CRISPR-Cas9 is a gene editing technique that utilizes the Cas9 enzyme to cut DNA at specific locations guided by CRISPR RNA. It allows scientists to precisely modify genes and has applications in medicine, agriculture, and scientific research. Some examples include developing disease-resistant crops and mosquitoes, growing human organs in pigs, and potentially curing genetic diseases. While promising, CRISPR also faces ethical concerns regarding safety, unintended effects, germline editing, and unequal access to treatment. Overall, CRISPR is a revolutionary new biotechnology but more research is still needed to fully realize its benefits and address ethical implications.

1. GENOME EDITING TECHNIQUE
CRISPR-CAS9
PRESENTED
ACADEMIC YEAR: 2022/2023
MODULE: CHROMOSOMAL AND
GENETIC ANOMALY
MINISTRY OF HIGHER EDUCATION AND SCIENTIFIC RESE ARCH
FACULT Y OF NATURAL AND LIFE SCIENCES
M1 BIOTECHNOLOGY AND MOLECUL AR PATHOLOGY

2. INTRODUCTION
Gene editing refers to the use of advanced
biotechnologies to make specific alterations
to an organism's DNA. The primary goal of
gene editing is to modify the genetic material
of an organism in a precise and targeted
manner, which can lead to changes in its
physical and biochemical characteristics. This
technology allows scientists to add, delete, or
modify the DNA sequence of an organism's
genome with great precision.
There are several techniques used for gene
editing, but the most widely used is the
CRISPR-Cas9 system. This system involves
using a bacterial protein called Cas9, which
can be programmed to target and cut specific
DNA sequences. Once the DNA is cut, the
cell's natural repair mechanisms can be
utilized to add, delete or replace genetic
material.
So how does this work mechanistically, and
what are its applications?

3. WHAT IS ?
CRISPR is a gene-editing technology that allows scientists to selectively modify specific genes within an
organism's DNA. The CRISPR system works by using an RNA molecule to guide an enzyme called Cas9.
CRISPR : Clustered Regularly Interspaced Short Palindromic Repeats
cas9 is a protein that is part of the
CRISPR gene editing system.
Specifically, Cas9 is an enzyme
that can cut DNA at specific
locations, as guided by a small
RNA molecule called a guide
RNA. The Cas9 protein is derived
from a type of bacteria that uses
the CRISPR system as a defense
mechanism against invading
viruses.
Cas9 : CRISPR Associated Proteins
2D AND 3D STRUCTURE OF
CAS9 PROTEINE
Cas
9 Target
DNA
sgRNA
Target
DNA
Cas9 gRNA

4. what does each word mean ?
Clustered mean that these repeating
sequences and spacers are usually found
together in a cluster (bound together)
Regularly refers to the fact that the repeating
sequences are usually of a consistent length
and pattern
In 1987, Yoshizumi Ishino and his team
of researchers from the Osaka University in Japan
first reported the presence of CRISPR, in the
Escherichia coli genome.
These refer to short, repeated sequences of DNA
nucleotides found within the genome of prokaryotes.
A BRIEF HISTORY OF CRISPR-CAS9 AS A GENOME-
EDITING TOOLS
REPORTE ABOUT THE PRESENCE
OF CRISPR IN BACTERIA
1987
Short / Repeats
same DNA sequence
Interspaced
different DNA sequence
Palindromic it mean these sequences are the same when read from 5' to 3' on one
strand of DNA and from 5' to 3’ on the other strand
genome of prokaryotes
spacers spacers
spacers
/
/
/
/
/
/
Clustered /Regularly
/
/

5. DISCOVERY OF CRISPR-
ASSOCIATED (CAS) 2002
in 2002, Ruud Jansen and his team coined the term
CRISPR and discovered CRISPR-associated (Cas) genes.
Scientists eventually reported that the CRISPR spacers were
derived from invading phage and extrachromosomal
DNA, which led to the discovery of the CRISPR/Cas
system's role in adaptive immunity in prokaryotes.
(Cas)
CRISPR Associated Proteins
The CRISPR system was originally discovered as a bacterial
immune system, and it works by using RNA molecules to
guide Cas proteins to specific DNA sequences where they can
make precise cuts or modifications.
There are several different types of Cas proteins, each with
their own unique properties and functions. For example, Cas9
is one of the most commonly used Cas proteins in gene
editing applications
Ruud Jansen 2002
even unicellular bacteria have a very basic immune
system. As an daptive immunity
BACTERIOPHAGE
CRISPR
CAS PROTEINS GENES
The CAS proteins in general are going to be:
HELICASES, those are proteins that unwind DNA. Or NUCLEASES, those that cut the DNA.

6. IDENTIFICATION OF THE CRISPER CAS9 SYSTEM
AS A DNA-EDITING TOOL 2012
In 2012, Jennifer Doudna, Emmanuelle Charpentier,
discovered that by designing guide RNA to target a specific
region in the genome, “the CRISPR-Cas9 system can be
used as a “cut-and-paste” tool to modify genomes. As a
DNA-editing tool, CRISPR-Cas9 can remove or introduce new
genes as well as silence or activate genes. CRISPR-Cas9 has
been used to switch off genes that limit the production.
George Church: he developed the first direct genomic sequencing
method, which resulted in the first genome sequence (the human
pathogen).
Feng Zhang(2013):Feng Zhang and his colleagues at the Broad
Institute of MIT and Harvard show that CRISPR-Cas9 can be used to
edit genes in human cells. They demonstrate that the technique can
be used to target multiple genes at once and to create knockout
mutations
After years of speculation over who would be recognized for
the pioneering work on the gene editing tool CRISPR–
Cas9, the Nobel Prize in Chemistry has finally been awarded
to Emmanuelle Charpentier and Jennifer Doudna.in 2020
Jennifer Doudna Emmanuelle
Charpentier
Feng Zhang
George Church Emmanuelle
Charpentier
Jennifer Doudna

7. CRISPR PROKARYOTIC ANTIVIRAL
DEFENSE MECHANISM
1.Adaptation
When a virus or other foreign DNA enters a bacterial
cell, the CRISPR system recognizes and cuts a short
fragment of the foreign DNA, called a spacer. This
spacer is then integrated into the bacterial genome
between the CRISPR repeats, providing a record of the
invading virus.
2.Expression
The CRISPR array is transcribed into a long precursor
RNA molecule, which is then processed into small
CRISPR RNA molecules (crRNAs) by cellular enzymes.
3.Interference
The crRNA molecules combine with a set of Cas
proteins to form a CRISPR-associated
ribonucleoprotein (crRNP) complex. This complex
scans incoming DNA and RNA for complementary
sequences to the crRNA. If a match is found, the Cas
proteins cut the target DNA or RNA, thereby
preventing viral replication.

8. CRISPR-CAS9 MECHANISM OF ACTION
The CRISPR-Cas9 system consists of two key molecules that
introduce a change (mutation?) into the DNA. These are:
 An enzyme called Cas9. This acts as a pair of ‘molecular
scissors’ that can cut the two strands of DNA at a specific
location in the genome so that bits of DNA can then be
added or removed.
 A piece of RNA called guide RNA (gRNA). This consists of a
small piece of pre-designed RNA sequence (about 20 bases
long) located within a longer RNA scaffold. The scaffold part
binds to DNA and the pre-designed sequence ‘guides’ Cas9 to
the right part of the genome. This makes sure that the Cas9
enzyme cuts at the right point in the genome.
 The guide RNA is designed to find and bind to a specific
sequence in the DNA. The guide RNA has RNA bases that are
complementary to those of the target DNA sequence in the
genome. This means that, at least in theory, the guide RNA
will only bind to the target sequence and no other regions of
the genome.
 The Cas9 follows the guide RNA to the same location in the
DNA sequence and makes a cut across both strands of the
DNA.
 At this stage the cell recognises that the DNA is damaged and
tries to repair it.

9. APPLICATIONS OF
CRISPR CAS9
To date, CRISPR-Cas9 has been commonly used
to create gene editing in plants, animal, and
human samples. This technique is widely used in
various scientific fields, including medical
science and therapeutics, as well as plant and
animal sciences.

10. 1.CREATE ANTI-MALARIA MOSQUITOES
Fighting malaria by eliminating the mosquitoes that carry out the disease genetically are modified to not
infect humans using crispr technology.
Mosquitoes performing
disinfection.
Genetically modified mosquitoes that do not infect
humans are inherited by nearly 100% of the offspring.

11. 2.CONCEIVING “IMPROVED”
BABIES
3.CULTIVATING HUMAN ORGANS
IN PIGS
4.BRING EXTINCT SPECIES BACK
TO LIFE
5.CURE DISEASES
6.BOOST PLANTS AND IMPROVE
THEIR QUALITIES
the first baby genetically modified to immunize him against the
AIDS virus by deactivating a gene called CCR5
Human organs grown in genetically modified pigs.
Bringing the mammoth, the Tasmanian tiger or the back to life
thanks to CRISPR-Cas9.
injecting genetically modified T cells into a patient with
lung cancer so that they recognize and attack tumor
modifying plants without integrating foreign genes, CRISPR
could revolutionize varietal improvement while avoiding the

12. DANGERS OF CRISPR CAS9
UNINTENDED CONSEQUENCES
INCOMPLETE EDITS
OFF-TARGET EFFECTS IMMUNE RESPONSE

13. E TH ICS O F C R ISPR CAS9
Safety:
One of the most pressing ethical concerns
related to CRISPR-Cas9 is safety.
Ethical limits:
Finally, there is a broader
ethical question about the
appropriate use of CRISPR-
Cas9. Some argue that there
should be limits on the use
of the technology
Access to treatment:
There is also concern
about unequal access
to CRISPR-Cas9
treatment.
Germline editing:
The use of CRISPR-Cas9 to
edit the genes of embryos or
sperm and eggs raises
concerns about the long-
term effects of such changes.
Informed consent:
Another important
ethical consideration is
informed consent.
Patients who receive
CRISPR-Cas9 treatment
must be fully informed
about the risks and
benefits of the
treatment

14. CONCLUSION
The CRISPR-Cas9 system offers several advantages over other genome editing
techniques, including its ease of use and high specificity, meaning it can target
specific genes without affecting other parts of the genome. As a result, it has become
a popular tool for biomedical research and is being explored as a potential treatment
for genetic diseases. With so many invigorating possibilities for this exciting new
technology, it will be fascinating to see which of these major diseases and issues
will be solved first, signaling the dawn of a new era in molecular biology

15. THANK YOU.