CRISPR CAS9 technique

principle and a basic description of the crispr cas9 technique

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 Introduction CRISPR mechanism in bacteria 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)

12. Ligase

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

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

15. Fertilized mouse egg injected with Cas9 and guide RNA

16. 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.

17. 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

18. 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.

19. 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

20. Thank You!