Case Study No.

1 – Editing DNA with CRISPR-CAS9

Executive Summary

In order to overcome issues with conventional gene editing, a team led by Waseem
Qasim of the University College London Great Ormond Street Institute of Child Health "used a
modified form of the CRISPR gene-editing protein that doesn't cut DNA, but instead changes
one DNA letter to another, a technique known as base editing." The article also stated that
Alyssa is the first person to ever be treated with base-edited CAR-T cells. Alyssa, a 13-year-old
child with severe leukemia, was treated with CRISPR base editing by Great Ormond Street
hospital, which made the announcement in the first few days of December of last year. She is in
remission and has no cancer cells that could be seen after the treatment.

Introduction

CRISPR (clustered regularly interspaced short palindromic repeats) refers to a naturally

occurring set of brief, repetitive nucleotide sequences that can be found in the genomes of many
bacteria. Researchers from across the Atlantic released the results of a clinical experiment in
November, demonstrating how CRISPR gene editing may be used to change immune cells so
they can recognize altered proteins unique to a person's tumors. The outcomes of 16 patients
were discussed at a recent meeting of the Society for Immunotherapy of Cancer in Boston and
were later published in Nature. In the meantime, scientists from the Medical Faculty of the
University of Bonn and the Institute of Structural Biology at the University Hospital Bonn
recently published findings on CRISPR-activated protein scissors in Nature.

Literature Review

For many years, the promise of CRISPR has been known. Professors Jennifer A. Doudna
and Emmanuelle Charpentier, two of its founders, received the Chemistry Nobel Prize in 2020.
The citation made reference to their discovery that CRISPR, a built-in anti-viral defense system
found in bacteria, could be instructed to cut any DNA molecule at a specific location. (Churi,
2023) There are a lot of patients who undergo CRISPR Treatment including the case of Alyssa, a
teenager in the UK who was identified as having a leukemia kind that attacks T cells, a category
of white blood cells. Both chemotherapy and a bone marrow transplant failed to be effective.
Consequently, medical professionals at London's Great Ormond Street Hospital tried a CRISPR-
based strategy. It involved using CRISPR to change donor-provided healthy T cells. The treated
cells were changed to prevent rejection by Alyssa's immune system and to enable them to find
and destroy Alyssa's own malignant T cells. Alyssa received these cells as a treatment after that.
It appears to have succeeded.

1. Advantages and Disadvantages of CRISPR

Genome editing is a remarkably potent technique that can assist you in answering a wide
range of scientific problems. Many CRISPR applications are being explored in the field of
disease, and CRISPR screening is already being used to find prospective therapeutic targets.
Unfortunately, it may not always be the most effective instrument for the job.

1.1 Pros

1.1.1 Modifying Your Target Area Is Easy

Well, so the first time you set up the CRISPR-Cas9 genome editing system, it's not easy.
Before you see any progress, your protocol needs to be optimized. The good news is that it is
quite easy to "cut" and tweak your setup after your technique is up and running in order to focus
on different genomic areas for editing. To introduce your new guide RNAs into your functioning
system, all you have to do is design and order them.

1.1.2 There are Lots of Publications using CRISPR

For a good reason, CRISPR-Cas9 has quickly established itself as a reliable genome-editing
technique. It functions. It's a quick and easy approach to find out how your gene or other
genomic region works. You can find out whether or not someone else has been successful in
genome editing your target cells by performing a quick PubMed search. This information will
both reassure you that it is doable and provide you with an experimental plan to follow.

1.1.3 It's affordable

A gene or area can be deleted, silenced, or altered in other ways using CRISPR-Cas9 editing.
If you're fortunate, you might be able to borrow Cas9 and guide RNA expression vectors from a
labmate or collaborator; all you'll need to buy then are the primers needed to make the guide
RNA vectors. You may complete the remaining parts of the preparation process in the lab, and
the only additional supplies you'll require are those you can find in any genetics lab with cell
culture capabilities: cloning tools, cells, media, and transfection supplies.

1.2 Cons

1.2.1 It Takes a Long Time to Set Up From Start

Not every facility has a functioning genome-editing pipeline. If you work in a lab without
such a pipeline but have determined that CRISPR-Cas9 genome editing is the best method to
advance your study, it's likely that your principal investigator (PI) will assign you the duty of
developing and perfecting the protocol.

1.2.2 It's not consistently effective

Your efforts in any genome-editing experiment may be significantly hampered by poor

editing efficiency, which is affected by a variety of variables. The percentage of cells in your
culture vessel that have been successfully edited is essentially what editing efficiency refers to.
Although an editing efficiency of less than 100% is by no means disastrous, it does indicate that
you should be cautious when interpreting your results. The unprocessed cells in your population
may hide any modest effects of your manipulation.

1.2.3 Off-Target Effects

The CRISPR-Cas9 technique is highly precise in theory, but it is not in actual use. It can
cause "off-target" changes, which are mutations in the genome that are not intended. Off-target
effects can negatively affect other genes or parts of the genome at random. This needs to be
taken into account while discussing your findings. By utilizing the modified Cas9 nickase, which
produces a single-stranded break rather than a double-stranded break, off-target effects can be
minimized. You can never be certain that you won't have some off-target impacts, though. The
results could perhaps be disastrous.
 2. Procedure of CRISPR

Alyssa was admitted to the Great Ormond Street Hospital's (GOSH) Bone Marrow
Transplant (BMT) Unit in May to receive genetically altered chimeric antigen receptor (CAR) T-
cells. The healthy donor provided the CAR T cells at first. New base-editing technology had
been used to modify these cells so they could search down and kill the malignant T-cells without
harming one another. She went on to have a second bone marrow transplant to rebuild her
immune system after going into remission just 28 days later. She is doing well at home
recovering with her family six months after her BMT, and she is continuing her post-BMT
follow-up at GOSH. Alyssa's sole alternative without this experimental therapy was palliative
care.

Conclusions

After addressing several difficulties, CRISPR/Cas9 has the potential to develop into a
dependable and simple genome editing technology. CRISPR/Cas9 opens the door for revealing
gene function in biology and fixing gene abnormalities in disorders thanks to its versatility and
simplicity. More research is required to better understand CRISPR characteristics, Cas9's
including its specificity, off-target effects, and delivery strategies.
 Case Study No. 2 – Editing DNA with CRISPR-CAS9

Executive Summary

Researchers can precisely modify any target DNA locus using the CRISPR system, which was
not possible with earlier gene editing methods. Opportunities for addressing illnesses that have
long eluded medical research are created by the potential to edit a disease mutation to fix genetic
mistakes. Efficiently treating esophageal cancer with this CRISPR-Cas9 PD-1 knockdown
treatment is great. A similar PD-1 knockdown treatment strategy was examined in a different
clinical trial to treat esophageal cancer.

Introduction

The gene-editing technique CRISPR-Cas9 (clustered regularly interspaced short palindromic

repeats) is positioned to transform medicine. For a variety of diseases, including inherited eye
problems, neurological conditions like Alzheimer's and Huntington's disorders, and non-inherited
diseases including cancer and HIV, researchers are developing CRISPR-Cas9 therapeutics. In
fact, many of these diseases already have CRISPR human studies running. Both esophageal
cancer and several types of lung cancer are difficult to treat. Poor disease outcomes are a result
of the fact that esophageal cancer is frequently detected late in its stage and that many lung
tumors are highly resistant to chemotherapy. According to projections, 235,760 new instances of
lung and bronchial cancer, as well as 19,260 new cases of esophageal cancer, will be identified
in 2021.

Literature Review

An investigation into CRISPR-Cas9 gene therapy in patients with advanced non-small-

cell lung cancer that just came to an end has demonstrated the feasibility and safety of using
CRISPR-edited T cells in clinical settings. The procedure entailed withdrawing blood from
patients with lung cancer, eliminating PD-1 from the immune cells in these blood samples, and
then reinfusing the immune cells back into the bloodstream.
 The research, which was published in Gene Therapy, suggests utilizing CRISPR to
disable or modify a master regulator gene to stop it from creating a protein that lessens the
effects of chemotherapy could enhance the treatment of lung cancer. The possibility of utilizing
CRISPR to deactivate a gene called NRF2 to change the synthesis of the protein that shields
squamous cell carcinoma lung cancer tumors from the effects of chemotherapy or radiation has
been studied by the researchers. Scientists have already demonstrated that they can selectively
target the NRF2 gene without harming normal cells, where the gene confers health advantages,
in trials with tumor cells and in animals.

By fully comprehending the effects of a CRISPR gene cut that permitted the NRF2 gene
to retain enough Genetic code to continue producing a version of the protein, albeit in an altered
or truncated form, the authors of the current study hoped to advance their findings. (Kakkad,
2022)

1. Advantages and Disadvantages of CRISPR in Lung Cancer Treatment

CRISPR has emerged as the preferred method for researchers looking at cancer because
of all of its advantages over other gene-editing techniques. There is also optimism that it will be
useful in the treatment of cancer. But CRISPR has flaws, and because of these drawbacks, many
scientists are hesitant to use it on humans. Off-target editing, or when CRISPR makes DNA cuts
outside of the target gene, is a serious drawback. As happened in a 2002 study of gene therapy,
scientists are concerned that such inadvertent changes could be detrimental and possibly cause
cells to become malignant. The possibility of cancer development was raised by Dr. Chavez. "If
[CRISPR] starts breaking random bits of the genome, the cell can start stitching things together
in extremely odd ways," he said. Yet, he continued, scientists have improved CRISPR's capacity
to exclusively cut the intended target by modifying the architectures of Cas and the guide RNA.

The challenge of getting CRISPR components into cells is another possible barrier.
Coopting a virus to carry out the task is the most popular method for doing this. The virus has
been altered to contain guide RNA and Cas genes rather than genes that cause disease. It's one
thing to introduce CRISPR into lab-grown cells, but quite another to do so in human bodily cells.
Certain CRISPR-carrying viruses can infect several cell types, so they might, for example, end
up modifying muscle cells while the intended target was liver cells.
 Researchers are looking into various strategies to precisely administer CRISPR to
particular cells or organs in the human body. Viruses that solely affect one organ, such as the
liver or brain, are being tested by some. Others have produced minuscule objects known as
nanocapsules that are intended to deliver CRISPR elements to particular cells.

There are also worries about how the body—in particular, the immune system—will
respond to CRISPR-carrying viruses or to the CRISPR components themselves because CRISPR
is only recently been tested in humans. Some people are concerned that CRISPR-edited cells
might be destroyed by the immune system if it attacks Cas, a bacterial enzyme that is foreign to
human bodies. A patient passed away twenty years ago as a result of his immune system's
enormous onslaught on the viruses carrying the gene therapy he had gotten. Newer CRISPR-
based strategies, on the other hand, rely on viruses that seem to be safer than those utilized in
earlier gene treatments.

Conclusions

After addressing several difficulties, CRISPR/Cas9 has the potential to develop into a
dependable and simple genome editing technology. CRISPR/Cas9 opens the door for revealing
gene function in biology and fixing gene abnormalities in disorders thanks to its versatility and
simplicity. More research is required to better understand CRISPR characteristics, Cas9's
including its specificity, off-target effects, and delivery strategies.
 Case Study No. 3 – Editing DNA with CRISPR-CAS9

Executive Summary

Beta-thalassemia symptoms are only momentarily relieved by blood transfusions, which

are frequently out of reach for many patients. In order to fix the genetic mutation that causes the
disease, CRISPR-Cas9 gene editing therapies are being pursued. Increasing the amounts of fetal
hemoglobin in blood is an alternate kind of treatment that is also appropriate for sickle cell
disease treatment in order to avoid disease symptoms.

Patient data demonstrated that CTX001 therapy, a treatment that employs this alternative
strategy, has successfully cured seven patients with beta thalassemia disease at the American
Society of Hematology annual meeting in 2020. These patients no longer experienced pain flare-
ups, had elevated fetal hemoglobin levels, and most significantly, no longer required blood
transfusions.

Introduction

Sickle cell disease (SCD) and transfusion-dependent beta-thalassemia (TDT) are severe
monogenic disorders with severe and possibly fatal symptoms. The most prevalent monogenic
disorders in the world with 60,000 and 300,000 patients, respectively, receiving a diagnosis each
year. The hemoglobin subunit gene is mutated in both disorders (HBB). The CRISPR(clustered
regularly interspaced short palindromic repeats) -based medicine has now been administered to
22 study participants who have been monitored for a minimum of three months. All 22 of them
responded to treatment and are now free of the pain crises and blood transfusions that are,
respectively, unique to beta thalassemia and sickle cell patients.

Literature Review

The fourth time that CRISPR and Vertex have presented findings from their beta
thalassemia and sickle cell investigations is at EHA. Their assertion that treatment with CTX001
could significantly alter the course of both diseases has appeared stronger with each cut of
clinical trial data. (Pagliarulo, 2021)

The most recent findings involved 15 patients with beta thalassemia, a condition that
causes anemia and other serious health issues. Vertex and CRISPR have gathered up to 26
months' worth of follow-up information on those patients thus far. Testing from 10 of them
revealed long-lasting effects of gene editing in the bone marrow, and none of them had needed
the blood transfusions they ordinarily needed before therapy.

The seven sickle cell disease patients whose data were included in Friday's study reported
comparable positive results. All seven have not gone through the painful and risky vaso-
occlusive crises typical of sickle cell patients since receiving treatment with CTX001. Up to this
point, the seven individuals had been monitored for a total of five to 22 months. Data from four
of those patients were edited, and this demonstrated a persistent effect.

The two companies with the most experience testing a CRISPR-based therapy on humans
are CRISPR and Vertex. Each patient's stem cells are modified as part of their therapy using
CRISPR gene editing. A version of the oxygen-carrying protein hemoglobin that is present at
birth but diminishes with age is produced by the cells through engineering. Both sickle cell and
beta thalassemia, which are both brought on by defects in the genes that code for hemoglobin,
may be cured by reintroducing these cells into the body, where they establish in the bone
marrow. Similar strategies are being used by several other businesses when developing their
gene editing tools.

1. Advantages and Disadvantages of CRISPR

Genome editing is a remarkably potent technique that can assist you in answering a wide
range of scientific problems. Many CRISPR applications are being explored in the field of
disease, and CRISPR screening is already being used to find prospective therapeutic targets.
Unfortunately, it may not always be the most effective instrument for the job.

1.1 Pros

1.1.1 Modifying Your Target Area Is Easy

 Well, so the first time you set up the CRISPR-Cas9 genome editing system, it's not easy.
Before you see any progress, your protocol needs to be optimized. The good news is that it is
quite easy to "cut" and tweak your setup after your technique is up and running in order to focus
on different genomic areas for editing. To introduce your new guide RNAs into your functioning
system, all you have to do is design and order them.

1.1.2 There are Lots of Publications using CRISPR

For a good reason, CRISPR-Cas9 has quickly established itself as a reliable genome-
editing technique. It functions. It's a quick and easy approach to find out how your gene or other
genomic region works. You can find out whether or not someone else has been successful in
genome editing your target cells by performing a quick PubMed search. This information will
both reassure you that it is doable and provide you with an experimental plan to follow.

1.1.3 It's affordable

A gene or area can be deleted, silenced, or altered in other ways using CRISPR-Cas9 editing. If
you're fortunate, you might be able to borrow Cas9 and guide RNA expression vectors from a
labmate or collaborator; all you'll need to buy then are the primers needed to make the guide
RNA vectors. You may complete the remaining parts of the preparation process in the lab, and
the only additional supplies you'll require are those you can find in any genetics lab with cell
culture capabilities: cloning tools, cells, media, and transfection supplies.

1.2 Cons

1.2.1 It Takes a Long Time to Set Up From Start

Not every facility has a functioning genome-editing pipeline. If you work in a lab without
such a pipeline but have determined that CRISPR-Cas9 genome editing is the best method to
advance your study, it's likely that your principal investigator (PI) will assign you the duty of
developing and perfecting the protocol.

1.2.2 It's not consistently effective

Your efforts in any genome-editing experiment may be significantly hampered by poor

editing efficiency, which is affected by a variety of variables. The percentage of cells in your
culture vessel that have been successfully edited is essentially what editing efficiency refers to.
Although an editing efficiency of less than 100% is by no means disastrous, it does indicate that
you should be cautious when interpreting your results. The unprocessed cells in your population
may hide any modest effects of your manipulation.

1.2.3 Off-Target Effects

The CRISPR-Cas9 technique is highly precise in theory, but it is not in actual use. It can
cause "off-target" changes, which are mutations in the genome that are not intended. Off-target
effects can negatively affect other genes or parts of the genome at random. This needs to be
taken into account while discussing your findings. By utilizing the modified Cas9 nickase, which
produces a single-stranded break rather than a double-stranded break, off-target effects can be
minimized. You can never be certain that you won't have some off-target impacts, though. The
results could perhaps be disastrous.

2. Procedure of CRISPR

Treatment choices for inherited disorders are being revolutionized by CRISPR

technology. With this technology, it is feasible to target a particular genomic region and change
that location more effectively than with other gene editing techniques.

There are two main CRISPR-based approaches being studied to cure beta thalassemia.
The first option is to fix the mutant version of the ß-globin gene. Given the wide range of
mutations that might result in beta thalassemia, this strategy is difficult and is restricted to the
repair of known HBB variants.

Hemoglobin F, a type of the protein that is typically inhibited after infancy, is used to
replace the damaged or depleted form of hemoglobin in beta thalassemia, which is the second
CRISPR-based method of treatment. The HBB gene does not encode hemoglobin F, which is
unaffected by the disease-causing mutation.

Conclusions

A new age in beta-thalassemia treatment has begun after years of research and will soon
provide many alternative alternatives as well as the potential for cure. Currently used gene
editing cannot be regarded as safer than virally-mediated gene addition since the long-term
effects of gene editing processes in HSCs are not yet known. The most promising approach
among innovative medicines remains the traditional gene addition perspective, and gene therapy
for beta-thalassemia may soon be made accessible to a sizable patient population following the
release of long-term data from Phase III trials.