1. Presented by –
Dwijottam Sarma Bordoloi
(17/BBT/20)
Ashish Pratim Mahanta
(17/BBT/60)
Course : Biotechnology
Department of Applied Biology
CRISPR CAS9 TECHNOLOGY
2. CONTENTS
1. Introduction
2. CRISPR mechanism in bacteria
3. CRISPR CAS9 technique
4. Executions till date
5. Ethical Concerns
6. Conclusion
7. References
3. 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.
4. 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
5. 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.
Repeats = 28-37 bp
Spacer DNA = 32-38 bp
Bacteriophage
Viral DNA
Assisting enzymes – Cas1 (endonuclease), and Cas2
(endoribonuclease)
6. (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
8. (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)
11. 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)
18. 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.
19. Jennifer Anne Doudna –
American Biochemist
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
Emmanuelle Marie Charpentier
– French researcher
20. 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.
21. 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