1.
CRISPR-CAS9
BY HASNAT TARIQ

2.
GENOME EDITING
• Genome editing is a group of technologies that give scientists the
ability to change an organism's DNA.
• These technologies allow genetic material to be added, removed, or
altered at particular locations in the genome.
• A number of genome editing techniques have been developed such
as :
• ZFNs (Zinc Finger Nucleases)
• TALENs (Transcription Activator Like Effector Nucleases)
• CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

3.
CRISPR
• CRISPR are sections of genetic code containing short repetitions of
base sequences followed by spacer DNA segments.
• It is a revolutionary technique that can modify any region of the
genome of any species with high precision and accuracy without
harming other genes.
• It was identified in a prokaryotic defence system.

4.
Terms to understand
• Palindromic Sequences
A palindromic sequence is a nucleic acid sequence on
double-stranded DNA or RNA wherein reading 5' (five-prime) to
3' (three prime) forward on one strand matches the sequence
reading 5' to 3' on the complementary strand with which it forms
a double helix.

5.
Terms to understand ( Continue..)
• Spacer DNA
Noncoding DNA that separates one gene from another is
called spacer DNA.
In bacteria, spacer DNA sequences are only a
few nucleotides long.

6.
Terms to understand ( Continue..)
• crRNA
Contains the guide RNA that locates the correct section of
host DNA along with a region that binds to tracrRNA forming an
active complex.
• tracrRNA
Trans activating crisper RNA (tracer RNA) is another
important molecule that plays a critical role in the processing of
pre-crRNA. It is a short RNA sequence and is complementary to
the CRISPR repeat. It binds to crRNA and and forms an active
complex.

7.
Terms to understand ( Continue..)
• sgRNA
Single guide RNA is a combination of tracrRNA and crRNA.
• PAM
Protospacer adjacent motif (PAM) is a 2-6 base pair DNA
sequence immediately following the DNA sequence targeted by
the Cas9 nuclease in the CRISPR bacterial adaptive immune
system. PAM is a component of the invading virus or plasmid,
but is not a component of the bacterial CRISPR locus.

8.
CAS9
• Cas9 (CRISPR associated protein 9) is an RNA-
guided DNA endonuclease enzyme associated with
the CRISPR.
• It is associated with the CRISPR adaptive immunity system
in Streptococcus pyogenes, among other bacteria.
• It catalyzes site-specific cleavage of double stranded DNA.

9.
CRISPR-CAS SYSTEM
• The CRISPR-Cas system is a naturally occurring, adaptive
microbial immune system for defense against invading phages
and other mobile genetic elements.
• It contains a cluster of CRISPR-associated (Cas) genes and its
corresponding CRISPR array.
• CRISPR arrays consist of repetitive sequences (direct repeats,
referred to as repeats) interspaced by short stretches of
nonrepetitive sequences (spacers) derived from short segments
of foreign genetic material.

10.
CRISPR systems in prokaryotic immunity

11.
CRISPR/Cas9 and Targeted Genome Editing

12.
CRISPR/Cas9 and Targeted Genome Editing

13.
CRISPR/Cas9 and Targeted Genome Editing

14.
CRISPR/Cas9 and Targeted Genome Editing

15.
CRISPR/Cas9 and Targeted Genome Editing

16.
What can we do with CRISPR?

17.
What can we do with CRISPR?

18.
Applications of CRISPR

19.
Advantages of CRISPR
 Target design simplicity
 Efficient
 Cheaper
 Multiplexed Mutations
 Quicker
 More accessible for researchers

20.
Limitations of CRISPR
 Target sequences may be limited due to PAM sequences.
 Off target effects could be possible.

21.
Future of CRISPR
• Human gene therapy
• Agriculture, crops, animals
• Screens for drug target ID
• Viral gene disruption
• Ecological vector control (Mosquito sterilization etc)
• Synthetic biology ( Pathway engineering)
• Programmable RNA targeting

22.
The First CRISPR Trial in Humans Will Target
Cancer
• The doctors plan to use CRISPR to edit human T cells, which play a
central role in the immune system, to target tumors.
• For human CRISPR trial, doctors plan to extract blood cells from the
patients and then edit the cells outside the body, an approach called ex
vivo gene therapy.
• Using CRISPR, the doctors will delete two specific genes from the T cells.
One is a so-called “checkpoint” molecule (PD-1) that cancer cells exploit
to halt immune system activity. The other is a receptor that T cells use to
detect dangers, such as germs or sickly tissue. They’ll replace that
receptor with an engineered one designed to direct T cells toward
tumors.

23.
Ethical Issues
• Balance of risks and benefit
• Ecological disequilibrium
• Regulations for consumers
• Application of CRISPR/Cas9 technique to human germline
• Genome editing for enhancement

24.
References
• Futurism. (2018). How CRISPR Works: The Future of Genetic
Engineering and Designer Humans. [online] Available at:
https://futurism.com/images/how-crispr-works-the-future-of-
genetic-engineering
• Futurism. (2018). The first American CRISPR trial in humans will
target cancer. [online] Available at: https://futurism.com/first-
american-human-crispr-trial-target-cancer