Department of Biochemistry and Microbiology

Course Title: Advanced Biochemistry (BBT609)

Assignment Topic:
CRISPR Cas9: A Versatile Genome Editing
Tool

An AI-generated 3D image of the @CRISPR-Cas9 complex targeting a

DNA strand.

Submitted to: Prof. M Anwar Hossain, PhD

Course Instructor: BBT609.1
Date of Submission: 3 August 2025
Assignment No.: 3
Submitted by: Group 5
Name Student ID Contribution
Raian Islam 2425341670 Introduction, Discovery and Development
Fatima Jahir 2425370670 Mechanism, Applications
Muhammad Mustahsin Mihrab 2517692070 Advantages, Challenges, Conclusion
 Safa Faria 2425026670 Future Perspectives, Ethical Concerns

INTRODUCTION

Genome editing technologies have transformed life sciences, with CRISPR-Cas9 emerging as the
leading tool due to its simplicity, efficiency, and adaptability. Unlike earlier editing methods
such as zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases
(TALENs), CRISPR-Cas9 is guided by a customizable RNA molecule, making it both cost-
effective and highly specific. This RNA-guided DNA endonuclease system allows precise
modification of genetic sequences in a wide range of organisms, revolutionizing research in
medicine, agriculture, and biotechnology. Its speed, programmability, and accessibility have
driven global innovation in disease modelling, therapeutics, and functional genomics.

DISCOVERY AND DEVELOPMENT

The origins of CRISPR-Cas9 trace back to 1987, when Ishino et al. identified unusual repeat
sequences in Escherichia coli. These repeats, later named CRISPR (Clustered Regularly
Interspaced Short Palindromic Repeats), appeared widely among bacteria and archaea, though
their purpose was initially unclear. The breakthrough occurred in the early 2000s when
independent research teams discovered that spacer sequences within CRISPR arrays matched
those of viruses and plasmids, suggesting a role in adaptive immunity. Researchers such as
Francisco Mojica and Ruud Jansen proposed that CRISPR, along with associated Cas (CRISPR-
associated) genes, protect microbes from invading genetic elements by integrating fragments of
foreign DNA as ‘spacers.’

The evolutionary milestone came with the identification of the Cas9 protein in Streptococcus
pyogenes. In 2012, Emmanuelle Charpentier and Jennifer Doudna reconstituted the Cas9 system
in vitro using a single guide RNA to direct DNA cleavage at precise locations. This innovation
rapidly paved the way for genome editing in eukaryotic cells, as later groups led by Feng Zhang
and George Church validated its efficiency across diverse organisms.

Major milestones include:

 1987: Discovery of CRISPR repeats (Ishino et al.)

 Early 2000s: Spacer sequence function and association with immunity established
 2012: Programmable DNA cleavage using Cas9 and guide RNA (Charpentier & Doudna)
 2013: Demonstration of genome editing in mammalian cells.

1
In 2020, Charpentier and Doudna received the Nobel Prize in Chemistry for their pioneering
work on CRISPR-Cas9.

MECHANISM

CRISPR-Cas9 genome editing works through 3 main steps: target recognition, DNA cleavage,
and repair.

1. Target Recognition and Binding:

 The single-guide RNA (sgRNA)
directs the Cas9 enzyme to a specific
DNA sequence by matching it via the
CRISPR RNA (crRNA) region.
 Binding requires a nearby PAM
(Protospacer Adjacent Motif)
sequence, typically 5ʹ-NGG-3ʹ.
 Once bound, Cas9 unwinds the DNA
and forms an RNA-DNA hybrid to
begin editing.
2. DNA Cleavage:
 Upon binding to target DNA, Cas9
undergoes a conformational change,
activating its HNH (Histidine-
Asparagine-Histidine) and RuvC Fig: A schematic overview of CRISPR-Cas9 gene editing.
domains.
 These domains cut both DNA strands, creating a double-stranded break (DSB) a few
bases upstream of the PAM site, usually producing blunt ends.
3. DNA Repair: There are 2 main pathways.
 Non-Homologous End Joining (NHEJ): A fast but error-prone repair process that may
introduce small mutations (insertions or deletions), often disrupting the gene.
 Homology-Directed Repair (HDR): A more accurate method that uses a template to
guide precise genetic changes.

APPLICATIONS

1. Infectious Disease Treatment

 HIV Therapy: Removing HIV DNA and modifying the CCR5 gene to block infection.
 Bacterial and Viral Infections: Targeting the genomes of pathogens directly.
2. Gene Regulation and Imaging

2
  CRISPRa and CRISPRi: Used to activate (CRISPRa) or suppress (CRISPRi) gene
expression without cutting DNA.
 Live-Cell Imaging: Attaching fluorescent tags to Cas9 to track gene activity in real-time.
3. Gene Therapy
 Down Syndrome: Targeting DYRK1A or APP genes
on chromosome 21.
 Sickle Cell Disease and β-thalassemia: By altering
the BCL11A gene to increase foetal haemoglobin.
 Cystic Fibrosis: By fixing mutations in the CFTR
gene.
 Duchenne Muscular Dystrophy: By restoring
dystrophin protein production.
4. Climate-Resilient Crops
 CRISPR can be used to develop crops capable of
withstanding extreme temperatures, salinity, and flooding.
 Examples include CRISPR-edited rice and wheat varieties tolerant to drought or high
heat.
5. Cancer Therapy
 T-cell Engineering: Modifying immune cells to better detect and kill cancer cells.
 Tumor Suppressor Reactivation: Turning back on genes that usually prevent cancer but
were previously silenced.

ADVANTAGES

1. Precision and Specificity: CRISPR-Cas9 offers remarkable precision by targeting specific

DNA sequences through the use of guide RNAs. This targeted approach ensures high
accuracy and significantly reduces the risk of unwanted mutations, making it a superior
alternative to older genome-editing technologies such as ZFNs and TALENs.
2. Efficiency: One of the key strengths of CRISPR-Cas9 is its efficiency. It enables rapid and
effective genome editing across a range of organisms, often yielding high rates of successful
gene modifications.
3. Versatility: CRISPR-Cas9 is highly versatile, functioning effectively in both prokaryotic and
eukaryotic systems. It supports a wide range of applications, including gene knockouts,
knock-ins, and modulation of gene expression through activation (CRISPRa) or repression
(CRISPRi).
4. Cost-effectiveness: CRISPR-Cas9 requires fewer resources and less time compared to older
methods like ZFNs or TALENs, making genome editing more affordable and accessible for
research and clinical applications.

3
5. Potential Therapeutic Use: CRISPR is being explored in clinical trials to treat various
genetic disorders, including sickle cell anemia, certain cancers, and inherited forms of
blindness, showing promise for future gene-based therapies.

CHALLENGES

1. Off-target Effects: Unintended edits at sites with sequence similarity to target DNA can
cause mutations.
2. Delivery Issues: Efficiently delivering CRISPR components into cells (especially in vivo) is
still a technical hurdle.
3. Ethical Concerns: Germline editing (e.g., in embryos) raises moral and societal concerns
about designer babies and heritable changes.
4. Immune Response: Cas9 protein (often derived from Streptococcus pyogenes) can trigger
immune responses in humans.
5. Regulatory and Legal Barriers: Differences in international laws and uncertainty in patent
rights limit CRISPR’s application.

FUTURE PERSPECTIVES

1. Next-Generation CRISPR Tools: New systems like TIGR-Tas are smaller, PAM-
independent, and offer high specificity, improving gene editing precision and delivery,
especially in therapeutic applications.
2. Prime and Base Editing: These advanced editors allow precise DNA changes (substitutions,
insertions, deletions) without double-strand breaks, reducing off-target effects and enhancing
safety.
3. RNA Editing with Cas13: Tools like Cas13 enable programmable and transient RNA
editing, useful for gene regulation and treating diseases without altering DNA permanently.
4. CRISPR-Associated Transposons (CASTs): Allow targeted gene insertions (up to 10 kb)
without inducing DNA breaks, advancing genome engineering in bacteria and potentially
eukaryotes.
5. AI Integration: Machine learning tools (e.g., CRISPR-GPT, DeepFM-CRISPR) help design
more accurate guide RNAs, predict editing efficiency, and minimize off-target risks.
6. Improved Delivery Methods: Nanoparticles, carbon nanotubes, and virus-free systems are
being developed for more efficient CRISPR delivery in both plants and human cells.
7. Agricultural Applications: CRISPR enables faster crop improvement for traits like drought
resistance, pest resistance, and nutritional enhancement through promoter and trait stacking
edits.
8. Synthetic Biology and Bioengineering: CRISPR is driving custom-built genetic circuits in
microbes for producing biofuels, pharmaceuticals, and environmentally friendly materials.

4
9. Diagnostics and Disease Models: CRISPR-based diagnostic tools (e.g., SHERLOCK) and
multiplexed genome editing in animals are advancing personalized medicine and disease
modelling.

ETHICAL CONCERNS

1. Lack of consent: Individuals born with edited genes cannot give consent, making it ethically
problematic to alter their DNA before birth.
2. Equity issues: High costs and limited access to CRISPR technology could increase social
and healthcare inequalities worldwide.
3. Weak global regulation: Inconsistent international laws and oversight create risks of
unapproved and unethical gene-editing experiments.
4. Environmental impact: Releasing genetically edited animals or gene drives could
unintentionally harm ecosystems and natural species balance.
5. Bioweapon potential: CRISPR could be misused to engineer biological weapons, raising
concerns about global security and misuse in warfare.

CONCLUSION

CRISPR-Cas9 has emerged as a revolutionary genome-editing tool, offering unprecedented

precision, efficiency, and simplicity in modifying genetic material. Its versatility has transformed
biological research and opened new possibilities in medicine, agriculture, and biotechnology.
From correcting genetic defects to engineering disease-resistant crops, CRISPR-Cas9 holds
immense promise. However, ethical considerations, potential off-target effects, and regulatory
challenges must be carefully addressed to ensure its responsible and safe application. As the
technology continues to advance, CRISPR-Cas9 is poised to remain at the forefront of genetic
engineering, shaping the future of science and human health.

REFERENCES

 Sandhya, D., et al. (2020). Journal of Genetic Engineering and Biotechnology, 18(1), Article
25.
 Wang, S. W., et al. (2022). Molecular Cancer, 21, 78.
 Huang, K., Qu, Y., Cousins, H., et al. (2024). bioRxiv.
 Gorovitz, A., et al. (2023). Journal of Molecular Biology Methods, 25(6), 45-59.

5
 Ceasar, S. A., Rajan, V., Prykhozhij, S. V., Berman, J. N., & Ignacimuthu, S. (2016).
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1863(9), 2333-2344.