CRISPR cas9: A versatile genome editing tool
Assignment no.: 03
Course Name: Advanced Biochemistry
Course Code: BBT609
Date: 3rd August, 2025
CRISPR-Cas9, a revolutionary gene-editing tool, was inspired by a natural immune system in bacteria.
It was co-developed by Jennifer Doudna and Emmanuelle Charpentier, who were awarded the Nobel
Prize in Chemistry in 2020—becoming the first all-female team to jointly receive a science Nobel.
Submitted to:
Prof. M Anwar Hossain
Department of Biochemistry and Microbiology, North South University.
Group no.: 02
Jeremiah Kevin Mondal – Introduction (1.0)
Shafaq Bintee Hasan – Mechanism of Action (2.0)
Mohammad Ushan Kabir Rythom– Applications of CRISPR-Cas9 (3.0) and Future
Perspectives and Conclusion (5.0)
Parthasarathi Roy – Ethical Considerations and Limitations (4.0)
1.0 Introduction
Over the past decade, advances in genetic engineering have revolutionized the biological sciences, and
among the most transformative tools is CRISPR-Cas9. CRISPR, short for Clustered Regularly
Interspaced Short Palindromic Repeats, is a naturally occurring system discovered in bacteria, where it
serves as an adaptive immune mechanism against invading viruses.
CRISPR-Cas9, first observed in 1987 as repeating DNA sequences in bacteria, was later identified (2005–
2007) as part of a microbial immune system that uses RNA-guided enzymes like Cas9 to cut foreign
DNA. Jennifer Doudna and Emmanuelle Charpentier created a synthetic CRISPR-Cas9 system in 2012
that makes genome editing more effective and accessible by enabling precise targeting and DNA
modification in various organisms. It was modified for accurate genome editing in human cells by 2013.
With uses in research, medicine, and agriculture, it has since transformed biotechnology and won its
creators the 2020 Nobel Prize in Chemistry.
The Cas9 protein, which functions as molecular scissors, and a guide RNA (gRNA), which points Cas9
toward a particular DNA sequence, are the two main parts of the CRISPR-Cas9 tool. Then the gRNA-
Cas9 complex attaches to the target DNA site, and Cas9 causes a double-strand break. The cell then uses
its natural repair processes to fix the break, either by homology-directed repair (HDR), which permits the
insertion of desired DNA sequences, or non-homologous end joining (NHEJ), which may result in
mutations.
The ease of use, affordability, and accuracy of CRISPR-Cas9 have revolutionized various disciplines,
from medicine and evolutionary biology to agriculture and environmental science. These days, it is
employed to create crops resistant to disease, create model organisms, study how genes work, and even
look into the potential of treating genetic disorders in humans.
2.0 Mechanisms of Action
The CRISPR-Cas9 system functions by mimicking a bacterial defense mechanism. In bacteria, the
CRISPR array stores fragments of viral DNA, called spacers, acquired during past infections. When the
same virus attacks again, the bacteria produce CRISPR RNAs (crRNAs) that match the viral DNA. These
crRNAs, in conjunction with a trans-activating crRNA (tracrRNA), form a complex that guides the Cas9
protein to the target sequence, where Cas9 introduces a double-strand break.
Scientists use a single guide RNA (sgRNA) in the laboratory, which fuses crRNA and tracrRNA into one
molecule. The sgRNA is engineered to match the target DNA sequence. The Cas9-sgRNA complex scans
the DNA until it finds a region that matches the guide RNA and is adjacent to a PAM sequence
(Protospacer Adjacent Motif), typically “NGG” for the commonly used Streptococcus pyogenes Cas9.
Once the double-strand break is created, the cell initiates repair. Researchers who want to disrupt a gene
rely on non-homologous end joining, which can cause small insertions or deletions (indels) that inactivate
the gene. If a specific change is desired, a donor template can be introduced, and the cell may use
homology-directed repair to insert the new sequence precisely. This mechanism allows unprecedented
control over genetic modification and has been adapted for gene knockouts, knock-ins, point mutations,
epigenetic editing, and more.
Figure 1: Mechanism of CRISPR-Cas9 Genome Editing
3.0 Applications of CRISPR-Cas9
Medicine and Human Health: Gene therapy is one of the most exciting uses of CRISPR-Cas9. By
fixing DNA mutations, it provides potential treatments for hereditary illnesses like sickle cell anaemia,
Huntington's disease, Duchenne muscular dystrophy, and cystic fibrosis. The first clinical trial that used
CRISPR to treat sickle cell disease and beta-thalassemia in 2020 showed encouraging results.
CRISPR is also utilized in cancer research because it enables researchers to precisely alter genes linked to
cancer, which helps with drug development and tumor biology comprehension. Developing CRISPR-
based diagnostics (like SHERLOCK and DETECTR systems) and investigating viral genome editing
(like HIV excision from host cells) are two examples of how CRISPR may be used to manage infectious
diseases.
Agriculture: CRISPR-Cas9 enables the creation of genetically modified crops without the introduction
of foreign DNA, addressing public concerns regarding genetically modified organisms. Researchers have
employed CRISPR to enhance the nutritional content, drought tolerance, and disease resistance of crops
such as rice, maize, and wheat. It can also be used in livestock to improve productivity, disease resistance,
and animal welfare.
Fundamental Research: Functional genomics greatly benefits from CRISPR. Researchers can
investigate the function of genes in development, physiology, and disease by knocking them out in cell
lines or animal models. Genes linked to drug resistance, cell division, or immune evasion can be found
through genome-wide screening made possible by high-throughput CRISPR libraries.
4.0 Ethical Considerations and Limitations
Although it could improve the world, CRISPR-Cas9 has several ethical and technical issues attached to it.
One of the most argued points is about germline editing, which is the modification of an embryo that can
be inherited. The He Jiankui case, which created the first gene-edited babies in 2018, was met with
furious backlash and showcased that more laws and policies are needed worldwide.
The lack of precision in CRISPR-Cas9 cutting still stands as an unsolved puzzle. There are still answers
to be found for unintended mutations. Advancements in the design of guide RNAs and the high-fidelity
Cas9 version are lessening the chances of unintentional mutation; still, the quest for precision will be a
constant.
There is also the problem of how CRISPR works, because PCR can be done with a viral or lipid vector,
which can cause the immune system to be unresponsive. Additionally, the immune system recognizing
Cas9 could be less effective in therapies targeted at humans.
There is still the case of inequality that needs to be solved. The therapies developed with CRISPR could
be limited to wealthier or more developed nations, creating an even greater divide in world healthcare.
Regardless of their philosophies, global annul still makes the same decision and draws international
boundaries towards gene editing technologies, calling for honesty, visibility, inclusiveness, and the hope
for the collective safety of humanity.
5.0 Future Perspectives and Conclusion
The CRISPR-Cas9 system is one of the most modern revolutionary biotechnological achievements of the
21st century and is developing startlingly rapidly. It is spurred on by new developments of revision
debugging, like 12 and 13, and prime editing, which allow for greater manoeuvrability in editing the RNA
strands and substituting specific parts without needing a double-strand break. The creation of activation
and inhibition CRISPR or CRISPRa/i systems allows for modification of the expression of a specific gene
without the need for transcription of the gene into a DNA sequence. This development of CRISPR opens
doors for the dissection of gene interactions in epigenetics and gene networks.
Modern technological advancements also aid in the development of multiplexed editing, which allows for
the alteration of gene sequences and the expression of several genes at once, which is beneficial in
treating chronic diseases like cancer or polygenic diseases. Other developments in enhanced biosafety
measures, like self-limiting Cas9 or controlled gene editing from within systems, also improve regulation
of gene editing events.
All in all, CRISPR-Cas9 is one of the most powerful bioweapons that can be used for the welfare of the
modern world. The precise nature and efficiency changes in modern medicine, agricultural development,
and scientific studies are even more rapid. Looking ahead, it is important to carefully evaluate the ethical
impact, improve the technology's safety, and ensure equitable distribution for all to realize the CRISPR-
Cas9 system's full potential.
6.0 References
1. Barrangou, R., & Doudna, J. A. (2016). Applications of CRISPR technologies in research and
beyond. Nature Biotechnology, 34(9), 933–941.
2. Cox, D. B. T., Platt, R. J., & Zhang, F. (2015). Therapeutic genome editing: prospects and
challenges. Nature Medicine, 21(2), 121–131.
3. Doudna, J. A., & Charpentier, E. (2014). The new frontier of genome engineering with CRISPR-
Cas9. Science, 346(6213), 1258096.
Hsu, P. D., Lander, E. S., & Zhang, F. (2014). Development and applications of CRISPR-Cas9
for genome engineering. Cell, 157(6), 1262–1278.
4. Jiang, Fuguo & Zhou, Kaihong & Ma, Linlin & Gressel, Saskia & Doudna, Jennifer. (2015). A
Cas9-guide RNA complex preorganized for target DNA recognition. Science (New York, N.Y.).
348. 1477-81. 10.1126/science.aab1452.
5. Ledford, H. (2020). CRISPR treatment inserted directly into the body for first time. Nature News.
