CRISPR in Medicine: A Review of the Challenges, Approaches,
and Clinical Trials for the Medical Applications of CRISPR
Technologies
Abstract
CRISPR-Cas9 came after ZFNs and TALENs, and gene-editing technology has been utilized in managing
genetic conditions affecting living organisms. It has a double-strand break (DSB), which helps trigger
DNA repair pathways in the cells, and exposing the DNA to radiation makes the pathways develop. The
limitations of the technology are that humans have an adaptive immune system that can easily identify
CRISPR Cas9 as different, and it has been in existence for a short duration. CRISPR Cas9 is a geneediting method that has been used to examine conditions such as Sickle Cell Anemia and Beta
Thalassemia, LCA, hATTR, Leukemia, Lymphoma, and Lung Cancer, among other genetic disorders.
Some clinical trials have been conducted, with some being successful and others showing areas that
researchers should improve. CRISPR-Cas9 should continue to be utilized in genetic studies as it will help
identify how to target mutant DNA at specific sites in human bodies. Correct utilization of the technology
will bring significant benefits to biological research and manage some of the existing complex conditions.
CRISPR-associated systems (Cas) are effective methods for focusing on certain genes for use in
biotechnology, agricultural engineering, laboratory research, and the treatment of human illness. The
most widely used gene-editing nuclease, Cas9, has showed considerable promise for the treatment of
malignancies, viral infections, genetic illnesses, and other conditions. According to recent research,
certain additional CRISPR-Cas system varieties may also have the unexpected potential to enter the fight
as gene-editing tools for a variety of purposes. Despite the quick advancements in basic research and
clinical trials, numerous fundamental issues—such as editing effectiveness, relative delivery difficulties,
off-target consequences, immunogenicity, etc.—present ongoing, major obstacles.
Keywords: CRISPR-Cas9, gene-editing technology, mutant DNA, Clinical Trials, Genetics.
Introduction:
CRISPR/Cas9 has developed significantly from its initial discovery as a bacterial immune system to its
multiplex genome-editing applications. Despite these amazing advancements, CRISPR/Cas9 multiplex
gene integration still faces a number of difficulties that must be resolved in order to increase the
technique's capacity and effectiveness. First, it's important to streamline the gRNA design and target
locus selection processes. For CRISPR/Cas9 gene editing to be effective, including multiplex gene
integration, designing gRNAs is a key step (1). Although numerous assisting tools for gRNA designs have
significantly improved for a variety of Cas proteins (7) (13) the effectiveness of these gRNAs in vivo still
needs to be examined. This is especially important for multiplex gene integration). Additionally, the choice
of target loci has a big impact on how effective the integration is. Baek et al. discovered, for instance, that
specific target locations in gene-sparse regions were very ineffective, which may be related to the
chromatin accessibility issue (3). Similar findings have also been shown in other places. The potential and
effectiveness of multiplex gene integration would be considerably enhanced by the characterisation of
effective gRNAs and target loci, as well as by the placement of synthetic landing pads. Second, HDR has
to be improved as yeast's DSB repair process. For repairing DSBs, Baker's yeast is known to favour HDR
over non-homologous end joining (NHEJ). This is shown by producing Cas9 and gRNA in a cell without
donor DNA, which may make yeast hazardous. However, multiplex gene integration tests revealed that
certain yeast colonies may still survive in the absence of donor DNAs, proving that NHEJ is active in
repairing the DSBs (3). As a result, there may be more false positive colonies and the multiplex genes
integration process may be less effective. It has been shown that deletions of several NHEJ pathway
genes, including POL4, DNL4, and Ku70, lower false positive rates and boost HDR success. Deleting the
NHEJ genes may thus improve the capacity and effectiveness of multiplex genome integration by
lowering the quantity of background colonies produced by NHEJ chromosomal repairs. The effectiveness
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of yeast transformation must be increased. Introducing a significant quantity of foreign DNA to yeast cells
is necessary for CRISPR/Cas9 multiplex gene integration. More donor DNA and/or gRNA cassettes will
be needed as there are more targets to be altered. To increase the effectiveness of the transformation,
transformation procedures may be used, such as electroporation or the addition of amino acids.
Figure: CRISPR/Cas9-based genome editing. gRNA interacts to the DNA around the PAM site
in a complex formed by Cas9 and gRNA. NHEJ or HDR may be used to repair a DSB in the
target location. As a general rule, the NHEJ repair process leads in alterations and deletions, as
well as a frameshift that results in a gene loss. The nucleotides may be inserted into the gene,
causing a frameshift or inserting cDNA, if a DNA donor with homology in the ends is given.
Genome-directed repair (HDR) is a term used to describe the process of repairing damaged
DNA using a guide RNA (gRNA) and a PAM (protospacer-adjacent motif).
Finally, integrating various techniques may enhance the development of CRISPR/Cas9 multiplex
genome integration in yeast. For instance, combining pre-installed target sites with RNA cleaving
mechanisms (3), RNA cleaving mechanisms with a sequential integration approach, or both, can reduce
the time needed and increase the number of genes for multiplex integration. The approaches that are
now being developed have been outstanding and have limitless potential. However, the potential for
multiplex genome editing will increase as these issues are successfully resolved and other approaches
are creatively combined. As a result, research into and improvement of intricate, specialised metabolic
pathways in yeast will proceed more quickly.
Background
With the establishment of the gene as the base unit in the field of heredity, the ability to make specific
modifications is essential in the quest to combat disease. Gene therapy is one of the most promising
methods to achieve such a goal, having potential to cure diseases, ranging from cancers to infectious
diseases. The Human genome contains ~25,000 genes, and mutations in specific genes can cause
genetic disorders, such as Huntington's disease, while mutations in oncogenes can cause severe
disease, such as cancer. Mutations occur ubiquitously and can occur at a higher rate due to
environmental factors, such as radiation or UV light. These mutations may also be inherited. Over 3,000
genes have been linked to disease phenotypes and with the completion of the human genome
sequencing, genetics has become a major focus of research in clinical medicine. One way to combat
these diseases is through gene therapy...An effective therapeutic technology with promise to treat genetic
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disorders. This can be done through the restoration of mutated genes through in-vivo or ex-vivo methods.
A popular method to do so involves recombinant DNA technology, in which healthy DNA fragments,
modified in a lab environment, are inserted into the genome via a vector. These vectors often pose large
challenges, as they must be efficient in the release of one or more genes, depending on the size, while
not invoking an immune response. These can be plasmidial, nanostructured, or viral, with viral most often
used. This is because of its efficacy in invading cells, when the infectious part of the virus is removed,
viruses can accurately and safely deliver the DNA fragments to the nucleus of cells. However, viral
vectors as the primary means of expressing gene therapy are not effective, since the potential for offtarget effects and immune response is very high. For instance, when first experimenting with the
technology, in 1999, 17 year old Jesse Gelsinger passed away when receiving a potential treatment for
OTCD. The death was due to the uncertainties involving gene therapy and hence the field took a major
step back for clinical applications.There are very few FDA approved treatments, to this day, using this
technology and they are all mostly ex-vivo, which makes it very exclusive and expensive. The gene
therapy medicine available is YESCARTA, which deals with B-cell lymphoma and costs $373,000 for a
single dose of the therapy.
Gene Editing:
The process and technologies surrounding gene editing revolves around the natural cellular process of,
which is the process by which cells repair their DNA after any sort of break. DNA, an integral part of
proper cellular health, is inherently unstable, as it is subject to constant mechanical stress and chemical
modifications that may lead to breaks in strands of the double helix. With the invention of CRISPR/Cas9,
a genetic modification tool that is derived from the defence mechanism of certain bacteria against
the viruses and plasmids, genome editing re-emerged in 2012. A broad range of experimental models,
along with cell lines, laboratory animals, plants, and human clinical trials, have been successfully tested
using this strategy. The Cas9 nuclease is directed to make a site-directed double-stranded DNA break by
employing a short RNA molecule as a guide in the CRISPR/Cas9 system. The damage done to DNA may
be repaired using a method that permanently alters the genomic target sequence. This can be caused
naturally, during DNA replication and Meiosis, and externally, from radiation and certain
chemotherapeutic drugs. By looking at an example from yeast cells (Scherer and Davis, 1979 using
Saccharomyces cerevisiae), it can be understood that DSBR, when done incorrectly, can directly lead to
gene alterations, gene deletions, chromosome aberrations, and even cell death. DSBR is typically carried
out by two means, which are homology-directed repair (HDR) and non-homologous end joining (NHEJ),
which are the mechanisms by which eukaryotic cells operate. In NHEJ, only a few base pairs of homology
are required between the two broken DNA ends. NHEJ occurs without a homologous DNA template,
hence the random nature of the repair and its characterization as error-prone.This means that repair is
extremely volatile, in that the cell can easily repair with an unwanted or incorrect strand of DNA, which
allows for mutations, which are oftentimes not very precise. Also, the cell can easily make unwanted
deletions. This can occur at any stage of the cell cycle and is the typically used DSBR mechanism for
cells. Homologous recombination, on the other hand, is a more precise means of repair, which involves
the use of a homologous repair template for reparation. In contrast to NHEJ, HDR takes place typically
during the late S or G2 phase of cell cycle, in which the sister chromatid itself serves as a repair template.
The process involves end resection, strand invasion, DNA synthesis, holliday junctions, ligation, and
precise repair. HDR is not performed often by cells, as NHEJ is more efficient. However, through HDR,
given that cells are able to make very precise repairs, safe gene editing technologies seek to activate
HDR as the DSBR process, simply giving the cell a different homologous DNA template, as opposed to
the typical sister chromatid template.
CRISPR/Cas9 may also be used to improve yeast strains using metabolic engineering tactics, such as
gene disruption, gene downregulation, and overexpression of indigenous yeast pathways, which can be
used to eliminate or reduce competitive pathways.
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NHEJ is naturally used for 2 gene edits, which include gene knockout and gene deletion. HDR is used for
gene correction and gene addition. Gene editing technologies typically seek to utilize the natural gene
correction mechanism for their repairs, and hence utilize HDR for repairs and additions. To carry this
process out, an exogenous DNA template, which can be either single-stranded or double-stranded, is
required and is typically introduced through gene editing technologies.
CRISPR Cas-9
CRISPR Cas-9 is utilized by scientists by creating a Cas-9 complex to create double-strand breaks in the
DNA. Gene editing using CRISPR–Cas9 has revolutionised life sciences, allowing for nearly infinite
genomic manipulation: Double-stranded breaks in DNA are repaired via one of the inherent repair routes
that Cas9 endonuclease may access. Imprecise double-strand repair introduces random changes like
indels or point mutations, while precise editing restores or selectively edits the locus as specified by an
endogenous or exogenously given template. The natural cellular DNA repair pathway, NHEJ or HDR, is
utilized by replacing the template DNA with a custom template. The Cas-9 complex consists of the Cas9
protein and guide RNA, with the protein itself consisting of six domains: REC I, REC II, Bridge Helix, PAM
Interacting, HNH, and RuvC.
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Caption: REC I’s primary function is for binding the guide RNA to the complex, as well as
structural properties. The role of the REC II domain is not yet well understood. The bridge helix
is rich in arginine and is essential in initiating the cut itself. The PAM Interacting aspect is
responsible for binding to the target DNA. The HNH and RuvC domains make the actual cut.
Ethical dilemmas
Germline editing refers to the practise of altering the genomes of gametes (eggs and sperm) and early
embryos in addition to somatic cells, which make up the majority of the body. Any such changes to
people would have an impact on both the person and their offspring. Theoretically, they may also be
utilised to improve positive characteristics rather than treat illness. So, until the grave moral and cultural
ramifications are better understood, scientists have advocated for a halt on human germline modification.
The moratorium is entirely supported by JAX and upheld by its researchers.
Technological Limitations
Although CRISPR/Cas is a very potent technology, it has significant drawbacks. It is:
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For many therapeutic applications, it is still challenging to deliver the CRISPR/Cas material to
mature cells in significant quantities. The most frequent form of delivery is by viral vectors.
Even cells that take up CRISPR/Cas may not have genome editing activity since the technology
is not 100% effective.
Rarely completely precise, and "off-target" modifications, albeit uncommon, may have serious
repercussions, especially in clinical applications.
In addition, pre-existing antibodies against Cas9 are a major concern, as seen in a study conducted with
human subjects showing more than 50% with immunity against the bacterial nucleases. In addition, the
gRNA triggers an innate immune response in human cells that can be concerning. These concerns are
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largely for in vivo gene therapy and as a result, ex-vivo options are being explored for the future of
CRISPR technologies.
Discussion
Figure: CRISPR-(d)Cas9 may be used to treat haematological diseases. CRISPR-Cas9 gene
therapy has great promise for treating inherited blood illnesses such hemoglobinopathies,
anemias, and coagulation disorders. CRISPR-Cas9 may also help us better understand the
molecular pathways that lead to the development of blood disorders, which in turn can lead to
the relief or even the cure of human illnesses.. Cellular stem and progenitor cells (HSPCs),
haematopoietic stem/progenitor (HSPCs) and induced pluripotent stem cells (iPSCs) are all
examples of cell types that may be reprogrammed to become any kind of cell.
Sickle Cell Anemia and Beta Thalassemia (ex-vivo)
Of all monogenic diseases, Sickle Cell Anemia is the most common and
most studied. Sickle Cell Anemia, also known as Sickle Cell Disease, is an inherited blood disorder in
which oxygen transport is inhibited by a lack of healthy red blood cells. Beta-Thalassemia is another
disease which is very similar to Sickle Cell Anemia.
CRISPR-Cas9 was used to target BCL11A enhancers, and when patients received autologous CD34+
cells, there was an increase in fetal hemoglobin (10). Gene editing using this technology helps identify the
disease-causing mutation, which will guide the decisions on how to manage the hematopoietic stem cells.
Different clinical trials have been conducted to establish a suitable treatment for the conditions. In order to
cure sickle cell disease and beta-thalassemia, this CRISPR cell therapy clinical study includes restoring
the expression of foetal haemoglobin. Patients' bone marrow stem cells are taken out and CRISPR-edited
to render BCL11A, a suppressor of foetal haemoglobin synthesis, inactive. Analysis of seven clinical trials
reveals that two doses of crizanlizumab can help enhance safety for vaso-occlusive crises patients, and
individuals who received L-glutamine drug group have less hospitalization rate in comparison to those
given place (2).The clinical trials will help explore ways to enhance the quality of life of patients with both
conditions. The advantage of the method is that it focuses on reducing the long-term progression of the
condition. However, the drawback is that the research provided findings for clinical trials that are still in
progress, and therefore the findings cannot be relied on to manage sickle cell anemia and Beta
Thalassemia.
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LCA (in-vivo)
Leber Congenital Amaurosis (LCA) is another monogenic inherited retinal
disease that is known to cause blindness or vision loss from early childhood. In 2017, the gene therapy
Luxturna was approved by the FDA, which was the first directly administered gene therapy approved in
the United States. Luxturna is only effective if the patient has a mutation in the RPE65 gene and viable
retinal cells. The drug currently sits at a price of $850,000 for a one-time treatment and works by
delivering a normal copy of the RPE65 gene through a viral vector. Using CRISPR/Cas-9 could prove to
be a cheaper and more effective solution to the disease.
Over the years, low vision aids have been utilized to correct the
abnormalities, but it does not offer a long-term solution. Chiu et al. (2021) (5) highlight that AAV-mediated
subretinal is used in gene editing, and it is done by introducing the CEP290 gene, and it utilizes the
CRISPR-Cas9 system. The operation involves normal splicing of the CEP290 protein, making it start
expressing itself. The CRISPR-Cas9methid ensures that there is better management of the GRK1
promoter. The technology has helped enhance retinal viral gene therapy, and better outcomes will be
expected in the coming years. Multiple clinical trials have been conducted for this condition. Clinical trials
since 2007 have focused on targeting epithelium-specific 65kDa, and the objective is determining issues
with retina pigments (30). CRISPR seeks to find a way of cutting and deleting exogenous DNA that makes
LCA persist. Triggering the enzyme will make individuals acquire immunity. The advantage of the
technology is that it could lead to new discoveries. Retinal viral gene therapy would become a standard
technique of delivering genes to the retina and eliminating the defective ones. That would help improve
vision and ensure individuals acquire immunity against eye disorders.
Nevertheless, its drawback is that there is no reliable delivery method for the procedure. There will be a
disruption in the delivery of genetic material, which would affect individuals' immune systems.
hATTR (in-vivo)
Hereditary transthyretin amyloidosis (hATTR) is a rare disease caused by
mutations in the gene encoding transthyretin (TTR). It is a fatal disease, given that it makes proteins fold
the wrong way and stick together, forming clumps known as amyloid fibrils. This process is known as
amyloidosis and these clumps can accumulate in organs and tissues which interferes with organ
functions and hence is fatal. The disease typically has severe effects on the nervous system and heart,
as nerve pain, vision loss, dementia, loss of movement control, and heart failure are common effects of
AATR. It is a monogenic disease and the use of CRISPR-Cas9 tools may reduce the amount of faulty
TTR protein in the body, hence alleviating pressure on organs. It is typically diagnosed at the age of 5065 and affects approximately 4000 people annually in the United States.
Ravichandran, Lachmann, & Wechalekar (2020) indicates that deposition
of the proteins results in multi-organ failure as they will develop to become insoluble fibrils. CRISPRCas9–mediated is ideal for targeting specific body organs, and it will prove to be an effective therapy. The
technology helps recognize the motifs and target a site despite the unwinding of the helix (25). Clinical trials
are using inotersen and tafamidis to test the effectiveness of the techniques in managing different types
of ATTR amyloidosis (15). The drugs are helping in dissolving the insoluble amyloid fibrils and eliminating
the misfolded proteins. The trials target different proteins, which will contribute to better insights. The
research is moving toward developing drugs that target affected sites and ensuring they correct the
genes. That will help provide commercially viable options that will be used to reduce the prevalence of the
condition. The advantage of the trials is that the treatment will apply to human beings and all the other
living organisms. Protein deposition affects many species, and finding the solution will help to enhance
their sustainability.
The drawback of the trails is that it is still expensive to carry out the procedure. That causes uncertainty
as it means only a few individuals will be able to afford the treatment once it has been marked as safe for
medical use.
Leukemia
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Leukemia is a type of blood cancer that affects the lymphatic system and bone marrow. Proliferation
occurs in the lymph nodes and spleen, making the body produce an excess amount of white blood cells
(4). CRISPR Cas-9 technology can provide a safe platform for treating cancerous cells, and it will achieve
that through exploiting mRNA and protein. When using the technique, there has been no proof of
genomic integration, but there are higher chances of delivering genes and protein to the target cells. That
would lead to gene recombination that can help in reducing the number of white blood cells produced in
the body. Low off-target effects will make it a preferred method of treating leukemia as there will be fewer
side effects for the patients. Research by Khalaf et al. (2020) reveals that CRISPR/Cas9 has been
employed in animal cancer models, and they have been shown to correct mutations in vivo (16). The genemodified animals will help provide critical insights utilized in leukemia biological research. Advanced
research in the future will focus on using CRISPR in patients with leukemia, and the findings will inform
future research in this field. The method's main advantage is that it would result in the treatment of
untreatable conditions. The animal cancer model's success would make the FDA and other bodies
approve the use of gene editing in leukemia patients. One drawback is that there is a lack of data about
the use of the technique in embryonic stem cells. That will limit its use in homologous recombination and,
therefore, management of leukemia.
Lymphoma
Lymphoma is a form of cancer that affects the lymphatic system. Lymphoma targets the bone marrow and
thymus gland and is characterized by enlargements of the lymph nodes and weight loss (11). Patients with
lymphoma have less than five years' survival rate. (9) Felce et al. (2020) indicate that CRISPR/Cas9 helps
in enhancing lymphoma immune evasion by targeting deregulated genes, including the B-cell marker
Cd19 in the tumor microenvironment. 15 of the 17 T-cell lymphoma patients could be examined as of
December 6. According to CRISPR, the overall response rate (ORR) was 71%, with a full recovery
occurring in 29% of patients. The business described the drug's safety profile as adequate. The success
rate proves to be higher than that of other immunotherapies, and it could be a potential technique to
utilize in the future. It achieves the results by reducing B-cell lymphoma cells sensitivity and preventing
drug dependencies among the patients. That would help knock out the lymphoma cells and prevent them
from spreading and affecting other human body organs. CRISPR/Cas9 has enhanced Lymphoma clinical
trials by preventing tumor formation after the engineering of T cells. (23) The cell-based therapy helps
repair genetic alternation, which will lead to stimulation of immune response. Quaz outlines that the
technique’s efficacy has moved researchers away from ZFNs, TALEN, and other gene-editing methods.
The CRISPR model will be critical in examining the contribution of proteins and how they express
themselves, therefore contributing to aggressive lymphoma. It will likely influence research direction in the
coming years as researchers continue to specialize in gene-editing techniques. The significant benefit of
the clinical trials is that it helps in rating the effectiveness of the cell-based therapy. The research shows
that there is a great potential for managing tumor malignancies such as Lymphoma in the future.
However, the main issue is that it is challenging to establish their effectiveness in graft-vs-host disease.
The trials are still at their initial stages, and they may end up producing different results in human
subjects.
Lung Cancer
It is a form of cancer that causes the uncontrolled growth of cells in the lungs. Lungs are critical in the
human respiratory system, and when there is an overgrowth, it will affect how the chest will take in
oxygen and exhale carbon dioxide. The conditions can affect people who have never smoked, but
smoking predisposes an individual to higher risks. Richards et al. (2020) indicate that there were 148,869
deaths associated with lung cancer, and it remains to be the most lethal form of cancer. Identifying the
condition in its early form is critical as it helps in reducing the chances of death (26). Lung cancer screening
is highly recommended, and it helps identify the early presence of the condition. CRISPR Cas9
technology helps prevent the multiplication of mutant cells and prevents them from destroying healthy
cells in the lungs (20). Higher efficacy makes the technique popular among the scientific community, and it
will be critical in combating lung cancer in the future. The technique leads to site-specific changes in the
lungs, which helps improve DNA recombination. By encoding the proteins, the cells can acquire adaptive
defense mechanisms and utilize memory signatures from the RNA to prevent any form of invasion from
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mutant cells. Recent research by Sarkar & Khan (2021) reveals that the first clinical trial to utilize
CRISPR/Cas9 shows that modifying T-cells can help treat cancer (27). The technique can be used in the
target cells, and it would help contribute to site-specific results. Despite the clinical trials not being
approved, it shows there are promising results, and the strong efficiency will lead to more human trials.
The research results will inform future research, and CRISPR Cas9 will be utilized in managing lung
cancer. The major advantage of the technique is that it allows the researchers to target the specific cells
in the site. That would help reduce the negative events when the technique penetrates to the neighboring
cells that may contribute to DNA breakage. Its drawback, however, is that the trials have not yet been
used in ongoing treatments. That will help provide more insightful results and inform the use of the
method in future lung cancer research.
A recent study from researchers at ChristianaCare's Gene Editing Institute in the US has demonstrated
how to recognise and assess the broad biological impact of gene editing on targeted tissues, where the
edits are intended to completely disable a particular sequence of genetic code. This study highlights the
safety and effectiveness of using CRISPR gene editing in patient treatments. The research, which was
published in Gene Therapy, suggests utilising CRISPR to deactivate or modify a master regulator gene to
stop it from creating a protein that lessens the effects of chemotherapy might enhance the treatment of
lung cancer.
Conclusion
In the past several years, CRISPR technologies have advanced dramatically, enabling accurate and
diverse genome modification. These adaptable technologies, which we now collectively refer to as
"universal tools," transformed the biological sciences and made fundamental research discoveries
possible for a wide range of applications. It is anticipated that CRISPR will be used in medical facilities to
provide a wide range of therapeutic options for treating human ailments, including cancer. These
technologies may be used for several therapeutic applications if work on improving and revolutionising
new methods of delivering genome engineering tools into cells and advancing their ability to edit
continues. Tumor research uses CRISPR/Cas systems extensively for a variety of in vitro and in vivo
applications. A number of clinical studies are now being conducted to speed up or improve the medicines'
dependability in order to more effectively cure cancer. To develop and use these technologies in clinics,
however, requires ongoing, intensive research. These technologies have the potential to provide several
options for precise and desirable genome editing and to flourish in the current age of medicine. The entire
potential of the CRISPR system to serve society in the near future will be ensured by ongoing work to
comprehend all of their drawbacks, enhance editing capabilities, and develop delivery methods.
Researchers have looked at the idea of using CRISPR to disable a gene called NRF2 to alter the
manufacturing of a protein that protects squamous cell carcinoma lung cancer tumours from the side
effects of chemotherapy or radiation. In tests with tumour cells and in animals, they have already shown
that they can selectively target the NRF2 gene without hurting normal cells, where the gene delivers
health benefits. The authors of the present study intended to enhance their findings by fully understanding
the implications of a CRISPR gene cut that enabled the NRF2 gene to retain enough DNA code to
continue manufacturing a version of the protein, although in an altered or truncated form. The CRISPRCas9 system was created by bacteria to defend them against different bacteriophages, but in recent
years, it has drawn substantial attention for its growing importance in the treatment of cancer and genetic
illnesses. CRISPR-Cas9 technology may be used to swiftly design oncolytic viruses and immune cells for
the treatment of cancer. It may be used for therapeutic analysis, which is more important, since it is
feasible to precisely change genes in model organisms and people. Furthermore, it is essential for the
development of thorough genetic libraries for cancer patients. Genetic illnesses are developing as a
consequence of several circumstances that result in gene mutation. The mutation impacts the signalling
pathways that result in various cancer types. DNA repair mechanisms are activated by double-strand
breaks (DSBs), which are crucial for repairing or replacing damaged DNA. The technique known as
CRISPR was developed to restore the damaged DNA by focusing on certain locations in various bodily
organs. It took the place of earlier gene editing technologies like TALENs and ZFNs. The process can
guarantee that the genes develop adaptive immunity and that they can defend against any foreign
substances. Among other genetic disorders, CRISPR Cas9 has been utilised to treat Sickle Cell Anemia,
Beta Thalassemia, LCA, hATTR, leukaemia, lymphoma, and lung cancer. For patients with Sickle Cell
Anemia and Beta Thalassemia, it detects the disease-causing mutation, and for those with LCA, it deletes
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exogenous DNA. The effectiveness of the gene-editing technique has been shown in clinical trials, and
additional research should be done. Although some of the experiments were conducted using animal
models, it is still unknown if they would be effective when applied to people. In addition, social and ethical
issues surrounding the use of CRISPR-Cas9 technology need to be discussed publicly. An ethical
controversy has been sparked by the birth in China of the first gene-engineered twins ever. It is debatable
whether human genomes can be modified in somatic cells or embryos to meet their needs. The birth of
children with CRISPR-Cas9 gene editing will result in further issues. For instance, undetected off-target
effects that have not yet been extensively researched will drastically alter human genomes over time and
may potentially have devastating impacts. Concerns about the newborns' social and mental development,
as well as their innate human qualities like intellect, personality, and attractiveness, are also included.
Worse even, the widespread and more serious societal problems that would result from the economic
usage of such technology. To reduce the aforementioned issues in the future, the development of
CRISPR-Cas9 technology must be supported with structured and strict supervision procedures.
Despite the previously mentioned ethical difficulties, translational study of CRISPR-Cas9 technology in
biomedicine is essential and should first focus on advanced malignancies and severe inherited illnesses.
The CRISPR-Cas9 system can be used as a flexible and convenient nucleic acid manipulation platform to
investigate the molecular mechanisms underlying disease genesis and progression and, more
importantly, to treat or reverse disease-causing gene mutations for clinical translational applications in
serious human diseases, particularly cancers. Preclinical research currently being conducted and
potential routes for translational applications principally focus on these three elements: (1) genetically
altering the tumour genome to reduce tumour cell growth and proliferation and increase necrosis and
apoptosis, or to increase the sensitivity of the tumour cells to radiotherapy and some chemotherapeutics;
(2) altering the genome of immune cells to release more toxic cytokines or to free immune cells from
functional inhibition; and (3) altering the genome of normal cells, such as bone marrow cells, to shield
them from the harmful effects of radiotherapy.
Any advancement in gene editing using CRISPR Cas9 would change biological research and aid in the
treatment of some of the world's most difficult genetic disorders, thus researchers should keep using this
technique.
Acknowledgement:
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