CRISPR Cas 9 TECHNOLOGY

CRISPR Cas 9 TECHNOLOGY is about the new technology by which we can treat many genetic disorders and so on.

1. CRISPR Cas 9 TECHNOLOGY Definition, Technology, Applications & Limitations Madiha Asad (0110) Sameera Jameel (0089) Sonia Amjad (0099)

2. CONTENT: Introduction History Mechanism & Technology Types Applications Limitations Future of CRISPR

3. INTRODUCTION: Genome Editing: Genome editing with site-specific nucleases is a type of genetic engineering in which DNA is inserted, replaced, or removed from a genome using artificially engineered nucleases . Process: The nucleases create a specific DSBs at desired locations in the genome and harness the cell’s endogenous mechanism to repair the induced break by natural processes of HDR and NHEJ. This allows reverse genetics, genome engineering and targeted transgene integration experiments to be carried out in an efficient way.

4. Until 2013 , the dominant genome editing tools were: ZFNs ( Zinc finger nucleases, engineered DNA-binding proteins ) TALENs ( Transcription activator-like effector nucleases, restriction enzymes). The Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) 9 protein system from Streptococcus pyogenes is the latest ground-breaking technology for genome editing.

5. HISTORY of CRISPR/Cas 9:

6. CRISPR Cas 9: “ CRISPR Cas 9 is a unique technology that enables geneticists and medical researchers to edit parts of the  genome  by removing, adding or altering sections of the  DNA   sequence .”   It is causing a buzz in the science world. It is a genome editing tool that is faster, cheaper and more accurate than previous techniques of editing DNA and has wide range of potential applications.

7. CRISPR Cas 9 SYSTEM in BACTERIA CRISPR/Cas 9 systems are part of the adaptive immune system of bacteria and archaea, protecting them against invading viruses by cleaving the foreign DNA in a sequence-dependent manner.

8. working

9. CRISPR Cas 9 systems are the part of adaptive immune system of bacteria. Types of CRISPR Cas 9 : There are three types of CRISPR Cas 9: Type I Type II Type III

10. IMPORTANT COMPONENTS IN MECHANISM: CRISPR Loci: A CRISPR locus is as an array of short direct repeats interspersed with spacer sequences. Cas Protein: . An RNA guided DNA endonuclease that target and cleave invading DNA in a sequence specific manner.

11. Mechanism: It consist of three main steps: Spacer acquisition. crRNA Processing. Interference.

12. Spacer Acquisition: In this step the viral DNA is cut into pieces These segments are then fixed into the spacer DNA

13. crRNA Processing: crRNA Processing in type I:

14. In type II: In type III: crRNA Processing:

15. Interference Phase: crRNAs which are formed in the previous step are then combine with cas protein and form a complex.

16. Interference in type I and II:

17. Interference in type III:

18. Techniques: Gene knocking expression . Genome engineering tool. Transporter of enzyme to specific DNA sequence. Beyond genome editing . Regulate gene expression. Visualize selective loci in living cell.

19. Application Of CRISPR Cas 9 CRISPR-Cas9 has ignited a revolution. It has enabled a simple and affordable way to manipulate and edit DNA (completely changing the face of genome engineering).

20. Applications CRISPR in Agriculture CRISPR in Gene Therapy & Medicine  Live Imaging of DNA/mRNA with CRISPR/Cas9   CRISPR/Cas9-mediated Chromatin Immunoprecipitation    Epigenetic Editing with CRISPR/Cas9  

21. Using CRISPR Libraries for Screening CRISPR in Other Fields   CRISPR Has Expanded Transgenic Animal Research  CRISPR-edited algae with high biofuel yield   Solving some of the biggest problems facing humanity, including malaria and extinction.    

22. Limitations of the CRISPR/Cas9 System  CRISPR/Cas is an extremely powerful tool, but it has important limitations. It is: Difficult to deliver the CRISPR/Cas material to mature cells in large numbers, which remains a problem for many clinical applications. Viral vectors are the most common delivery method. Not 100% efficient, so even the cells that take in CRISPR/Cas may not have genome editing activity. Not 100% accurate, and “off-target” edits, while rare, may have severe consequences, particularly in clinical applications.

23. Ethical Issues: In addition to editing somatic cells. I t’s possible to edit the genomes of gametes and early embryos, called germline editing. Any such edits in humans would not only affect an individual but also his or her progeny . They could also theoretically be used to enhance desirable traits instead of curing disease. Scientists have therefore called for a moratorium on human germline editing until the serious ethical and societal implications are more fully understood.

24. PROS Reverse respectively all the mutations Fast than others Utilize in many different species Excellent ability to target any genomic region. Off target effects Mosaic effects Ethical effects Social effects on society CONS PROS & CONS

25. DRAWBACKS, PROBLEMS & SOLUTIONS: Mosaic problem Safety Efficiency In this unwanted gene sequence form during embryonic stage and causes cancer. Its solution is adding new CRISPR machinery in the cell and double stranded DNA. Wrong attachment (Double stranded DNA in Solution). Ethical Barriers Bio-Weapons

26. CRISPR CAS 9 Current use: The current treatment options merely address symptoms of sickle cell disease, but CRISPR-Cas9 has demonstrated the potential to cure the underlying genetic cause of the disease Scientists have also used CRISPR to detect specific targets, such as DNA from cancer-causing viruses and RNA from cancer cells.  Most recently, CRISPR has been put to use as an experimental test to detect the novel coronavirus.

27. Future Advances: CRISPR-edited cells could also be used to test new therapies and discover which work at the molecular level. Researchers are also now modeling patient cancers more efficiently by editing specific genes using CRISPR-Cas9 in vitro, providing large-scale biomass whereby functional and drug studies can be performed.

28. CONCLUSION: So, CRISPR has its advantages and disadvantages ranging from ethical concerns. It is fastest, cheapest and most precise way of editing genes. By this, the scientists are able to eliminate diseases, reduce poverty, provide unlimited clean energy but at the same time get out of hand easily. CRISPR has potentially given us direct access to source code of life and at the same time gives a great amount of hope to millions of people.