CRISPR/Cas Technology
An Amazing Method for Disease Treatment.
K.P.M.P. Gunathilaka
Figure 01 (A.Vidyasagar and N. Lanese. (2021, Oct).
“What is CRISPR?,” [Online Article]. Available:
https://www.livescience.com/58790-crispr-explained.h virus. [2], [3], [4]
tml)
is used in disease treatment. In this article,
what is CRISPR/Cas technology, the
development and applications of it in disease have developed CRISPR/Cas technology.
treatment are discussed.
CRISPR/Cas technology is derived from
an actual process which is occurring
inside the bacteria and archaea cells.
When a viral infection happens, the
proteins which is called as Cas in the
bacterial cell, take bits of that viral DNA
and insert that sequence into the bacterial
genome. Because of the pattern of this
added DNA sequence, they are called as
clustered regularly interspaced short
palindromic repeats (CRISPR). Then,
this DNA sequence makes a RNA strand
that is complementary to it. This RNA
strand is then attached to a protein known
as Cas9. When the particular virus attacks
the bacteria again, this Cas9 protein
When it is heard about the word
“Genetics”, not many people are
well aware about what is actually
going on this field. Actually, it is an
amazing field with full of new
researches and findings that deals
with genetics of organisms. This
field contributes for lot of findings
in many fields. Disease treatment is
one section that gets benefits from
genetics. CRISPR/Cas technology
is one such method in genetics that
identifies it with the help of the RNA
strand that it contain and destroy that
Once the scientists have identified this
process, they found out, this method can
be applied to any DNA type. Then they
When a specific DNA sequence is
inserted to a Cas9 protein and when it is
inserted to particular cell, this protein
reads the DNA sequence until it finds a
similar site to its’ RNA sequence with
about 20 nucleotides. Then that target
sequence of the DNA is cut by the Cas9
protein. Once it is repaired, the
mutations may occur. This leads to
disable that particular gene of the
genome. This is called as gene or
genomic editing. And also for replacing
genes with errors, turning on or off genes
without changing its’ sequences this can
be used. These basic things have been
improved for disease treatment. [2], [3],
[4]
 Figure 02 – Genome editing by CRISPR/Cas9 system (A.Vidyasagar and N. Lanese. (2021, Oct).
“What is CRISPR?,” [Online Article]. Available: https://www.livescience.com/58790-crispr
explained.html)
Development
The CRISPR system was first discovered in
1987. This discovery helped to find out new
treatment methods for some severe disease
types [5]. But its’ genome editing ability
has confirmed in 2012. Since then, this
technology becomes the most widely using
genome editing method. This capability has
used to achieve many victories in
pathogenic mutation correction, solutions
for organ xenotransplantation and in cancer
immunotherapy etc. But there are still some
limitations of this method. Scientists are
working continuously to develop this
technology day by day. [1]
As the results of the researches of
CRISPR/Cas technology different new
systems have been developed. A system has
been developed with the ability of
activation or inhibition of the transcription
of target genes which is called as dead
Cas9 system. This was done by
introducing two point mutations to HNH
and RuvC nuclease domain. This system
doesn’t have the
ability to cleave target DNA, but it remains
its’ binding ability to DNA. This system is
also useful for genome imaging due to the
use with fluorescent proteins, simultaneous
manipulation of multiple genes of a cell.
[1]
Base editing system is another developed
system that have the ability to do point
mutations without cleaving the double
stranded DNA. It highly avoids the
formation of the random insertions and
deletions. But these systems cannot be used
for any target sequence due to its point
mutation restriction. Still scientists are
working to overcome this problem. [1]
To recognize and cleave the target DNA
using Cas9, at the 3’ end of the target
DNA, there should be NGG PAM. Instead
of this PAM, there are some other type of
PAMs can be seen in some with low
efficiency. To overcome this, scientists
have developed many variants. These are
called as Cas9 variant systems. Previously
inaccessible disease relevant genetic
variants
have been repaired using them. But still
these systems should need further
improvements due to their low efficiency.
[1]
CRISPR/Cas technology has been also
improved to edit RNA. They are called as
RNA editing systems. Like in DNAs, here
the target RNAs are recognized and are
cleaved. For that RNA guided Cas13
protein is used. Since this systems are
highly efficient and specific and can
overcome the ethical issues and potential
risks, they are widely used for preclinical
studies of many diseases. [1]
Another development of CRISPR/Cas
technology is prime editing system, which
has the ability to control all base
substitutions, insertions and deletions of the
DNA sequence without breaking the double
strand or with the use of donor DNA
templates. This system is contained with
prime editing guide RNA and Cas9 protein
with the catalytic ability that combined
with reverse transcriptase. This system has
high efficiency for genomic editing. But
still, further researches should be required
to understand its’ function well. [1]
Applications
With those developments, CRISPR/Cas
technology is used for many applications in
disease treatment. One of the widely using
application is, animal models for human
diseases using CRISPR/Cas technology.
Rather than traditional animal models,
these models have helped to get more
efficient results about human diseases.
Because without limiting to mice as an
animal model, larger animals like pigs,
rabbits and non-human primates can be
used for animal modeling for human
diseases thanks to CRISPR/Cas
technology. These larger animals have
closer characteristics in anatomy,
physiology and evolution than mice. So
more accurate results about diseases can be
obtained. The treatment methods for
metabolism, cardiovascular and pulmonary
diseases have developed using this method.
And models for neurodegenerative,
xenotransplantation, cancer and
immunodeficiency have also been
developed from this. Now it has identified
that the best models are given by non
primates due to they have more similar
characteristics to humans. [1]
With the use of induced pluripotent stem
cells (iPSCs), CRISPR/Cas technology gets
the ability to create new disease models.
This combined technology is allowed to do
cellular replacement therapies and precise
therapies for diseases like β-thalassemia,
neurodegenerative diseases and acquired
immunodeficiency syndrome etc. [1]
Applications in gene therapy includes
another range of applications of
CRISPR/Cas technology in disease
treatment. Monogenetic diseases like β
thalassemia, sickle cell disease and
hemophilia B are treated using
CRISPR/Cas technology. Using this
technology in β-thalassemia, the defective
β-globin gene of iPSCs cab ne repaired.
Then the red blood cells get the ability to
produce them normally. Another method
for treating this disease is reactivating fetal
hemoglobin by knocking out BCL11A
gene. These two methods are also used to
treat for the sickle cell disease. [1]
Other hereditary diseases like Duchenne
muscular dystrophy, Retinitis pigmentosa,
hereditary tyrosinemia and cyctic fibrosis
have also been shown success in diseases
treatment using this technology. [1]
And also gene therapy using CRISPR/Cas
technology is used for viral infectious
disease treatments. It can be used for
treating HIV AIDS. CRISPR/Cas system
targets the long terminal repeats and
destroy HIV – 1 provirus. So, the HIV – 1
can be completely removed from the
genome of the patient. The HIV
co-receptor CCR5 gene in CD4+ T cells
can be knocked out to induce the
resistance for HIV – 1 infection. Human
papilloma virus that causes to cervical
cancer can also be treated using this
technology. This is done by cutting and
blocking the expression of E6 and E7
genes by sgRNAs. [1]
CRISPR/Cas technology is also used for
cancer treatment. This causes to inhibit
tumor cell growth and promote cell
apoptosis. That may lead to tumor growth
inhibition. So the oncogenic
genome/epigenome mutations can be
corrected by this. Universal CAR-T cells
are produced using CRISPR/Cas to do
immunotherapy for cancer treatment as
well. [1]
Therefore it can be seen that CRISPR/Cas
technology would become a more vast
technology in future than today. By
improving researches more and more, this
technology will give more benefits for the
world in the future with better treatment
methods to save many lives of the world.

References

[1] Y. Xu and Z. Li. (2020, Aug). CRISPR-Cas systems: Overview, innovations and
applications in human disease research and gene therapy. Computational and
Structural Biotechnology Journal [Online]. 18, 2401-2415. Available:
https://www.sciencedirect.com/science/article/pii/S2001037020303846
[2] A.Vidyasagar and N. Lanese. (2021, Oct). “What is CRISPR?,” [Online Article].
Available: https://www.livescience.com/58790-crispr-explained.html [3] “CRISPR,”
[Online Article]. Available: https://www.newscientist.com/definition/what-is-crispr/
[4] A.M. Henle. (2019, Jan). “How CRISPR lets you edit DNA.” [Online Video].
Available: https://www.youtube.com/watch?v=6tw_JVz_IEc
[5] M. Alagoz and N. Kherad. (2020, May). Advance genome editing technologies in the
treatment of human diseases: CRISPR therapy (Review). International Journal of
Molecular Medicine [Online]. 521-534. Available: https://www.spandidos
publications.com/10.3892/ijmm.2020.4609