
genome.
Proposal:
When the skin is exposed to UV light for too long, the
probability that the UV light causes pyrimidine dimers
becomes bigger. DNA-polymerase puts an A-nucleotde
on the other strand where a pyrimidine dimer is cre-
ated. When this mutaton takes place in the p53 gene,
a tumor suppressor gene, the cell is no longer able to
control other genes that could be damaged. This means
that damaged cells will contnue to divide, so next
generaton cells will also be damaged. Approximately,
when there are several mutatons in a cell, the cell will
become a cancer cell. Because the cell will divide with-
out control, a tumor will arise. When this happens in
the skin, we call it skin cancer.
1
The soluton to the problem:
Tardigrades are very tny animals
2
(about 1 mm) that
can withstand massive amounts of radiaton. For exam-
ple, they can survive 1000 tmes more radiaton than a
full grown elephant. [E]
Actve protecton (through DNA repair):
phrA, a DNA photolyase protein which repairs DNA us-
ing light
3,4
RAD51, which glues broken strands of DNA back to-
gether, therefore irrelevant
5,6
Tardigrades also have passive protecton, but that is
impossible for humans.
phrA separates pyrimidine dimers into separate nucle-
obases
7
phrA is especially useful because it is coded by only
one gene
8
DNA extracton
The frst step includes breaking the cells open, also
called cell disrupton or cell lysis. Afer that we re-
move the cell organelles and the cell membrane, than
all the proteins will be demolised. The third step in-
cludes that we add another enzyme called RNase to
the remaining DNA and RNA. Finally we will flter the
soluton. Afer these fve steps you can split the DNA by
using a restricton enzyme. The remaining DNA can be
replicated in a PCR device.
How to insert the DNA in the right place using gene
therapy?
The modifcaton with the phrA gene must be done on
zygotes. Therefore we use transgenesis. The vector we
use to insert the gene in the human DNA is the CRIS-
PR-Cas9 enzyme. [SUPERSCRIPT] Afer the transgenesis
is succeeded, the cell will treat this new DNA as part of
its own genome, translatng and transcribing the new
gene phrA along with the cells own genes.
Plan:
We insert the gene coding for the phrA protein in a
specifc number of rat cells by transducing the cells
and seeding them at single cell density. Afer letng
the cells multply for a while, we irradiate them with
UV-B radiaton. Then we lyse all the cells. Afer a specif-
ic tme we are going to check if the phrA protein really
did its job by countng the amount of pyrimidine dimers
by placing isolated DNA on a gel and applying specifc
antbodies against these dimers. If this experiment suc-
ceeds, we will contnue with apply our soluton to rats.
We will observe their ordinary behavior. If the trans-
gene rats show a signifcant (more than 50%) decrease
in tumor development, we assume our soluton works.
If that is the case, we will contnue our project by also
applying this method to isolated human cells. If also
that works, we can test our soluton on humans.
Biology: Find the phrA gene and insert it into the rats
and study the efect of pyrimidine dimers in relaton to
skin cancer.
Physics: Get a good understanding of the working of ra-
diaton to prevent the damage caused by this.
Mathematcs: Radiaton damage is a very stoch
astc process
9
, because the probability of a photon
causing a pyrimidine dimer is controlled by random
chance. If mathematcal distributons are not ap-
plied, we could base our conclusion on an exceptonal
chance, therefore making our results unreliable.
Ethical problems with genetc engineering:
Our soluton will spark controversy when it would be
used as a medicine. The main argument against GE for
religious people is that we will be playing god. But to
us this is like saying someone cant eat cookies because
youre on a diet. They are not in a positon to work
against a small change that can help so many. To us its
not ethical to deny people this right.
Then theres the ethical problem with testng. To avoid
human testng we will use mice and single cells for test-
ing before implementng our change.
-Image of a tardigrade
Graph relatng the irradiance of the source to the percentage of actve
tardigrades over tme, added human MED value for comparison
Safety issues:
The single cell testng phase is very important, here
we can see if our method and alteraton (de)actvates
genes that arent supposed to. If we are to alter the
human genome we must be sure that we only make
changes that are supposed to happen. Because this is
a big safety issue we will have to do a lot of single cell
testng.
Conclusion:
We can conclude that we have found a soluton to our
problem concerning the negatve efects of radiaton.
Once we have fnished our experiments and human
DNA has been manipulated and improved, we will be
able to withstand higher doses of radiaton which will
decrease the risk of skin cancer.
The CRISPR Cas9 enzyme is targeted to DNA by a guide RNA, once in place the enzyme makes a
double stranded cut
Thymine dimer formed by ultraviolet radiaton