CRISPR-Cas9 is a gene editing technology that uses the bacterial immune system to cut DNA at specific locations. It allows researchers to understand, characterize, and control DNA. CRISPR-Cas9 uses an RNA-guided DNA endonuclease enzyme called Cas9 that is directed by guide RNA to cleave target DNA. It has numerous applications including modifying genes in plants and animals, developing disease resistant crops, and potentially curing genetic diseases in humans by precisely editing genes. While revolutionary, it also raises ethical concerns that must be considered and addressed.

1. CRISPR
CRISPR-Cas9

2. INTRODUCTION
“Clustered Regularly Interspaced Short Palindromic
Repeats”
 New gene editing technology with the potential to revolutionize
genetic engineering and the biotechnology industry.
 Use to understanding, characterizing, and controlling DNA.
 Based on bacterial immune system.
 Single solution to many problems

3. HISTORY
 1987 Researcher youshizmi Ishano find CRISPR sequence in E.coli but they don’t
characterized their functions.
 2000 CRISPR sequence were found by moica to be common in other microbes.
 2002 mr. Jansen Coined CRISPR name, defined, signature cas gene.
 2007 First experimental evidence for CRISPR adaptive immunity.
 2012 Idea of using CRISPR- Cas9 as a genomic engineering tool, was published
by Jennifer and Emmanuelle.
 2013 First demonstration of Cas9 genome engineering in Eukaryotic cell.
 2014 First CRISPR patent was granted to Feng Zheng.

4. In bacterial immune system
In 2000 spacer DNA matched with bacterial DNA and identified nearby CRISPR associated
Cas genes.

5. Cas9 / CRISPR-Cas9
“CRISPR associated system protein 9”
 an RNA-guided DNA endonuclease enzyme associated with
the CRISPR.
 Cas9 protein is responsible for locating and cleaving target DNA,
both in natural and in artificial CRISPR/Cas systems.
 a prominent tool in the field of genome editing.
 Advance in Investigation, prevention, treatment of diseases, to
increase crop yield, and to understand the function of the gene.

6. STRUCTURE
 Cas9
 Cas9 protein has six domains
 The Rec I domain is the largest and is
responsible for binding guide RNA.
 The role of the REC II domain is not yet
well understood.
 The arginine-rich bridge helix is for
initiating cleavage activity.
 The PAM-Interacting domain responsible
for initiating binding to target DNA.
 The HNH and RuvC are nuclease domains
that cut single-stranded DNA.

7. Guide RNA
 The guide RNA is engineered to
have a 5′ end that is
complimentary to the target DNA
sequence.
 This artificial guide RNA bind
Cas9, induces some
conformational changes, and make
it active.
 Then both act on target DNA.

8. GENOME EDITING
Gene editing technique is Inserting , removing, or modifying DNA in a
genome
By Using
Radiation, chemicals, Nucleases or Molecular Scissor.
Being Used
In Mega nucleases, ZFN, TALEN and CRISPR-Cas.
With CRISPR-Cas9 multiple genes can be targeted simultaneously, referred to as
genome editing.
The nucleases create Double Strand Break (DSB) at desired location, which is repair
through NHEJ or HDR mechanism.

9. MECHANISM
Cas9 PROTEIN
Searches for target DNA by binding with sequences
that match its protospacer adjacent motif (PAM)
sequence.
GUIDE RNA
Have a 5′ end that is complimentary to the target
DNA sequence.
If the complementary region and the target region pair properly,
the RuvC and HNH nuclease domains (domains of Cas9) will cut the
target DNA.

10. WORKING
An enzyme
that cuts DNA
A guiding piece
of genetic
material
The guide RNA targets and
binds to a specific DNA
sequence and the attached
Cas9 enzyme

11. 11
Repair
mechanism

12. Applications of CRISPR in different
Industries:
Microbiology
Biomedicine
Animals
Agriculture

13. Microbiology
 CRISPR/dCas9-mediated protein imaging
 Fluorescence in situ hybridization (FISH) use for labeling genetic loci.
 Mechanism of action of CRISPR:
 dCas9+yellow/green FP Multicolor CRISPR Complex
 This complex bind with genetic loci matching sgRNA
 Labeling done

14. Why protein imaging done?
Direct visualization of genomic loci facilitates deep
understanding of spatial organization of microbial genome
and gene expression.
This technique is also effective to
Identify repetitive sequences such as Isoenzymes and
RNA
Substantially effect biosynthesis of desired metabolites

15. Biomedicine:
 Epstein Barr Virus (EBV)
 CRISPR eradicate viral DNA of EBV
 CRISPR is an Anti-herpesvirus removing cancer causing EBV from
tumor cells
 Transplantation
 Gene editing of mismatched humans or even non human mammals
as potential organ donors
 Editing will reduce risk of immune responses and rejection when
using mismatched organ

16. Animals:
 Cashmere goat
CRISPR/Cas9 system FGF5 Gene
 Increased number of second hair follicles
 Enhanced fiber length
 More cashmere produce
 This change induced at
a. morphological level
b. genetic level

17. Examples of crops modified with
CRISPR Technology
 Potential tool for developing virus
resistance crop variety
 Can be used to eradicate unwanted
species like herbicide resistance
weeds, insect pest
 Developing biotic and abiotic
resistant traits
in crop plants
Agriculture:

18. REVOLUTIONARY ASPECTS OF CRISPR Cas9
It was stated that
“CRISPR is dramatically accelerating the pace of
research in nearly every biological field.”
 CRISPR Cas9 tool use in the Fixing of the Donor Organ
Shortage which one is a revolution in medical sciences.
An Alternative to Petroleum.
Designer Pets and Service Animals.

19. PROS AND CONS
CONS
 Off target effects
 Mosaic effects
 Ethical
&
 Social effects on the
society
PROS
 Reverse respectively all the
mutations
 Fast then others
 Utilize in many different
species
 Excellent ability to target
any genomic region .

20. DRAWBACKS, PROBLEMS AND THEIR
SOLUTIONS
 MOSAIC PROBLEM
 Safety
 Efficiency
in this unwanted gene sequence form during embryonic stage and causes
cancer.
Its solution is adding new CRISPR mechaniery in the cell add double
standard DNA.
 Wrong attachment (double stranded DNA is solution).
 Ethical barriers
 Bio weapons

21. CONCLUSION
 Summing it all up, CRISPR has its advantages and disadvantages ranging from
ethical concerns
 to being known as the fastest, cheapest and most precise way of editing genes.
 This scientific breakthrough has the ability to eliminate diseases, solve world
hunger, provide unlimited clean energy but at the same time get out of hand very
easily.
 CRISPR has potentially given us direct access to the source code of life and at the
same time given a great amount of hope to billions of people

22. REFERENCES
 https://www.nbcnews.com/storyline/the-big-questions/4-ways-revolutionary-
gene-editing-tool-could-change-world-n726371
 https://en.wikipedia.org/wiki/CRISPR
 https://www.broadinstitute.org/what-broad/areas-focus/project-
spotlight/questions-and-answers-about-crispr