The document summarizes key aspects of CRISPR genome editing technology. It describes how CRISPR uses Cas9 and guide RNA to precisely target and edit DNA sequences. It provides a brief history of CRISPR discovery and outlines its components and mechanism of action. The document also discusses several medical applications of CRISPR including treating Duchenne muscular dystrophy, beta-thalassemia, and testing for viruses. It concludes that CRISPR is a flexible and accurate gene editing tool being explored for various applications in agriculture, biotechnology, and medicine.

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CRISP
R :A
NEW
BREAK
THRO
UGH
Dr. SHERIN SHAJI
Dr. VEDICA SETHI
Dr. ZEENATH GHOUSKHAN

2. INTRODUCTION
•Genome editing is a form of genetic engineering
in which DNA sequences are inserted , deleted or
replaced in the genome of living organisms using
molecular scissors like nucleases.
The four main families of nucleases is
Meganucleases
Zinc finger nucleases(ZFNS)
Transcription activator like effector based
effector nucleases (TALEN)
CRISPR

3. CRISPR : CLUSTERED
REGULARLY INTERSPACED
SHORT PALINDROMIC REPEAT
•It is a prokaryotic immune system that
confers resistance to foreign genetic
elements such as those present within
plasmids and phages that provides a form of
acquired immunity.
•It forms the basis of a genome editing
technology known as CRISPR-Cas9 that
allows permanent modifications of genes
within organisms.
•CRISPR-Cas system consist of two key
molecules that introduce a change into the
DNA sequence
1. Cas 9 - act as molecular scissors
2. gRNA – guides Cas9 to the right part
of the genome
gRNA = crispr rRNA + tracrRNA

4. 4HISTORY: Key Events
•1987- CRISPR sequences were first
discovered in Escherichia coli.
•2002- Coined CRISPR name, defined
signature Cas genes.
•2007- First experimental for CRISPR adaptive
immunity.
•2012- Idea of using CRISPR/Cas 9 as a
genome engineering tool by Jennifer Doudna
and Emmanuelle Charpentier.
•2013- Demonstration of Cas 9 genome in
eukaryotic cells.
•2015- The CRISPR-Cas system was selected
by Science as 2015 Breakthrough of the Year.

5. Different CRISPR-Cas
system in Bacterial
Adaptive Immunity-
•Type 1 – contain Cas3 gene which
encodes large proteins with
separate helicase and DNAase
activity, in addition to genes
encoding protien that form cascade
like complexes with different
compositions.
•Type 2 – includes HNH type
system in which Cas9, a single very
large protein is sufficient to
generate crRNA and cleaving the
target DNA in addition to
ubiquitous Cas 1 and Cas 2.
•Type 3 - contain polymerase and
RAMP modules in which at least
some of the RAMPs seem to be
involved in the processing of the
spacer–repeat transcripts,

7. CRISPR LOCUS
Spacer : the direct repeats in a
CRISPR locus are seperated by
short stretches of nonrepetitive
DNA called spacers that are derived
from invading plasmid or phage
DNA.
Protospacers : the nucleotide
sequence of the spacers which is
similar to a region in the phage
genome which block phage
replication.
Leader sequence : is a conserved
sequence associated with CRISPR
locii located upstream of CRISPR.
CRISPR array : composed of series
of repeats interspaced by spacer
sequences acquired from invading
genome.

8. KEY COMPONENTS OF
CRISPR
crRNA :
contains the guide RNA that locates the
correct section of host DNA along with a
region that binds to tracrRNA (in a hairpin
loop form) forming an active complex.
tracrRNA :
binds to crRNA and forms an active
complex.
gRNA :
a combined RNA consisting of tracrRNA
and at least one crRNA.
Cas 9 :
they are RNA guided DNA endonuclease
associated with CRISPR to interrograte
foreign DNA.

9. MECHANISM OF ACTION OF
CRISPR

10. WHAT MAKES CRISPR
SYSTEM
THE IDEAL GENOME
EDITING TOOL?
•High potency and specificity
•Broadly applicable to both invivo and
exvivo application
•Simple editing tools, allow unique
ability to scale and optimize speed
•Potential one time curative treatment
for genetic disease
•Ability to target multiple DNA site
simultaneously which makes it different
from other genome editing tools like
ZFNs and TALENs
•Multifunctional programability to insert,
delete or repair gene

12. APPLICATION OF CRISPR IN
MODERN MEDICINE
•To understand the role that specific
mutations in specific genes influence a
particular trait of an organism.
•To recreate known stable mutations in cell
lines that can serve as models of a particular
disease.
•To create stable mutations in whole
organisms (plants / animals) and create
strains that can be used in research or
commerce.
•To create gene therapies in order to treat or
diagnose congenital diseases, infections, or
cancers especially new outbreaks like ZIKA
virus
•To create gene drives that can modify
populations of organisms in a specific way

14. CRISPR TREATMENT FOR
DUCHENE MUSCULAR
DYSTROPHY
•New gene editing enzyme CRISPR cpf1
corrected DMD in human cell
•DMD mutation in dystrophin gene
•CRISPR cpf1 differ from CRISPR Cas9 :
much smaller than Cas9 enzyme
which make it easier for the delivery to
muscles
• skipping a mutation region or
precisely repairing a mutation in gene
CRISPR cpf1 mediated genome editing
corrects DMD mutation but also muscle
contractility and strength

15. CRISPR targets point mutation in exon 23
it creates a stop codon and serve as a
model of DMD
Efficacy by using a two vector system of
CRISPR rather than single vectors for the
guide RNA and Cas9 which will restore
the dystrophin positive fibers
Recovery: cell level- decrease infiltration,
inflammatory cells and decrease fibrosis
and reversal of necerosis.

16. Final outcome:
•Improved grip strength
•Force generation,
resistance against eccentric
contractions
•Decreased blood levels of
creatinine kinase
•Dystrophin expression on
vascular smooth muscle
•Important source of
oxygenation during activity
and cardiomyocytes-
restored dystrophin.

18. TREATMENT FOR
MYOTUBULAR
MYOPATHY
•Is a X-linked genetic disease
affecting new born boys.
•Caused by mutation in
MTM1 gene encoding
myotubularin, a protein
involved in functioning of
muscle cells.
•Research conducted by
University of Washington and
Harvard Medical School in
USA achieved a new method
of treatment of MM by gene

19. CORRECTION OF BETA-
THALASSEMIA MUTATION USING
CRISPR CAS9
•Beta- Thalassemia caused by mutation in
human Hb heritage.
•Creation of human induced pluripotent stem
cells (iPSC) from Beta Thalassemia patients
could offer an approach to cure the disease.
•Correction of disease causing mutation in
iPSC’s restore normal function and provide a
rich source of cells in transplantation.
•CRISPR/Cas9 correct the HBB mutation in
patient derived iPSC without leaving any
residual foot print.

21. CRISPR-Cas13a:
DIAGNOSTIC TOOL
FOR ZIKA AND
DENGUE VIRUS “The
Sherlock”
•SHERLOCK uses an RNA guide that
gloms onto RNA, not DNA, and an
enzyme called Cas13a cuts the
genetic material. Once Cas13a
snips the target, it starts
indiscriminately cutting any RNA it
encounters.
• A team lead by bioengineer James
Collins and CRISPR genome-editing
pioneer Feng Zhang, both from the
Broad Institute in Cambridge,
Massachusetts, has now shown
that these “collateral” cuts can
form the basis for the SHERLOCK

22. •The researchers demonstrate that
SHERLOCK can detect viral and bacterial
infections, cancer mutations found at low
frequencies, and subtle DNA sequence
variations known as single nucleotide
polymorphisms that are linked to other
diseases.
•SHERLOCK, a somewhat strained acronym
coined by the team, stands for specific high
sensitivity enzymatic reporter unlocking.
•To exploit SHERLOCK for detecting small
amounts of virus, the researchers spiked
samples containing Zika or dengue virus with
so-called fluorescent reporter RNA. When
this RNA is cut, it effectively shoots off
fluorescent flares.
•The team then unleashed Cas13a connected
to a bit of RNA that targeted genetic
sequences from either Zika or dengue. Once
Cas13a found and sliced even a few viral
sequences, it subsequently snipped the
fluorescent reporters and created a
detectable signal indicating the presence of
the virus.

23. The
detectio
n
sensitivit
y of the
new
CRISPR-
Cas13a
system
for
specific
genetic
material
is 1
million
times
better
than the
most
common
ly used
diagnost

24. CRISPR: TARGETS
CANCER IN FIRST
HUMAN TRIAL
•Normally, T cells survey the body to seek
out and destroy abnormal cells that may
be turning cancerous. These cells often
have strange proteins on their surface
that alert T cells that they’re up to no
good. However, in an evolutionary
approach, cancer cells often gain the
ability to “switch off” any T cell that gets
in their way, effectively blocking the
attack.
•Many of the most successful cancer
therapies try to circumvent this response
by boosting the immune system. A 2015
study led by Dr. Carl June of UPenn, who
is advising the new trial, used an older,
less efficient gene engineering technique
called zinc finger nucleases to give T cells
better ability to fight off HIV. The therapy

25. The Plan;
•In all, scientists will recruit 18 patients
with three types of cancer (myeloma,
sarcoma or melanoma) who have
stopped responding to existing
treatment. The two-year trial will take
place at three centers that are members
of the Parker Institute For Cancer
Immunotherapy, including UPenn, UC
San Francisco and the University of
Texas.
•The researchers will remove T cells from
the patients and, using a harmless virus
to deliver the CRISPR machinery into the
cells, perform three gene edits on them.
The first edit will insert a gene for a
protein called the NY-ESO-1 receptor.
This protein gives T cells the power to
better recognize and home in on
cancerous cells.
•Unfortunately, T cells have two native
proteins that interfere with this process,
so the second edit will remove these

26. •The third edit gives T cells staying power:
it will remove a gene that allows cancer
cells to recognize the immune cell and
prevent the cancer from shutting off the
attack.
•Because CRISPR doesn’t work every time,
not all the cells will get every modification.
In the end, the engineered cells will be a
mixture with various combinations of the
proposed changes. Only 3-4% may contain
all three .After the edits, the researchers
will infuse the cells back into the patients
and closely monitor for any issues.
•One of the biggest worries is that CRISPR
might inadvertently snip other genes,
potentially creating new cancer genes or
triggering existing ones. Using various
tests, the team plans to carefully measure
the growth rate of the engineered T cells
and test for genomic abnormalities.But the
outlook is bright.
•In a test run using T cells from healthy

27. •Another worry is that
the technique itself
could activate the
body’s immune
response. CRISPR uses
an enzyme called Cas9,
which originates from
bacteria, to do the
snipping. Although
there are ways to
protect the edited cells
from the immune
system, they could still
be attacked.
• The last concern isn’t
scientific, but pertains
to UPenn’s potential
conflict of interest.
June, who will serve as
an advisor to the trial,
has several patents on
T cell engineering for
cancer and is involved
in several companies

30. CONCLUSION:
•The CRISPR/Cas 9 technique is one of a number of
gene-editing tools. Many favor the CRISPR/Cas9
technique because of its high degree of flexibility
and accuracy in cutting and pasting DNA.
•One of the reasons for its popularity is that it makes
it possible to carry out genetic engineering on an
unprecedented scale at a very low cost.
•How it differs from previous genetic engineering
techniques is that it allows for the introduction or
removal of more than one gene at a time.
•This makes it possible to manipulate many different
genes in a cell line, plant or animal very quickly,
reducing the process from taking a number of years
to a matter of weeks.
• It is also different in that it is not species-specific,
so can be used on organisms previously resistant to
genetic engineering.

31. •The technique is already being explored
for a wide number of applications in fields
ranging from agriculture through to human
health.
• In agriculture it could help in the design
of new grains, roots and fruits. Within the
context of health it could pave the way to
the development of new treatments for
rare metabolic disorders and genetic
diseases ranging from hemophilia through
to Huntingdon's disease.
• It is also being utilized in the creation of
transgenic animals to produce organs for
transplants into human patients.
• The technology is also being investigated
for gene therapy. Such therapy aims to
insert normal genes into the cells of people
who suffer from genetic disorders such as
cystic fibrosis, hemophilia or Tay Sachs.
•Several start-up companies have been
founded to exploit the technology
commercially and large pharmaceutical
companies are also exploring its use for
drug discovery and development purposes.

33. REFERENCES:
https://futurism.com/images/how-crispr-works-the-future-of-genetic-
engineering/
https://musculardystrophynews.com/crispr-cas9-treatment-dmd/
cgen.ucla.edu/2016/06/umms-scientists-use-crispr-to-discover-zika-and-
dengue-weaknesses/
https://musculardystrophynews.com/2017/03/27/x-linked-myotubular-
myopathy-boys-focus-recensus-study/
https://www.nature.com/articles/srep32463
https://singularityhub.com/2016/06/26/75-crispr-targets-cancer-in-first-
human-trial-what-you-need-to-know/
https://en.wikipedia.org/wiki/CRISPR