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CRISPR Cas9

It is Assignment file on CRISPR Cas9, history, mechanism, future, uses, limitations It is a gene editing tool

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Areeba Ghouri
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37 views5 pages

CRISPR Cas9

It is Assignment file on CRISPR Cas9, history, mechanism, future, uses, limitations It is a gene editing tool

Uploaded by

Areeba Ghouri
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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Bahauddin Zakariya University, Multan

Institute of Zoology

Assignment
Topic

CRISPR Cas-9
Submitted To

Dr. Tahira Ruby


Submitted By

Areeba Naeem
Roll Number 01
MPhil Zoology-I
Bahauddin Zakariya University, Multan

CRISPR Cas9
A powerful and precise genome editing tool derived from a bacterial immune system.
 CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats): A guide RNA that
targets specific DNA sequences.
 Cas9: A protein that cuts DNA at the targeted site, allowing for modification.
This system is derived from a natural defense mechanism found in bacteria, which use CRISPR
sequences and Cas proteins to recognize and cut the DNA of invading viruses.
Discovery of CRISPR
o Yoshizumi Ishino and colleagues at Osaka University in Japan first identified unusual
repetitive DNA sequences in the genome of E. coli bacteria. At that time the significance
was unclear.
o CRISPR as Part of the Bacterial Immune System (2005-2007) In 2005, Mojica and
Alexander Bolotin suggested that these sequences acted as a form of immune memory,
where bacteria stored fragments of viral DNA to recognize and defend against future
infections. By 2007, Philippe Horvath and colleagues at Danisco showed experimentally
that CRISPR was indeed part of a bacterial adaptive immune system that could target and
cut viral DNA.
o Development of CRISPRCas9 as a Gene Editing Tool (2012) Jennifer Doudna and
Emmanuelle Charpentier demonstrated that the CRISPRCas9 system could be adapted for
precise gene editing in other organisms.
o First Use of CRISPRCas9 in Living Cells (2013) In 2013, Feng Zhang and George Church
reported the successful use of CRISPRCas9 to edit the genomes of living cells, specifically
human and mouse cells.
o Ethical concerns have also arisen, particularly regarding the editing of human embryos, as
seen in the controversial case of Chinese scientist He Jiankui, who in 2018 claimed to have
created the first gene edited babies.

Components of CRISPR Cas9 system:


CRISPR: These are repetitive sequences of DNA found in bacteria. In between these repeats are
short fragments of "spacer DNA" derived from viruses that have previously attacked the bacterium.
This forms the bacterial "immune memory."
Cas9 (CRISPR associated protein 9): This is an enzyme that acts as molecular scissors, cutting
DNA at a location specified by a guide RNA (gRNA).
Guide RNA (gRNA), A synthetic RNA molecule that combines two regions:
 CRISPR RNA (crRNA), which is complementary to the target DNA sequence.
 Trans activating CRISPR RNA (tracrRNA), which binds to the Cas9 protein, forming an active
complex.

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Bahauddin Zakariya University, Multan

Mechanism of CRISPR Cas9


1. Recognition and Binding of Target DNA:
The guide RNA (gRNA) is designed to be complementary to the target sequence of the DNA that
needs to be edited. It acts as a homing device, guiding the Cas9 protein to the specific location in
the genome.
Cas9 cut the DNA, the target sequence must be located next to a Protospacer Adjacent Motif
(PAM). The PAM sequence, usually ‘NGG’ (where ‘N’ can be any nucleotide), is essential
because Cas9 can only recognize and bind to DNA that has this sequence adjacent to the target.
Once the PAM sequence is recognized, the Cas9 protein undergoes a conformational change,
allowing it to unwind the double stranded DNA and
match the guide RNA to the complementary DNA
strand.
2. DNA Cleavage:
After the Cas9 protein is guided to the target DNA
sequence, it creates a double stranded break (DSB)
in the DNA. The Cas9 protein has two active sites,
one in each of its two nuclease domains:
 The RuvC domain cuts the non-target strand
of the DNA.
 The HNH domain cuts the target strand (the
strand complementary to the guide RNA).

3. DNA Repair: Figure 1 Mechanism of CRISPR Cas9

After the Cas9 induced cut, the cell will try to repair
the broken DNA. There are two main pathways the cell uses for repair:
a. Non Homologous End Joining (NHEJ):

NHEJ is the cell’s default, error prone repair mechanism that directly joins the broken DNA ends.
This process often introduces small insertions or deletions at the site of the cut, potentially
disrupting the gene. This method is particularly useful for knocking out genes, as it can cause
frameshift mutations that lead to the inactivation of the gene.
b. Homology Directed Repair (HDR):

HDR is a more precise repair mechanism, used when a donor template DNA is present. This
template is usually provided by the researchers and contains the desired genetic modification
flanked by sequences that match the DNA around the break.
HDR allows for precise gene editing, such as replacing a faulty gene sequence with a corrected
version, adding new genes, or making specific point mutations.

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Bahauddin Zakariya University, Multan

The choice between NHEJ and HDR depends on the experimental design and the phase of the cell
cycle. HDR is generally less efficient than NHEJ and occurs primarily during the S and G2 phases
when the cell is actively replicating DNA.
Optimization of the CRISPR Cas9 System
Design of Guide RNA: The efficiency of the system largely depends on the design of the guide
RNA. Researchers design gRNAs to specifically target the region of DNA they wish to edit,
ensuring minimal off target effects.
Minimizing Off Target Effects: One major challenge in using CRISPRCas9 is the risk of off target
effects, where the Cas9 protein may cut DNA at unintended sites. To reduce this, scientists
optimize guide RNA design, use Cas9 variants with higher specificity, and employ advanced
computational tools to predict potential off target sites.

Applications in Animal Genetic Modification


1. Gene Knockout:
Disrupting or knocking out specific genes to study their functions.
Example: Knocking out genes related to immune response in mice to study disease mechanisms.
2. Gene Insertion:
Introducing new genes into an animal’s genome to create transgenic animals.
Example: Inserting human disease related genes into animals to create models for studying
diseases like Alzheimer’s.
3. Gene Correction:
Correcting mutations associated with genetic disorders.
Example: Correcting a mutation causing Sickle Cell Anemia.
CRISPRCas9 in Livestock
 Improving Productivity, modifying genes that enhance growth rates, meat quality, or milk
production.
 Disease Resistance, engineering animals with enhanced resistance to diseases.
 Animal Welfare, reducing traits that cause harm or discomfort.
CRISPRCas9 in Model Organisms
 Mice: Widely used in biomedical research, CRISPR allows the creation of precise gene
knockouts or knock ins. E.g. Creating mouse models for human diseases like cancer,
obesity, and neurological disorders.
 Zebrafish: CRISPR is used to study developmental biology and genetics in zebrafish due
to their transparent embryos and rapid development. E.g. By inducing specific mutations
that mimic human congenital heart defects.
 Using CRISPR modified animals to test potential drug treatments or therapies.

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Bahauddin Zakariya University, Multan

CRISPRCas9 in Conservation and Wildlife


 CRISPR can be used to help save endangered species by enhancing resilience to
environmental stressors.
Regulatory Challenges:
CRISPR can sometimes cut DNA at unintended sites, leading to off target mutations.
Ethical Concerns:
Using CRISPR for conservation raises ethical questions about unintended ecological consequences
and the long term impact on ecosystems.
Genetic modification of animals raises ethical questions about animal welfare and the long term
ecological effects.
Concerns over “designer animals” are the potential for misuse of CRISPR technology.
Regulations:
Varies by country; in some regions, CRISPR modified animals face stricter regulations than
traditional genetically modified organisms (GMOs).
Carrying out gene editing in germline cells is currently illegal in the UK and most other countries.
Reference:
Redman, M., King, A., Watson, C., & King, D. (2016). What is CRISPR/Cas9?
Ran, F., Hsu, P., Wright, J. et al. Genome engineering using the CRISPR-Cas9 system. Nat
Protoc 8, (2013)

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