NORTH SOUTH UNIVERSITY

Department of Biochemistry and Microbiology

Program: MS in Biotechnology
Course Title
Advanced Biochemistry (BBT609.1)
Assignment on
CRISPR-Cas9: A versatile genome editing tool
Submitted to
Professor Dr. M. Anwar Hossain, Ph.D
Submitted by
Group 3
Individual name with coverage portion of assignment ID
Wridny Haque (Cover Page) 2517407670
Kashfia Nehrin Zaman (Introduction) 2517149670
Sharmin Akter Sumi (Body) 2517374070
Md. Riad Uddin (Conclusion) 2517065670

1
CRISPR-Cas9: A versatile genome editing tool

Introduction

Genome editing is a technique used to precisely and efficiently modify DNA within a cell. It
involves making cuts at specific DNA sequences with enzymes called ‘engineered nucleases’.
Genome editing can be used to add, remove, or alter DNA in the genome. By editing the
genome the characteristics of a cell or an organism can be changed. These nucleases create site-
specific double-strand breaks (DSBs) at desired locations in the genome. The induced double-
strand breaks are repaired through nonhomologous end-joining (NHEJ) or homologous
recombination (HR), resulting in targeted mutations ('edits').

There are currently four families of engineered nucleases being used: meganucleases, zinc
finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and
the CRISPR-Cas system.

CRISPR-Cas9 is a genome editing tool that significantly impacts the scientific community. It is
faster, more inexpensive, accurate, and reliable than previous DNA editing techniques and offers
a wide range of potential uses. “CRISPR” stands for Clustered Regularly Interspaced Short
Palindromic Repeats. CRISPR is the DNA-targeting part of the system which consists of an RNA
molecule, or ‘guide’, designed to bind to specific DNA bases through complementary base-
pairing. Cas9 stands for CRISPR-associated protein 9, and is the nuclease part that cuts the DNA.
The CRISPR-Cas9 system was originally discovered in bacteria that use this system to destroy
invading viruses.

CRISPR-Cas9 was developed from a naturally occurring genome editing mechanism that bacteria
use to defend themselves. When bacteria become infected with viruses, they capture little
fragments of the viruses' DNA and insert them into their DNA in a specific pattern to form
CRISPR arrays. CRISPR arrays enable bacteria to "remember" viruses (or viruses that are closely
related to them). If the viruses strike again, the bacteria generate RNA segments from the
CRISPR arrays that detect and bind to specific sections of the viruses' DNA. The bacteria then
employ Cas9 or a similar enzyme to cleave the DNA apart, rendering the virus inoperable.

2
 Figure: Illustration showing the components of CRISPR-Cas9

History of CRISPR-Cas9: From Discovery to Genome Editing

1. Initial Discovery (1987) – Yoshizumi Ishino

Japanese scientist Yoshizumi Ishino and his team accidentally discovered clustered
repeats in E. coli DNA while studying a gene. The function was unknown at the time, and
they called it "CRISPR" (Clustered Regularly Interspaced Short Palindromic Repeats).

2. CRISPR’s Role in Bacterial Immunity (2000s)

Researchers (Mojica, Pourcel, others) found that spacers matched viral DNA, suggesting
CRISPR was part of a bacterial defense system. In 2007, Rodolphe Barrangou (Danisco)
proved CRISPR provides adaptive immunity in bacteria by storing viral DNA to fight
future infections.

3. Discovery of Cas9 & Guide RNA (2011-2012)

Emmanuelle Charpentier (Umeå University) discovered tracrRNA, a small RNA that helps
CRISPR target DNA. Jennifer Doudna (UC Berkeley) & Charpentier collaborated
and combined crRNA + tracrRNA into a single guide RNA (sgRNA). They showed
that Cas9 (CRISPR-associated protein 9) could be programmed with sgRNA to cut any
DNA sequence if followed by PAM (NGG).

4. Adaptation of Genome Editing (2012-2013)

Feng Zhang (MIT/Broad Institute) & George Church (Harvard) independently
demonstrated CRISPR-Cas9 editing in human and mouse cells.

This breakthrough made CRISPR a versatile, precise, and easy-to-use gene-editing tool.

3
5. Nobel Prize & Modern Applications (2020-Present)
2020 Nobel Prize in Chemistry awarded to Doudna & Charpentier for CRISPR-Cas9.

Today, CRISPR is used in medicine (gene therapy), agriculture (GMO crops), and research
(disease modeling).

How does CRISPR- Cas9 works:

1. The "Search" Function: The Guide RNA (gRNA)

The first part is a molecule called guide RNA (gRNA). Think of this as your specific search query.

 The Target Address: Scientists design this short piece of RNA to perfectly match the
sequence of the specific gene they want to edit. It's like giving the system a very precise
address or a unique sentence to look for within the entire DNA instruction book.
 The Guide: This gRNA molecule acts as a scout, scanning the vast library of DNA until it
finds the exact spot that matches its own sequence.

2. The "Cut" Function: The Cas9 Enzyme

The guide RNA is loaded into the second part of the system, a protein called Cas9.

 Molecular Scissors: The Cas9 protein is an enzyme that acts like a pair of highly precise
molecular scissors. It holds onto the guide RNA.
 Guided to the Target: The guide RNA directs the Cas9 scissors to the exact location on
the DNA that it matches. The Cas9 protein cannot cut just anywhere; it is completely
reliant on the guide RNA to tell it where to go.
 Making the Snip: Once the guide RNA has lined up perfectly with the target DNA
sequence, the Cas9 enzyme activates and cuts both strands of the DNA. This creates a
clean break in the genome.

3. The "Edit" Function: The Cell's Own Repair Crew

The cell naturally doesn't like having broken DNA, so its own repair mechanisms immediately
rush to the site of the cut to fix it. This is where scientists can cleverly introduce changes. There
are two main ways this can happen:

 Option A: Disrupt a Gene (The "Knockout") If scientists do nothing else, the cell's repair
machinery will try to glue the two cut ends of the DNA back together. However, this

4
 repair process is often imperfect and tends to introduce small errors, like adding or
deleting a few DNA letters. These small errors can be enough to scramble the genetic
sentence, effectively disabling or "knocking out" the gene. This is very useful for
researchers who want to understand what a specific gene does by observing what
happens when it's turned off.
 Option B: Replace or Correct a Gene (The "Edit") This is the most powerful part of the
technology. Along with the CRISPR-Cas9 system, scientists can also supply a new,
custom-made piece of DNA. This new piece acts as a template for the repair. The cell's
repair crew sees this template and uses it to fix the break, inserting the new sequence in
the process. This allows scientists to:
o Correct a mutation that causes a genetic disease.
o Insert a new gene to give the cell a new ability.
o Change a gene in a very specific way.

Application of CRISPR-Cas9:

Correcting Genetic Disorders: Scientists are actively researching the use of CRISPR-Cas9 to
correct the genetic mutations that cause inherited diseases. Significant progress has been made
in preclinical studies for conditions like sickle cell anemia, beta-thalassemia, Duchenne muscular
dystrophy, and cystic fibrosis.

Cancer Research and Therapy: CRISPR-Cas9 is a powerful tool in the fight against cancer. It is
being used to understand the genetic drivers of cancer by systematically knocking out genes in
cancer cells to identify those essential for their growth and survival. Furthermore, researchers
are exploring CRISPR-based therapies to engineer immune cells (T-cells) to better recognize and
attack cancer cells.

Diagnostics: The technology is also being adapted for diagnostic purposes. CRISPR-based
diagnostic tools are being developed for the rapid and sensitive detection of infectious diseases,
such as those caused by viruses, and for identifying specific genetic markers associated with
diseases like cancer.

Agricultural Advancements: In agriculture, CRISPR-Cas9 is being employed to improve crop

characteristics and enhance food production.

Crop Improvement: Scientists are using CRISPR to develop crops with desirable traits such as
increased yield, enhanced nutritional value, and resistance to pests, diseases, and
environmental stresses like drought and salinity. For example, researchers have successfully
created gluten-reduced wheat, non-browning mushrooms, and tomatoes with a longer shelf
life.

5
Challenges and limitations:
Off-target effects: Sometimes the Cas9 enzyme cuts the wrong part of the genome, which can
lead to unintended consequences.
Ethical concerns: Especially in human germline editing, where changes could be passed to
future generations. This raises serious moral questions about the potential for "designer
babies."
Delivery issues: Getting CRISPR components into cells, especially in living organisms, is still a
complex task.
Immune response: In some cases, the human body may recognize the Cas9 protein as foreign
and launch an immune attack.

How does CRISPR-Cas9 compare to other genome editing tools?

CRISPR-Cas9 is proving to be an efficient and customizable alternative to other existing genome
editing tools. Since the CRISPR-Cas9 system itself is capable of cutting DNA strands, CRISPRs do
not need to be paired with separate cleaving enzymes as other tools do. They can also easily be
matched with tailor-made “guide” RNA (gRNA) sequences designed to lead them to their DNA
targets. Tens of thousands of such gRNA sequences have already been created and are available
to the research community. CRISPR-Cas9 can also be used to target multiple genes
simultaneously, which is another advantage that sets it apart from other gene-editing tools.

Conclusion

CRISPR-Cas9 has dramatically changed the landscape of genetic engineering. Its ability to
precisely and efficiently edit DNA makes it a valuable tool in both scientific research and real-
world applications. From curing inherited diseases to improving food production, CRISPR holds
immense potential. However, with great power comes great responsibility.

References

Doudna, J.A., & Charpentier, E. (2014). The new frontier of genome engineering with CRISPR-
Cas9. Science, 346(6213), 1258096. https://doi.org/10.1126/science.1258096

Hsu, P.D., Lander, E.S., & Zhang, F. (2014). Development and applications of CRISPR-Cas9 for
genome engineering. Cell, 157(6), 1262–1278. https://doi.org/10.1016/j.cell.2014.05.010

Jinek, M., et al. (2012). A programmable dual-RNA–guided DNA endonuclease in adaptive

bacterial immunity. Science, 337(6096), 816–821. https://doi.org/10.1126/science.1225829

6
National Institutes of Health (NIH). (2023). What are genome editing and CRISPR-Cas9?
https://www.genome.gov/about-genomics/policy-issues/Genome-Editing/what-is-genome-
editing