This document discusses the CRISPR-Cas9 genome editing technique. It begins with an introduction to CRISPR as an adaptive immune system in bacteria. The CRISPR mechanism involves acquiring DNA from invading viruses and using CRISPR RNA and Cas9 proteins to cut matching viral DNA. Scientists now use the Cas9 nuclease guided by a synthetic single guide RNA to make targeted cuts in DNA for genetic engineering. Some applications include modifying crop plants and research in mice embryos. However, using CRISPR in human embryos raises ethical concerns about germline editing and unintended consequences.

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

7. tracrRNA
crRNAs

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)

9. Effector Complex
PAM
Genomic DNA
Break apart

10. Adaption
Expression
Interference
Fig: Mechanism Flowchart

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)

14. Ligase

15. EXECUTIONS TILL DATE
Tomatoes with stronger stems being able to carry heavier fruits

16. Disrupting the gene responsible for black colour by making a change

17. Fertilized mouse egg injected with Cas9 and guide RNA

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