CRISPR CAS9 TECHNOLOGY

Presented by –

Dwijottam Sarma Bordoloi

(17/BBT/20)
Ashish Pratim Mahanta
Course : Biotechnology
(17/BBT/60)
Department of Applied Biology
 CONTENTS
1. Introduction
2. CRISPR mechanism in bacteria
3. CRISPR CAS9 technique
4. Executions till date
5. Ethical Concerns
6. Conclusion
7. References
 INTRODUCTION
CRISPR refers to a type of adaptive immune system found in
bacterial cells, prevalent in bacterial genome.

Scientists discovered that a lot of bacteria have repetitive

DNA sequences in their genome – CRISPRs

CRISPR – Clustered Regularly Interspaced Short Palindromic

Repeats, termed by Francis Mojica in 1990.

CRISPR CAS9 Technology is the advantageous utility of this

mechanism for carrying out genetic engineering.
CRISPR COMPONENTS
crRNA/ CRISPR RNA : Guide RNA
present in Host DNA that bind
with tracrRNA and form hairpin
complex.

tracrRNA : Trans active RNA that

bind with crRNA form active
complex.

sgRNA : Single guide RNA

( crRNA + tracrRNA )

Cas9 : Protein / nuclease that

can modify DNA
 CRISPR MECHANISM IN BACTERIA
 Takes place after the invasion of virus followed by injection of
viral genome into the bacterial cell.

 Immune system:
(a) Adaption – Acquisition of spacer DNA with the insertion of
pieces of viral genome in between CRISPR repeats.
Bacteriophage

Repeats = 28-37 bp
Spacer DNA = 32-38 bp
Viral DNA

 Assisting enzymes – Cas1 (endonuclease), and Cas2

(endoribonuclease)
(b) Expression -

Transcription into mRNA that is complementary to Template.

tracrRNA bind to CRISPR repeats.

mRNA gets chopped off by Cas9 and RNAse III into individual
crRNAs.

Coding Strand

Template
tracrRNA

crRNAs
(c) Interference –

crRNAs get integrated with a Cas9 protein to form effector

complexes.

CRISPR repeats take hairpin like looped form.

[Fact: The bacterial genome preferably takes in a part of the viral

genome only from the portion adjacent to PAM
sequence(Protospacer Adjacent Motif) which is targeted by the
Cas9 nuclease]

On finding a viral genome containing a strand complementary to

the crRNA itself, it binds to viral DNA.

Cas9 induces a double strand cut in the viral DNA (assisting

domains: HNC and RuvC)
 PAM

Effector Complex

Genomic DNA
Break apart
 Adaption

Expression

Interference

Fig: Mechanism Flowchart

 CRISPR – CAS9 TECHNIQUE
Technique that can be used to make targeted cuts in DNA,
applicable in genetic engineering.

Can be delivered in embryonic stem cells of animals.

Requirements:

Design a plasmid that

can transcribe every
single piece of the
required components

Can be induced through – (a) Electroporation

(b) Viruses (lentivirus, adenovirus)
Ligase
 EXECUTIONS TILL DATE

Tomatoes with stronger stems being able to carry heavier fruits

Disrupting the gene responsible for black colour by making a change
Fertilized mouse egg injected with Cas9 and guide RNA
 ETHICAL CONCERNS

 Raises various ethical issues because of its possible

employment not only in adult cells but also in embryos of
humans.

 Also can be used for engineering humans with enhanced

properties (stronger bones, eye colour etc)

 Rising concerns of unintended consequences in clinical

applications in humans in future.
Jennifer Anne Doudna – Emmanuelle Marie Charpentier
American Biochemist – French researcher

 Proposed the use of CRISPR/Cas9 in programmable edit of

genomes in 2012

 Received 2015 Breakthrough Prize in Life Sciences for

CRISPR/Cas9 genome editing technology
 CONCLUSION

 Bacteria also contains an adaptive immune system like

humans although it has a different pathway.

 Bacteria executes this mechanism to fight viruses but we can

use it for genetic engineering.

 Various ethical issues have been raised along with different

ideas that can executed for human welfare.
 REFERENCES
Jennifer Doudna : CRISPR Basics
Channel name : Innovative Genomics Institute – IGI
Youtube link : http://youtu.be/47pkFey3CZ0

How CRISPR lets us edit our DNA | Jennifer Doudna

Channel name : TED
Youtube link : http://youtu.be/TdBAHexVYzc

The principle of CRISPR System and CRISPR – CAS9

Technique
Channel name : Biomedical and Biological Sciences
Youtube links : http://youtu.be/VtOZdThl6dM
http://youtu.be/KIMsVSQGBqw