This document provides an overview of the CRISPR-Cas9 genome editing system. It discusses the history and mechanism of genome editing, describes the key components and mechanism of CRISPR-Cas9, and outlines its applications in research including in animal models like zebrafish. The document also discusses advantages and disadvantages of CRISPR-Cas9 as well as ethical issues and future prospects of this genome editing technique.

1. CRISPR-Cas9 system
Arkaprava Roychaudhury
RA1612024010001
Department of Genetic Engineering

2. Contents
• Forward and Reverse Genetics
• Genome Editing
• History of Genome Editing
• Types of molecular scissors
• Mechanism of genome editing
• Timeline of CRISPR-Cas9 genome editing
• Patent Issues
• CRISPR-Cas9 Mechanism
• Key Components
• CRISPR- mediated defense mechanism in bacterial genome
• CRISPR as a genomic tool
• Step by Step Protocol
• Recent advances
• CRISPR in animal model system (Danio rerio)
• Advantages
• Disadvantages
• Ethical Issues
• Future Prospects
• Conclusion
• References
Department of Genetic Engineering

3. Forward and Reverse Genetics

4. Genome Editing
• A technique used by molecular biologists to insert, delete or replace,
a single or a couple of nucleotide bases in the genome, with help of
endonucleases commonly known as molecular scissors.
• These endonucleases create site specific double strand breaks at the
desired location of the genome.
• The induced double strand breaks are repaired through Non-
Homologous End Joining (NHEJ) or homologous recombination.
• Genome editing was selected by Nature Methods as the 2011
Method of the Year.[1]
Department of Genetic Engineering

5. History of Genome Editing
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1998
• HR-Mediated targeting
• First study of Genome Editing via HR in mouse ES cells.
1992
•Cre-lox
• Cre-lox mediated Genome Editing was successfully used for site specific
recombination
1998
• Zinc Finger Nucleases (ZFNs)
• Discovery of Zinc finger proteins that can target specific DNA
sequences.
2000
• Bacterial CRIPSER/Cas defense Mechanism
• CRISPER/Cas mediated Genome Editing discovered in Prokaryotes.
2009
• Transcription like effector nucleases (TALENs)
• DNA binding protein discovered in Xanthomonas bacteria.
2013
• CRISPR/Cas9 Genome Editing
• CRISPR/Cas9 system used for mammalian genome editing

6. Types of Molecular Scissors
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Figure adapted from : Yu, L., Batara, J. and Lu, B., 2016. Application of Genome Editing Technology to MicroRNA Research in Mammalians.
In Modern Tools for Genetic Engineering. InTech.

7. Mechanism of Genome Editing
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8. Timeline of CRISPR-Cas9 Genome Editing
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9. Patent Issues
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10. Patent Issues
Department of Genetic Engineering

11. CRISPR-Cas9 Mechanism
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12. CRISPR-Cas9 Mechanism
• Clustered Regularly Interspaced Short Palindromic Repeats
• Segments of prokaryotic DNA containing, repetitive base
sequences.
• These play a key role in a bacterial defence system,
• form the basis of a genome editing technology known as CRISPR-Cas9
that allows permanent modification of genes within organisms.
• CRISPRs are found in approximately 40% of sequenced bacterial
genomes and 90% of sequenced archaea.

13. CRISPR-Cas9 Mechanism

14. CRISPR-Cas9 Mechanism
Adapted from : Anders, C., Niewoehner, O., Duerst, A. and Jinek, M., 2014. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature, 513(7519),
p.569.

15. CRISPR-Cas9 Mechanism

16. Key Components
• CrRNA (CRISPR RNA) : Contains the guide RNA that locates the
correct section of host DNA along with a region that binds with the
tracrRNA (generally a hairpin loop) forming an active complex.
• TracrRNA (trans-activating crRNA) : Binds to CrRNA and forms an
active complex.
• SgRNA (Single guide RNA) : CrRNA + TracrRNA
• Cas9 Endonuclease : Protein that can modify DNA. Many variants
exist with different functions (SSB, DSB etc) due to Cas9’s DNA
recognition function.

17. CRISPR Locus
Picture adapted from : Horvath, P. and Barrangou, R., 2010. CRISPR/Cas, the immune system of bacteria and archaea. Science, 327(5962), pp.167-170.

18. CRSIPR-Mediated defense mechanism in bacterial
genome
Picture adapted from : Wiedenheft, B., Sternberg, S.H. and Doudna, J.A., 2012. RNA-guided genetic silencing systems in bacteria and
archaea. Nature, 482(7385), p.331.

19. CRISPR as a genomic tool

20. CRISPR as a genomic tool
Picture adapted from : Ablain, J., Durand, E.M., Yang, S., Zhou, Y. and Zon, L.I., 2015. A
CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish. Developmental
cell, 32(6), pp.756-764.

21. Step by step protocol
Image adapted from Schier lab, Harvard Medical School

22. Recent advances
Live cell imaging of low- and non-repetitive chromosome loci using CRISPR-Cas9[2]

23. Recent advances
Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening[3]

24. Recent Advances
CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome. [4]

25. Recent advances

26. CRISPR in animal model system (Danio rerio)
High-throughput gene targeting and phenotyping in
zebrafish using CRISPR/Cas9[5]

27. CRISPR in animal model system (Danio rerio)
Efficient genome editing in zebrafish
using CRISPR-Cas system [6]

28. CRISPR in animal model system (Danio rerio)

29. Advantages
• Easy to design.
• High specificity for KO and KI experiments.
• Can be done anywhere in the genome.
• Multiplexing is possible.
• Specific to Cas9.

30. Disadvantages
• OFF TARGET INDELS
• Limited Choice of PAM sequences
Solutions to the disadvantages
• Proper selection of SgRNA
• Use dCas9 over cas9
• Smaller SgRNAs of 17nts instead
of 20nts
• Use NHEJ inhibitor to boost HDR
• If possible, conduct a whole
genome sequencing of the
organism.

31. Ethical Isuues

32. Future Prospects

33. Future Prospects

34. Future Prospects
• CRISPR/Cas9-mediated Chromatin Immunoprecipitation
• CRISPR Technologies for Transcriptional Activation and Repression
• Epigenetic editing with CRISPR/Cas9
• LIVE Imaging of DNA/mRNA using CRISPR/Cas9
• CRISPR/Cas9 therapeutic applications

35. Conclusion
Undoubtedly this process caught most attention for their potential
in medical applications and numerous other biotechnological
applications like crop editing, gene drives and synthetic biology
Despite the enormous potential that lies within the CRISPR-Cas9
technology, further investigation is required to make the system an
applicable and safe tool for therapeutically useful approaches

36. References
[1] Chen, F., Pruett-Miller, S.M., Huang, Y., Gjoka, M., Duda, K., Taunton, J., Collingwood, T.N., Frodin, M. and Davis, G.D., 2011. High-frequency
genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nature methods, 8(9), pp.753-755.
[2] Qin, P., Parlak, M., Kuscu, C., Bandaria, J., Mir, M., Szlachta, K., Singh, R., Darzacq, X., Yildiz, A. and Adli, M., 2017. Live cell imaging of low-
and non-repetitive chromosome loci using CRISPR-Cas9. Nature Communications, 8.
[3] Joung, J., Konermann, S., Gootenberg, J.S., Abudayyeh, O.O., Platt, R.J., Brigham, M.D., Sanjana, N.E. and Zhang, F., 2017. Genome-scale
CRISPR-Cas9 knockout and transcriptional activation screening. nature protocols, 12(4), pp.828-863.
[4] Shin, H.Y., Wang, C., Lee, H.K., Yoo, K.H., Zeng, X., Kuhns, T., Yang, C.M., Mohr, T., Liu, C. and Hennighausen, L., 2017. CRISPR/Cas9 targeting
events cause complex deletions and insertions at 17 sites in the mouse genome. Nature Communications, 8.
[5] Varshney, G.K., Pei, W., LaFave, M.C., Idol, J., Xu, L., Gallardo, V., Carrington, B., Bishop, K., Jones, M., Li, M. and Harper, U., 2015. High-
throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9. Genome research, 25(7), pp.1030-1042.
[6] Hwang, W.Y., Fu, Y., Reyon, D., Maeder, M.L., Tsai, S.Q., Sander, J.D., Peterson, R.T., Yeh, J.J. and Joung, J.K., 2013. Efficient genome editing in
zebrafish using a CRISPR-Cas system. Nature biotechnology, 31(3), pp.227-229.