GROUP 4: AUTHOR

CONTRIBUTIONS

Asfia Tasnim Moon ID: 2527109670

Introduction and CRISPR-Cas9's mode of
action (1-2).
Jarin Tabassum ID: 2527003670
Structural and functional diversity among
Cas9 variants for tailored genome editing
and Multidimensional impact of CRISPR-
Cas9 across sectors (3-4).

Zarifah Chowdhury ID: 2415503670

Therapeutic Potential (Rewriting
Medicine's Future) and CRISPR’s
Unrivaled Advantages (A Gene-Editing
Revolution) (5-6).
CRISPR Cas9: A versatile
Shamsi Ornob Proma ID: 2527110670
genome editing tool Ethical considerations and challenges,
The Next CRISPR Era, and Conclusion (7-
9).
Assignment 3
Department of Biochemistry and Microbiology
Advanced Biochemistry (BBT608/609) MS in Biotechnology

Submitted To: Prof. M. Anwar Hossain Ph.D.

Submission Date: 3rd August, 2025
1. Introduction: CRISPR-Cas9 a nature's scalpel for rewriting life's code
CRISPR-Cas9 revolutionizes medicine by enabling precise DNA edits, curing genetic diseases,
and irreversibly altering cell lineages. Simpler than Zinc fingers or TALENs, it uses guide RNA and
Cas9 to form a ribonucleoprotein (RNP) complex, inducing targeted double-strand breaks.
Discovered in the 1980s as mysterious bacterial repeats, CRISPR’s role remained unclear until
the 2000s, when scientists recognized it as a bacterial immune defense against viruses (phages).
Cas9, a key protein, cuts viral DNA using CRISPR-stored sequences as guides 1.
In 2012, Jinek et al. reprogrammed CRISPR-Cas9 with synthetic RNA, proving its genome-editing
potential. Doudna and Charpentier (2014) highlighted its precision and versatility, transforming
it into a groundbreaking genetic tool—nature’s scalpel for rewriting life’s code1 ,2.

Figure 1: CRISPR through the years, deep dive into timeline of discovery and innovation

2.CRISPR-Cas9's mode of action

The genome-editing technique CRISPR-Cas9 targets a
particular DNA sequence with the help of a guide
RNA (gRNA). The gRNA attaches itself to a
complementary DNA target next to a PAM sequence,
usually "NGG." At the target site, Cas9 causes a
double-stranded break (DSB) after binding. The DNA
strand complementary to the gRNA is cut by the HNH
domain. On the other strand, the RuvC domain cuts.
The cell fixes the breach after that via NHEJ (non-
homologous end joining) involves the possibility of
introducing mutations. If given a DNA template,
Homology-Directed Repair (HDR) can be employed
for precise editing. This process makes it possible to Figure 2: CRISPR-Cas9's mode of action
specifically disrupt or fix genes2.

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3. Structural and functional diversity among Cas9 variants for tailored genome editing
The CRISPR-Cas9 system has evolved far beyond its original form through strategic protein
engineering, creating specialized variants that address key limitations while unlocking new
capabilities. These innovations fall into three transformative categories:
1. Precision Editors:
 Base editors (2016-2019) enable single-nucleotide changes without double-
strand breaks, with CBEs converting CG to TA and ABEs editing AT to GC.
 Prime editors combine Cas9 nickase with reverse transcriptase for precise
insertions, deletions, and all base-to-base conversions3.

Figur
e 3: Comparative Schematic of Base Editing vs Prime Editing Mechanisms
2. Optimized Cas9 Variants:
 High-fidelity versions (SpCas9-HF1, eSpCas9) minimize off-target effects
 Compact variants (SaCas9) facilitate viral delivery.
 Alternative nucleases (Cas12a, Cas13) target DNA and RNA with unique cutting
patterns.
3. dCas9 Applications:
The schematic highlights dCas9's evolution into a
precision gene regulation tool. Engineered by
inactivating Cas9's nuclease domains (D10A/H840A),
dCas9 maintains DNA-binding ability while enabling
targeted transcriptional control when fused to effector
domains. Advanced systems further enhance its
versatility, including MS2/rapamycin-recruited
chromatin modifiers and split-dCas9 designs for
modular effector delivery. These innovations transform
dCas9 from a simple DNA-targeting scaffold into a
programmable platform for epigenetic editing and
gene regulation,
Figure with wide-ranging
4: Development research
andand
therapeutic potential. The multi-panel
applications of dCas9 for epigenetic schematic
visuallyediting
captures these key engineering breakthroughs
in dCas9 technology4. While these advancements have

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 significantly expanded CRISPR's capabilities, challenges remain in delivery efficiency, off-
target effects for certain applications, and immune responses.

4. Multidimensional impact of CRISPR-Cas9 across sectors

From CRISPR-Cas9, once a bacterial immune system, has become a

revolutionary tool reshaping medicine, agriculture, and industry. In
healthcare, it’s moved from theory to FDA-approved therapies for
sickle cell disease and promising treatments for HIV and inherited
blindness. Agriculture benefits from disease-resistant and drought-
tolerant crops, tackling global food security. Industrial applications
include bioengineered microbes for pharmaceuticals and
sustainable biofuels, while CRISPR-based SHERLOCK systems
transform pathogen detection. These breakthroughs stem from
decades of research, evolving into precise gene-editing platforms.
As CRISPR advances, safer delivery methods and smarter variants
expand its reach, proving how basic science can address
humanity’s greatest challenges—from curing diseases to Figure 5: CRISPR-Cas9 a multifaceted
securing food supplies. The question has shifted from "what can tool transforming science and society.
CRISPR do?" to "what can't it?"

5. Therapeutic Potential: Rewriting Medicine's Future

CRISPR-Cas9 demonstrates remarkable potential across
diverse medical fields. In genetic disorders, it corrects
mutations causing sickle cell anemia, cystic fibrosis, and
Duchenne muscular dystrophy. For viral infections, it targets
latent HIV and HPV genomes, potentially eradicating
persistent infections. Neurological applications include
modifying disease-causing genes in Huntington's and
Alzheimer's, while ophthalmological trials successfully treat
inherited blindness like Leber's amaurosis. The technology Figure 6: CRISPR screening for cancer drug
targets
revolutionizes cancer therapy through engineered CAR-T cells
and oncogene knockout strategies, with CRISPR screens
identifying critical tumor targets and resistance mechanisms
(Figure 6). In regenerative medicine, CRISPR-edited stem cells
enable patient-specific therapies through corrected iPSCs.
Agricultural applications (Figure 7) leverage CRISPR to develop
stress-resistant, high-yield crops with enhanced nutritional
value, showcasing the technology's versatility from clinic to
farm. These advancements highlight CRISPR's transformative
impact across healthcare and biotechnology sectors. Figure 7: CRISPR-generated crops via
precise gene editing

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6. CRISPR's Unrivaled Advantages: A Gene-Editing Revolution
Unlike its predecessors ZFNs and TALENs, CRISPR-Cas9 has democratized genome editing
through its elegant simplicity and precision. The secret lies in its two-component system:
The RNA Guide: A customizable 20-nucleotide RNA sequence leads Cas9 to its target like a
molecular GPS. This replaces the complex protein engineering required by older technologies,
reducing design time from months to days.
Universal Molecular Scissors: The same Cas9 protein can be reprogrammed endlessly - simply
swap the guide RNA to target new genes. This modularity has spawned an entire industry of
CRISPR reagents and kits.
Recent advances have pushed CRISPR further ahead:
• Base Editors: Perform single-letter DNA changes without cutting (2016)4.
• Prime Editors: Offer search-and-replace functionality for DNA (2019)4.
• Epigenetic Editors: Silently turn genes on/off without altering sequence.

7. Ethical considerations and challenges

As CRISPR technology advances from research to real-world applications, it faces significant
ethical and practical hurdles. The precision paradox remains a critical concern—while improved
Cas9 variants achieve remarkable accuracy, even rare off-target edits could have severe
consequences in clinical use. The germline editing debate intensified after the controversial
2018 human embryo experiments, prompting global restrictions but unresolved questions
about therapeutic potential versus ethical boundaries. Meanwhile, the staggering $2.2 million
price tag for the first approved CRISPR therapy highlights urgent accessibility challenges.
Researchers are pursuing innovative solutions like nanoparticle delivery systems and open-
source platforms to democratize access, ensuring these breakthroughs benefit all of humanity
rather than becoming exclusive medical privileges5.

8. The Next CRISPR Era: Science Fiction Becoming Reality

CRISPR technology is entering a transformative phase where cutting-edge innovations mirror
speculative fiction. Artificial intelligence now accelerates CRISPR design, with algorithms that
predict optimal guide RNAs in seconds and engineer custom Cas proteins, bringing
unprecedented precision to gene editing. This AI-CRISPR synergy enables researchers to model
complex genetic interventions before laboratory testing.
Meanwhile, bioengineered "living factories" demonstrate CRISPR's industrial potential.
Microbes edited with CRISPR now manufacture malaria medications, ultra-strong spider silk
proteins, and sustainable biofuels through efficient biological processes. These applications
showcase how genetic reprogramming can revolutionize production paradigms6.
The critical challenge of delivery is yielding to ingenious solutions. Advanced vectors including
brain-penetrating lipid nanoparticles, organ-specific viral carriers, and microscopic DNA

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nanosyringes are overcoming biological barriers to precise therapeutic delivery. These
breakthroughs are translating CRISPR's potential into clinical reality.
Emerging visualization technologies will help communicate these complex advances, from
interactive CRISPR workflow comparisons to molecular animations of novel Cas variants. As
these innovations converge, CRISPR is evolving from a gene-editing tool into a platform
technology reshaping medicine, industry, and our fundamental relationship with biology 7.

9. Conclusion
CRISPR-Cas9 has emerged as one of the
most transformative breakthroughs in
modern biology, fundamentally changing
our approach to genetic engineering.
From its origins as a bacterial immune
mechanism to its current applications in
medicine, agriculture, and biotechnology,
CRISPR offers unprecedented precision in
genome editing. While challenges such as
off-target effects, ethical dilemmas, and
accessibility remain, innovations like AI-
guided design, base editing, and
advanced delivery systems continue to Figure 8: Past and future with CRISPR
refine its potential. The technology's
ability to cure genetic diseases, enhance
crops, and revolutionize diagnostics
underscores its vast societal impact.
However, responsible governance and equitable access will be crucial as CRISPR advances. As
we stand at the frontier of genetic engineering, CRISPR-Cas9 represents not just a tool, but a
paradigm shift—one that redefines humanity's ability to rewrite the code of life itself.

References
1. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive
bacterial immunity. Science. 2012 Aug 17;337(6096):816-21.
2. Jiang F, Doudna JA. CRISPR-Cas9 Structures and Mechanisms. Annu Rev Biopsy’s. 2017 May 22; 46:505-529.
3. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A., & Liu, D. R. (2016). Programmable editing of a target base in genomic DNA without
double-stranded DNA cleavage. Nature, 533(7603), 420-424.
4. Gaudelli, N. M., Komor, A. C., Rees, H. A., Packer, M. S., Badran, A. H., Bryson, D. I., & Liu, D. R. (2017). Programmable base editing of
AT to GC in genomic DNA without DNA cleavage. Nature, 551(7681), 464-471.
5. Frangoul, H., et al. (2021). CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia. New England Journal of Medicine,
384(3), 252-260.
6. Wang, X., Huang, X., Fang, X., Zhang, Y., & Wang, W. (2016). CRISPR-CAS9 system as a versatile tool for genome engineering in
human cells. Molecular Therapy — Nucleic Acids, 5, e388.
7. Zhang, B. (2020). CRISPR/Cas9: A Robust Genome-Editing Tool with Versatile Functions and Endless Application. International
Journal of Molecular Sciences, 21(14), 5111.