Hana Khattab
19100434
Farida Swaify
19100411
Abdelrahman
Beshr 19100125
Adham Khaled
19100612
GROUP 3 and 4
Introduction
Genome editing, also known as gene editing, is a set of technologies that enables scientists to
modify the DNA of an organism. These technologies allow for the addition, removal, or
alteration of genetic material at specific locations in the genome. One well-known approach
to genome editing is CRISPR-Cas9, which stands for clustered regularly interspaced short
palindromic repeats and CRISPR-associated protein 9. CRISPR-Cas9 has gained significant
attention in the scientific community due to its advantages over other genome editing
methods, including its speed, cost-effectiveness, accuracy, and efficiency.
Types
There are several types of CRISPR-Cas systems, each with its own unique features and
applications.
CRISPR-Cas9:
CRISPR-Cas9 is the most well-known and widely used system. It consists of the Cas9
nuclease enzyme guided by a single-guide RNA (sgRNA) to target and cut specific DNA
sequences. This system is primarily used for gene editing applications.
CRISPR-Cas12 (Cpf1):
CRISPR-Cas12, also known as Cpf1, is another RNA-guided genome editing tool. It has
distinct features from Cas9, such as different PAM (Protospacer Adjacent Motif) sequences
and cleavage patterns. Cas12 offers potential advantages in certain applications.
CRISPR-Cas13:
CRISPR-Cas13 is unique in that it targets RNA rather than DNA. Cas13 systems can be used
for RNA manipulation, such as gene regulation and RNA editing, providing a tool for
studying and modifying RNA molecules.
Base Editors (BE):
Base editors are a newer class of CRISPR tools that can enable precise point mutations by
directly converting one DNA base to another without causing double-strand breaks.
Examples include BE3 (base editor 3) and ABE (adenine base editor).
Prime Editors:
Prime editors are another recent advancement in CRISPR technology that offer enhanced
precision and versatility. They combine a catalytically impaired Cas nuclease with a reverse
transcriptase to precisely rewrite target DNA sequences.
Applications of CRISPR/CAS-9
In just a few years of its discovery, the CRISPR/Cas-9 genome editing tool has already being
explored for a wide number of applications and had a massive impact on the world in many
areas including medicine, agriculture, and biotechnology. In the future, researchers hope that
this technology will continue to advance for treating and curing diseases, develop more
nutritious crops, and eradicating infectious diseases.
Role in Gene Therapy
More than 6000 genetic disorders have been known so far. But the majority of the diseases
lack effective treatment strategies. Gene therapy is the process of replacing the defective gene
with exogenous DNA and editing the mutated gene at its native location. Since its discovery
in 2012, CRISPR/Cas-9 gene editing has held the promise of curing most of the known
genetic diseases such as sickle cell disease, β-thalassemia, cystic fibrosis, and muscular
dystrophy.CRISPR/Cas-9 for targeted sickle cell disease (SCD) therapy and β-thalassemia
have been also applied in clinical trials. SCD occurs due to point mutation in the β-globin
chain of hemoglobin leading to sickle hemoglobin (HbS). Either direct repairing the gene
of hemoglobin S or boosting fetal γ-globin are the two main approaches that
CRISPR/Cas-9 is being used to treat SCD. However, the most common method used in
a clinical trial is based on the approach of boosting fetal hemoglobin. First bone marrow
cells are removed from patients and the gene that turns off fetal hemoglobin production,
called B-cell Lymphoma 11A (BCL11A) is disabled with CRISPR/Cas-9. Then, the gene-
edited cells are infused back into the body. BCL11A is a 200 base pair gene found on
chromosome 2 and its product is responsible to switch γ-globin into the β-globin chain by
repressing γ-globin gene expression. Once this gene is disabled using CRISPR/Cas-9, the
production of fetal hemoglobin containing γ-globin in the red blood cells will increase,
thereby alleviating the severity and manifestations of SCD.
Scientists have been also investigating CRISPR/Cas-9 for the treatment of cystic
fibrosis. Furthermore, Duchenne muscular dystrophy (DMD), which is caused by a
mutation in the dystrophin gene and characterized by muscle weakness, has been
successfully corrected by CRISPR/Cas-9 in patient-induced pluripotent stem cells. .
Deletion/excision of intragenic DNA and removing the duplicated exon by CRISPR/Cas-
9 are the new and promising approaches in correcting the DMD gene, which restores
the expression of dystrophin protein.
Therapeutic Role of CRISPR/Cas-9
The first CRISPR-based therapy in the human trial was conducted to treat patients
with refractory lung cancer. Researchers first extract T-cells from three patient’s blood and
they engineered them in the lab through CRISPR/Cas-9 to delete genes (TRAC, TRBC,
and PD-1) that would interfere to fight cancer cells. Then, they infused the modified T-cells
back into the patients. The modified T-cells can target specific antigens and kill cancer
cells. Finally, no side effects were observed and engineered T-cells can be detected up to 9
months of post-infusion. CRISPR/Cas-9 gene-editing technology could also be used to treat
infectious diseases caused by microorganisms. One focus area for the researchers is
treating HIV, the virus that leads to AIDS. Replication can be completely shut down and
the virus eliminated from infected cells through excision of HIV-1 genome using
CRISPR/Cas-9 in animal models. In addition to the approach of targeting the HIV-genome,
CRISPR/Cas-9 technology can also be used to block HIV entry into host cells by editing
chemokine co-receptor type-5 (CCR5) genes in the host cells. For instance, an in vitro trial
conducted in China reported that genome editing of CCR5 by CRISPR/Cas-9 showed no
evidence of toxicity (infection) on cells and they concluded that edited cells could effectively
be protected from HIV infection than unmodified cells.
Role in Agriculture
As the world population continues to grow, the risk of shortage in agricultural resources is
real. Hence, there is a need for new technologies for increasing and improving natural food
production. CRISPR/Cas-9 is an existing addition to the field since it has been used to
genetically modify foods to improve their nutritional value, increase their shelf life, make
them drought-tolerant, and enhance disease resistance.There are generally three ways that
CRISPR is solving the world’s food crisis. It can restore food supplies, help plants to
survive in hostile conditions, and could improve the overall health of the plants.
Role in Gene Activation and Silencing
Beyond genome editing activity, CRISPR/Cas-9 can be used to artificially regulate
(activate or repress) a certain target of a gene through advanced modification of Cas-9
protein. Researchers had performed an advanced modified Cas-9 endonuclease called dCas-9
nuclease by inactivating its HNH and RuvC domains. The dCas-9 nuclease lacks DNA
cleavage activity, but its DNA binding activity is not affected. Then, transcriptional activators
or inhibitors can be fused with dCas-9 to form the CRISPR/dCas-9 complex. Therefore,
catalytically inactive dCas-9 can be used to activate (CRISPRa) or silence (CRISPRi) the
expression of a specific gene of interest. Moreover, the CRISPR/dCas-9 can be also used
to visualize and pinpoint where specifically the gene of interest is located inside the cell
(subcellular
localization) by fusing a
marker such as Green
Fluorescent Proteins
(GFP) with dCas-9
enzyme.
Challenges for CRISPR/Cas-9 Application
Despite its great promise as a genome-editing system CRISPR/Cas-9 technology had
hampered by several challenges that should be addressed during the process of application.
Immunogenicity, lack of a safe and efficient delivery system to the target, off-target
effect, and ethical issues have been the major barriers to extend the technology in
clinical applications. Since the components of the CRISPR/Cas-9 system are derived
from bacteria, host immunity can elicit an immune response against these components.
Researchers also found that there were both pre-existing humoral (anti-Cas-9 antibody) and
cellular (anti-Cas-9 T cells) immune responses to Cas-9 protein in healthy humans.
Therefore, how to detect and reduce the immunogenicity of Cas-9 protein is still one of the
most important challenges in the clinical trial of the system.
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