BIOLOGY CBSE PROJECT

CRISPR
TECHNOLOGY

Revolutionizing
Cancer Treatment
and Research

Dhruva Abhale
XII-D
 TABLE OF
CONTENTS

Introduction 01

Primary Mechanism 02

Key Applications 03

CRISPR-Based 04
Cancer Therapy

Case Study 05

Challenges and Future 06

Directions

Conclusion 07
Bibliography 07
INTRODUCTION
CRISPR (Clustered Regularly Interspaced Short Palindromic
Repeats) technology is at the forefront of genetic engineering,
enabling scientists to make precise and targeted
modifications to DNA sequences. Originally discovered as a
defense mechanism in bacteria against viral infections,
CRISPR has rapidly evolved into a powerful tool for genetic
manipulation across a wide range of organisms. Its ability to
target specific DNA sequences with high precision has
revolutionized the field of genetic research, particularly
cancer.

Cancer, characterized by uncontrolled cell growth and the

ability to invade other tissues, is driven by genetic mutations
and alterations in the expression of oncogenes and tumor
suppressor genes. Traditional cancer treatments, such as
chemotherapy and radiation, often lack specificity, leading to
damage to healthy cells and severe side effects. In contrast,
CRISPR offers a more targeted approach, enabling precise
correction. By directly altering the genetic underpinnings of
cancer, CRISPR holds the promise of developing highly
effective and personalized cancer therapies.

Moreover, the integration of CRISPR technology in cancer

research is paving the way for a new era of precision
oncology. This approach aims to tailor treatment strategies to
the individual genetic profile of each patient's cancer,
maximizing therapeutic efficacy while minimizing adverse
effects. As researchers continue to explore and refine
CRISPR-based therapies, the potential for this technology to
transform cancer treatment and improve patient outcomes is
becoming increasingly apparent.
Since the inception of Cas9 gene editing in mammalian
cells, there has been a rapid expansion in the field of
CRISPR technology. This expansion has led to the
development of various specialized CRISPR variants
designed to tackle specific challenges.

Scientists have created these variants through deliberate

design and evolutionary processes, resulting in improved
flexibility and increased precision in target selection.

A significant advancement known as prime editing has

emerged, which involves fusing a dCas9 with a reverse
transcriptase, enabling the engineering of various mutation
types, such as missense mutations, insertions, and
deletions.

Additionally, to facilitate unbiased proteome mapping,

researchers have employed engineered ascorbate
peroxidase (APEX2) tethered to dCas9, enabling targeted
biotinylation at specific genomic locations.
 KEY
APPLICATIONS

Cancer Diagnosis and

Genetic Research

Cancer
Treatment
 CANCER
DIAGNOSIS AND
GENETIC RESEARCH
CRISPR technology is revolutionizing cancer diagnosis and
genetic research by enabling more precise and comprehensive
studies of the genetic alterations that drive cancer. One of the
key applications of CRISPR in this area is in precision
medicine, where the technology is used to identify and validate
oncogenes—genes that have the potential to cause cancer when
mutated or overexpressed. By targeting specific genes, CRISPR
allows researchers to study their function in the context of
cancer development, helping to identify new therapeutic targets
and develop more effective treatments.

Genetic screening is another critical application of CRISPR in

cancer research. High-throughput CRISPR screens involve the
systematic disruption of thousands of genes to identify those
that are essential for cancer cell survival. These screens can
uncover genetic vulnerabilities unique to cancer cells, providing
valuable insights into potential drug targets. For example,
CRISPR screens have been used to identify genes that, when
knocked out, make cancer cells more susceptible to existing
therapies, paving the way for combination treatments that
enhance therapeutic efficacy.

CRISPR is also being used to develop more accurate models of

cancer. Traditional animal models, such as genetically
engineered mice, often fail to fully recapitulate the complexity
of human cancers. CRISPR allows for the rapid generation of
more accurate models by enabling the precise editing of
multiple genes simultaneously. These models are invaluable for
studying the genetic interactions that drive cancer and for
testing new therapies in a preclinical setting.
 CANCER
TREATMENT
CRISPR technology is transforming cancer treatment by
enabling the development of more targeted and effective
therapies. One of the most significant applications of CRISPR
in cancer treatment is in the field of immunotherapy,
particularly in the enhancement of CAR-T cell therapy. CAR-T
cells are a type of immune cell that has been genetically
modified to express a chimeric antigen receptor (CAR), which
enables them to recognize and attack cancer cells. By using
CRISPR to edit the genes of T-cells, scientists can improve their
ability to target and destroy cancer cells, leading to more
effective treatments with fewer side effects.

Another promising application of CRISPR in cancer treatment

is in gene therapy. This approach involves the direct genetic
modification of cancer cells to correct mutations or introduce
therapeutic genes. For example, CRISPR can be used to repair
mutations in tumor suppressor genes, restoring their ability to
inhibit cancer growth. Alternatively, CRISPR can be used to
introduce genes that trigger cancer cell death or enhance the
effectiveness of other treatments, such as chemotherapy or
radiation.

In addition to these applications, CRISPR is also being

explored as a tool for personalized cancer therapy. By analyzing
the genetic profile of a patient's cancer, researchers can use
CRISPR to develop customized therapies that target the
specific genetic mutations driving the disease. This approach
has the potential to significantly improve treatment outcomes
by tailoring therapy to the unique characteristics of each
patient's cancer.
The various mechanisms employed for gene editing.

(a) Genome editing:

Zinc‐finger nucleases (ZFNs), transcription activator‐like
effector nucleases (TALENs), and CRISPR‐Cas systems
are capable of generating double‐strand breaks (DSBs) at
precise locations within the genome.

(b) Generations of indels and precise mutations:

the introduction of DNA sequences or mutations into the
DNA can be achieved by means of homology‐directed
repair (HDR) or non‐homologous end joining (NHEJ)
processes with the aid of a donor template.

In mammalian cells, CRISPR‐induced DSBs are generally

mended via NHEJ, which can result in the incorporation
of small insertions and/or deletions, leading to gene
inactivation due to frameshift mutations.

When two DSBs occur on the same chromosome, a

substantial segment can be deleted, whereas DSBs on
different chromosomes
can give rise to chromosomal rearrangements.
Different strategies for editing cells using
CRISPR technology in patients.

On the left, ex vivo applications involve first

isolating cells, then expanding and editing them
before transplanting them back. On the right, in
vivo editing occurs by administering CRISPR‐Cas9
locally or systemically using viral packaging or
nanoparticles. The figure also highlights specific
clinical trials.
 CRISPR-BASED
CANCER THERAPY
PRECLINICAL
STUDIES
Before CRISPR-based therapies can be applied
in the clinic, they must undergo rigorous
preclinical testing to evaluate their safety and
efficacy. Preclinical studies involve the use of cell
lines and animal models to test the therapeutic
potential of CRISPR in targeting cancer cells.
These studies are essential for understanding the
mechanisms by which CRISPR can be used to
disrupt oncogenes, repair tumor suppressor
genes, and introduce therapeutic genes. They
also help to identify potential off-target effects,
where CRISPR inadvertently edits genes other
than the intended target, which could lead to
unintended consequences.

One of the major challenges in preclinical studies

is the delivery of CRISPR components to cancer
cells. Effective delivery is crucial for the success
of CRISPR-based therapies, as the Cas9 enzyme
and guide RNA must be delivered to the correct
cells and tissues without being degraded or
triggering an immune response.
Cancer Models:

Employing CRISPR for creating cancer models in cells and

mice. In the initial case (a), cultured cells or organoids
undergo genome editing through CRISPR, which
facilitates the incorporation of alterations or adjustments
in transcriptional control at one or more phases. In the
latter case (b), CRISPR mechanisms can be transferred to
animal models using diverse methods, thereby enabling
them to target numerous tissues and organs. One such
approach involves utilizing adeno‐associated viruses
(AAV) for delivery. Reprinted from [15] with permission
from Springer Nature
 CRISPR-BASED
CANCER THERAPY
CLINICAL STUDIES
Following successful preclinical studies, CRISPR-based
therapies move into clinical trials, where they are tested in
human patients. Clinical trials for CRISPR therapies
typically progress through several phases, each designed to
answer specific questions about the safety, efficacy, and
optimal use of the therapy.

Phase 1 trials:
focuses on evaluating the safety of the therapy in a small
group of patients and assesses the appropriate dosage and
adverse effects associated with the treatment.
Phase 2 trials:
expand the patient population to determine the efficacy of
the therapy. These trials also continue to monitor safety and
begin to assess the therapy's impact on cancer progression.
Phase 3 trials:
involves a larger group of patients and compare the
CRISPR-based therapy to existing standard treatments. The
goal is to confirm the therapy's efficacy, monitor side effects,
and collect information that will allow the therapy to be used
safely in the general population.

Despite the promising potential of CRISPR-based cancer

therapies, only a few have progressed to clinical trials due to
the complexity of the technology and the need to address
safety concerns. These trials represent a critical step forward
in the development of CRISPR-based cancer therapies, and
their outcomes will provide valuable insights into the
feasibility and effectiveness of this approach.
Applications of CRISPR in immune-oncology:

Illustrates the application of CRISPR in immuno‐

oncology. In scenario (a), primary T cells extracted and
purified from cancer patients can have a chimeric antigen
receptor (CAR) inserted using CRISPR technology, instead
of lentiviral‐ mediated transduction. CRISPR can also be
employed to deactivate immune‐checkpoint genes, such as
PD‐1 and CTLA‐4, within these T cells.

Alternatively, scenario b involves the isolation and

purification of primary T cells from healthy donors not
diagnosed with cancer. CRISPR systems are used to
introduce a CAR into these cells, and they can also be
utilized to inactivate the genes responsible for T cell
receptor (TCR) and HLA components. This process
generates ’universal’ allogeneic CAR T cells, which can be
infused into cancer patients.
 CASE STUDY
CRISPR-Edited T Cells in Cancer Treatment
Background:
A 50-year-old man with advanced lung cancer tried several treatments
like chemotherapy and radiation, but none worked. His cancer spread
to his liver and bones, leaving him with few options. He joined a
clinical trial to try a new treatment using CRISPR technology.

CRISPR Treatment:
Doctors used CRISPR to edit patients' T-cells (immune cells) to help
them fight cancer better. They removed a gene called PD-1 from the T-
cells because this gene can help cancer cells hide from the immune
system. By knocking out PD-1, the T-cells became better at attacking
the cancer.

Procedure:
1. Collecting T-Cells: The patient’s T-cells were taken from his
blood.
2. Editing the Cells: CRISPR was used to remove the PD-1 gene from
the T-cells in the lab.
3. Reinfusion: The edited T-cells were multiplied and then put back
into the patient’s body.

Results:
Positive Response: Within two months, the patient’s tumors
shrank, and his symptoms improved.
Stable Condition: Six months later, the cancer had not grown
back, and the patient was feeling better.
Mild Side Effects: The patient had mild flu-like symptoms after
the treatment, but nothing serious.

Conclusion:
This case shows how CRISPR technology can be used to improve
cancer treatment by enhancing the immune system’s ability to fight
tumors. It’s a promising new approach that could help patients with
difficult-to-treat cancers.
 CHALLENGES AND
FUTURE DIRECTIONS
While CRISPR/Cas9 technology holds immense promise for
cancer treatment, several challenges must be addressed before it
can be widely adopted in clinical practice. One of the most
significant challenges is the potential for off-target effects, where
the CRISPR system inadvertently introduces mutations in
unintended regions of the genome. These off-target effects can
lead to harmful side effects. Researchers are developing more
precise CRISPR systems with improved specificity and reduced
off-target activity to minimize these risks.

Another major challenge is the effective delivery of CRISPR

components to target cells. Ensuring that the Cas9 enzyme and
guide RNA reach the correct cells and tissues without being
degraded or triggering an immune response is critical for the
success of CRISPR-based therapies. In addition to technical
challenges, ethical and regulatory considerations are associated
with using CRISPR technology in cancer treatment. The ability to
edit the human genome raises important ethical questions about
the potential for unintended consequences, the possibility of
germline editing, and the need for informed consent.

Looking to the future, the continued development of CRISPR

technology is likely to lead to new and improved cancer therapies.
Advances in CRISPR precision, delivery, and safety will be critical
in overcoming current challenges and expanding the potential
applications of this technology.

In conclusion, while there are still significant challenges to

overcome, CRISPR technology represents a paradigm shift in
cancer treatment. Its ability to precisely target and modify the
genetic drivers of cancer offers the potential for more effective,
personalized therapies that could revolutionize the way we treat
this complex disease. Continued research and collaboration across
disciplines will be essential to fully realize the potential of CRISPR
and bring these promising therapies to patients.
 CONCLUSION

CRISPR technology is poised to revolutionize the field of

cancer treatment by offering new and innovative approaches
to targeting the genetic underpinnings of the disease. From
its applications in precision medicine and genetic research to
its potential in developing more effective cancer therapies,
CRISPR is transforming the landscape of oncology. While
significant challenges remain, including the need for more
precise editing techniques, effective delivery systems, and
robust ethical frameworks, the future of CRISPR-based
cancer treatment is bright.

As we continue to explore the potential of CRISPR

technology, it is clear that this powerful tool will play an
increasingly important role in the fight against cancer. By
enabling the precise manipulation of the genome, CRISPR
offers the potential to develop highly targeted and
personalized therapies that can improve patient outcomes
and reduce the burden of this devastating disease. As
research progresses and new discoveries are made, CRISPR
technology will undoubtedly continue to push the
boundaries of what is possible in cancer treatment, offering
new hope to patients and paving the way for a new era of
precision oncology.
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