Department of Biochemistry and Microbiology (BMD)
North South University
ASSIGNMENT
‘CRISPR-Cas9’ - A Versatile Genome Editing Tool
Course Title: Advanced Biochemistry and Molecular Biology
Course Code: BBT609
Assignment No.: 3
Submitted To: Professor Dr. M Anwar Hossain, PhD. (AWN)
Submitted By: Group 1
Submission Date: Sunday, 3rd August 2025
Name ID Contribution
Masfura Rahman Muna 2415056670 Introduction, History
Arifa Jahan Shupty 2325509670 Mechanism of Action, Application of CRISPR-Cas9
2415523670 Future of Genetic Engineering and Modern Biotechnology,
Doha Mohammad Ishaque
Current Research and Advancements in Genome Editing
Mansura Islam Supti 2415508070 Future Prospects and Conclusion
1
Introduction
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a natural built-in
defense system of bacteria, which, along with the Cas9 enzyme, can function as a powerful
“Molecular Scissor”.With the advancement in the research field of Genetic Engineering and
Biotechnology, the manipulation of a model organism as a vector to edit or repair genes is
undeniable. To meet this growing advancement in Biological research, CRISPR-Cas9 has
proven to be the most powerful gene editing tool as it can carry genes in any form, such as
DNA, mRNA, or protein. Moreover, DNA form can harbor all the necessary elements for
editing, such as a selectable marker, a single-guide RNA expression cassette (sgRNA), a
donor DNA sequence, and the Cas9 gene, which makes it more efficient as an editing tool. 1
This powerful tool has been widely used since 2013 to modify genes-whether insertion or
knocking out, or over-expression of a particular gene. It is more advantageous to manipulate
genes in comparison to the old gene-editing strategies, such as zinc finger nucleases and
effector nucleases (transcription activators). This tool can also repair double-stranded breaks
of DNA either by non-homologous end joining (NHEJ) or by homology-directed repair
(HDR).
History
1987 – Discovery:
Japanese scientist Yoshizumi Ishino identified palindromic DNA sequences (spacer-derived
from bacteriophage ) in Escherichia coli. At that time, the function of these DNA sequences
was unknown, and it was later named ‘CRISPR’.
2005 – Role of CRISPR Identified:
Spanish scientist Francisco Mojica found that CRISPR sequences, which are known as
spacers, are derived from viral DNA. He proposed that CRISPR is a part bacterial immune
system, which helps bacteria to fight against viruses. These sequences are inherited from
bacteriophages.
2007 – Confirmation of Immune Role:
Philippe Horvath and his team at a yogurt company proved CRISPR can protect bacteria from
viruses by recruiting ‘Cas’, a type of protein, to fight against viral pathogenesis by destroying
viral DNA.
2012 – CRISPR as Gene Editing Tool:
Jennifer Doudna (USA) and Emmanuelle Charpentier (France) turned CRISPR-Cas9 into a
programmable tool guided by RNA, which can cut DNA at precise sites. This milestone of
achievement made the gene editing pathway precise and easy.
2
2013 – Used in Human Cells:
Feng Zhang and George Church successfully used CRISPR-Cas9 in human cells. This opened
great opportunities for Medical Science.
2020 – Nobel Prize:
Doudna and Charpentier were awarded the Nobel Prize in Chemistry for developing
CRISPR-Cas9 as a gene-editing tool. 2
Mechanism of Action
Figure: Mechanism of Action of CRISPR-Cas9 Genome Editing.
1. Recognition and Binding:
The gRNA binds to Cas9, forming the CRISPR-Cas9 complex. The gRNA scans the
DNA for a complementary sequence adjacent to a Protospacer Adjacent Motif (PAM),
usually NGG in Streptococcus pyogenes Cas9. Once the target DNA is found, the gRNA
binds via Watson-Crick base pairing.
2. DNA Cleavage:
Cas9 undergoes a conformational change, activating its nuclease domains (HNH and
RuvC). The HNH domain cuts the target (complementary) DNA strand. RuvC domain
3
cuts the non-target DNA strand. This results in a double-strand break (DSB) at the target
site.
3. DNA Repair and Genome Editing:
The cell repairs the DSB via one of two pathways:
- Non-Homologous End Joining (NHEJ) – Error-prone repair causing
insertions/deletions (indels), leading to gene knockout.
- Homology-Directed Repair (HDR) – Precise repair using a donor DNA template for
gene insertion or correction. 3
Application of CRISPR-Cas9: A Versatile Genome Editing Tool
CRISPR-Cas9 has revolutionized molecular biology and biotechnology due to its precision,
efficiency, and versatility. Its main applications include:
1. Gene Therapy:
CRISPR-Cas9 is used to correct genetic mutations responsible for inherited diseases such
as Sickle Cell Anaemia, Cystic Fibrosis, and Duchenne Muscular Dystrophy.
Repairing or replacing faulty genes offers potential cures for genetic disorders. CRISPR
is used to study gene functions by systematically knocking out genes in a genome and
understanding pathways involved in development or disease.
2. Agricultural Biotechnology:
CRISPR is applied to develop disease-resistant crops and improve yield and nutritional
quality, and drought- or pest-tolerant plant varieties. Example: CRISPR-edited rice and
tomatoes with enhanced shelf life and productivity.
3. Drug Discovery and Development:
CRISPR is used to create disease models (e.g., in mice or cell lines) and identify drug
targets by gene knock-out or knock-in studies to study gene function in health and
disease.
4. Cancer Research and Treatment:
CRISPR is used to engineer immune cells (like CAR-T cells) to target cancer, which
helps in identifying oncogenes and tumor suppressor genes.
Future of Genetic Engineering and Modern Biotechnology
The future of genetic engineering is being redefined by advanced CRISPR-based
technologies, including engineered Cas9 variants and novel tools such as bridge RNAs. These
RNA-guided recombinases, as demonstrated by Hsu’s lab, enable precise insertions,
deletions, and rearrangements of DNA without inducing double-stranded breaks, unlike the
conventional CRISPR-Cas9 system. This technique significantly reduces the risk of off-target
4
effects and enhances control over genomic modifications, making genome editing more
predictable and safer. Often referred to as functioning like a “word processor for the
genome,” this technology holds the potential for designing modular and scalable genomic
architectures, which could be instrumental in both therapeutic and synthetic biology
applications.
Furthermore, the integration of CRISPR with artificial intelligence (AI) and machine learning
is accelerating the development of more efficient and specific genome editing tools. AI-
driven platforms are now capable of predicting the behavior of guide RNAs and engineering
novel Cas enzymes with improved specificity, efficiency, and thermal stability traits essential
for both clinical use and industrial biotechnology. As these hybrid systems evolve, they are
expected to revolutionize several sectors: from enabling customized gene therapies for
individuals to developing crops tailored to withstand extreme climates, and even creating
synthetic microorganisms capable of producing sustainable fuels, bioplastics, or cleaning up
environmental pollutants. This synergy between biotechnology and computational tools
marks a transformative shift in how we interact with and engineer life. 4
Current Research and Advancements in Genome Editing
Significant strides have been made in the clinical application of CRISPR-Cas9 technology.
One of the most notable advancements is Casgevy (CTX-001), a therapy developed for
patients with sickle cell disease and β-thalassemia. In clinical trials, patients treated with
Casgevy have shown durable increases in fetal hemoglobin levels and, in many cases, have
become transfusion-independent.
Similarly, EDIT-101, a pioneering in vivo CRISPR-based therapy aimed at treating Leber
congenital amaurosis (a genetic form of blindness), has shown encouraging results in safety
and efficacy. Unlike traditional ex vivo gene editing, EDIT-101 is delivered directly to the
retina, providing a practical demonstration of CRISPR's potential for direct in-body
applications.3 In addition to therapeutic progress, ongoing research is pushing the boundaries
of precision and versatility in genome editing. Modified versions of Cas9 with higher fidelity,
as well as tools like base editors and prime editors, are enabling single-nucleotide changes
without cutting the DNA strand, greatly reducing the risk of unwanted mutations. In
agriculture, multiplex genome editing, where multiple genes are edited simultaneously, is
being used to develop crop varieties with enhanced resistance to drought, pests, and diseases,
while also improving nutritional value. These agricultural innovations are particularly vital in
addressing food security challenges in the face of climate change.
Overall, the current wave of CRISPR-related research is setting the stage for a future where
genetic engineering becomes safer, more efficient, and more deeply integrated into medicine,
food production, and sustainable development. 5
Future Prospects
5
Among the most promising new technologies is CRISPR-Cas9, which can be used on genes
to modify tissues, and has an enormous scope in many fields, especially in medicine and
agriculture.
Healthcare:
To correct genetic errors like sickle cell anemia, cystic fibrosis, and muscular dystrophy,
CRISPR-Cas9 is being studied, and related research is being done to try and deliver CRISPR-
Cas9 to the body to modify and treat diseases like cancer.
Agriculture:
With CRISPR-Cas9 technologies, agriculture can be improved as new crops can be
developed to be more resistant to pests and diseases, and also have higher yields and
improved nutritional value.
Vaccine Development:
Vaccine development for infectious diseases like COVID-19 utilized CRISPR-Cas9, and we
hope that in the future, it will be used for other diseases as well.
Conclusion
In conclusion, we can say that the excitement in the scientific community is due to the
CRISPR-Cas9 simplicity and accuracy, especially in genetic modification, as it is the most
flexible technique available today. It is a remarkable and innovative invention that gives
geneticists and medical researchers the ability to change, remove, and insert segments of an
organism's DNA. 6
References
1. Tannous, J. et al. Establishment of a genome editing tool using CRISPR-Cas9
ribonucleoprotein complexes in the non-model plant pathogen Sphaerulina musiva.
frontiersin.orgJ Tannous, C Sawyer, MM Hassan, JL Labbe, C EckertFrontiers in genome
editing, 2023•frontiersin.org 5, (2023).
2. (Moscow), I. G.-B. & 2022, undefined. CRISPR–cas9: A history of its discovery and ethical
considerations of its use in genome editing. SpringerI GostimskayaBiochemistry (Moscow),
2022•Springer 87, 777–788 (2022).
3. Doudna, J. A. & Charpentier, E. The new frontier of genome engineering with CRISPR-Cas9.
science.orgJA Doudna, E CharpentierScience, 2014•science.org 346, 6213 (2014).
4. Durrant, M. G. et al. Bridge RNAs direct programmable recombination of target and donor
DNA. nature.comMG Durrant, NT Perry, JJ Pai, AR Jangid, JS Athukoralage, M Hiraizumi,
JP McSpedonNature, 2024•nature.com 630, 984–993 (2024).
5. Frangoul, H. et al. CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia. Mass
Medical SocH Frangoul, D Altshuler, MD Cappellini, YS Chen, J Domm, BK Eustace, J
FoellNew England Journal of Medicine, 2021•Mass Medical Soc 384, 252–260 (2021).
6
6. Maeder, M. L. et al. Development of a gene-editing approach to restore vision loss in Leber
congenital amaurosis type 10. nature.comML Maeder, M Stefanidakis, CJ Wilson, R Baral,
LA Barrera, GS Bounoutas, D BumcrotNature medicine, 2019•nature.com 25, 229–233
(2019).
