genome editing with crispr-cas9

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  1. 1. Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR- associated (Cas) proteins CRISPR/Cas9 System LOPAMUDRA NAYAK 04ABT/14
  2. 2. What is Gene/Genome Editing? A process whereby researchers can introduce a modification into an endogenous gene Disruption, Insertion, Replacement at a locus in the genome Control gene expression Create SNP Create Reporter fusions while maintaining endogenous gene regulation
  3. 3. What is CRISPR (CRISPR-Cas; CRISPR- Cas9)? Mechanism of adaptive immunity in bacteria and archaea Evolved to adapt and defend against foreign genetic material (e.g. phage) Several different types of CRISPR pathways in bacteria and archaea Type II: CRISPR-Cas9. Creates a double- strand break in the targeted DNA
  4. 4. Two major repair pathways of DSBs
  6. 6. crRNA + tracrRNA
  7. 7. Applications using CRISPR/Cas9 system Non-protein Coding Gene disruption
  8. 8. Applications using CRISPR/Cas9 system Conditional knockout - For essential genes or tissue-specific study inserting LoxP sites around the exon to be knocked-out Large chromosomal deletions - using two sgRNAs to induce DSBs at sites that flank the region of interest Nucl. Acids Res. June 6 (2013)
  9. 9. Methods of Transfection
  12. 12. LIPOSOME
  13. 13. Pro- High efficiency Con- Optimization required
  14. 14. CALCIUM PHOSPHATE TRANSFECTION Pro- Inexpensive Con- Cytotoxic to many cell types
  15. 15. Electroporation Pro- Fast; No limitation of DNA size Con- Substantial cell death
  16. 16. VIRAL DNA DELIVERY METHOD -Modified replication deficient viruses
  17. 17. Lentivirus
  18. 18. Adenovirus
  19. 19. Adenovirus Associated Virus(AAV)
  20. 20. COMPONENTS OF CRISPR/Cas9 SYSTEM The CRISPR /Cas9 system requires several components, the nuclease, the guide RNA, and usually a selectable marker to enable plants containing the components to be identified. These components can be delivered in a similar way to the introduction of genes in genetic modification. Components using Agrobacterium-mediated transformation is a preferred delivery method because it leads to introduction of single copies of the genes in 50% of cases.
  21. 21. Sanger sequencing 197 nt deletion 1 nt insertion 68 (A) (B)
  22. 22. Why is there a CRISPR Craze? Cas9 can be programmed to perform gene editing in mammalian cells. Changing a short RNA sequence can easily target to a different site in the genome Simpler and easier than other genome editing technologies (ZFN, TALENs) unprecedented efficiency and stunning ease of use ~ Science (2014) 344(6185):707-8 Gene therapy is back! SCIENCE VOL 341 23 AUGUST 2013
  23. 23. 2 0 | N AT U R E | VO L 5 2 2 | 4 J U N E 2 0 1 5 The patent war intensied
  24. 24. CASE STUDY
  25. 25. Background: The CRISPR/Cas9 system provides bacteria and archaea with molecular immunity against invading phages and conjugative plasmids. Recently, CRISPR/Cas9 has been used for targeted genome editing in diverse eukaryotic species. Results: The CRISPR/Cas9 system could be used in plants to confer molecular immunity against DNA viruses. The delivery of sgRNAs specific for coding and non-coding sequences of tomato yellow leaf curl virus (TYLCV) into Nicotiana benthamiana plants stably overexpressing the Cas9 endonuclease, and subsequently challenge these plants with TYLCV. Data demonstrated that the CRISPR/Cas9 system targeted TYLCV for degradation and introduced mutations at the target sequences. All tested sgRNAs exhibit interference activity, but those targeting the stem-loop sequence within the TYLCV origin of replication in the intergenic region (IR) are the most effective. N. benthamiana plants expressing CRISPR/Cas9 exhibit delayed or reduced accumulation of viral DNA, abolishing or significantly attenuating symptoms of infection. Moreover, this system could simultaneously target multiple DNA viruses. Conclusions: These data establish the efficacy of the CRISPR/Cas9 system for viral
  26. 26. An anti-browning mushroom developed by plant pathologist Yinong Yang using CRISPR-Cas9 gene-editing technology will have a longer shelf life and resist blemishes from handling and mechanical harvesting. USDA has ruled that the mushroom is not subject to the agency's regulatory process
  27. 27. DNA-free CRISPR Conventionally, researchers get CRISPR/Cas9 working in a plant cell by first shuttling in the gene that codes for the Cas9 enzyme. The gene is introduced on a plasmid a circular packet of DNA which is usually carried into a plant by the bacterial pest Agrobacterium tumefaciens. As a result, Agrobacterium DNA can end up in the plants genome. Even if the pest is not used, fragments of the Cas9 gene may themselves be incorporated into the plant's genome. Avoiding gene-shuttling altogether. Assembling the Cas9 enzyme together with its guide RNA sequences (which the enzyme requires to find its target) outside the plant, and use solvents to get the resulting protein complex into the plant. The technique works efficiently to knock out selected genes in tobacco plants, rice, lettuce etc. thus reported in Nature Biotechnology.
  28. 28. conclusion The CRISPR/Cas 9 technique is one of a number of geneediting tools. Many favour the CRISPR/Cas9 technique because of its high degree of flexibility and accuracy in cutting and pasting DNA. One of the reasons for its popularity is that it makes it possible to carry out genetic engineering on an unprecedented scale at a very low cost.
  29. 29. How it differs from previous genetic engineering techniques is that it allows for the introduction or removal of more than one gene at a time. Cas9 system is efficient , site- specific, can be multiplexed. This makes it possible to manipulate many different genes in a cell line, plant or animal very quickly, reducing the process from taking a number of years to a matter of weeks. It is also different in that it is not speciesspecific, so can be used on organisms previously resistant to genetic engineering.
  30. 30. References Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (August 2012). "A programmable dualRNAguided DNA endonuclease in adaptive bacterial immunity". Science 337 (6096): 816821. Bibcode:2012Sci...337..816J. doi:10.1126/science.1225829. PMID 22745249. Upadhyay, S.K., Kumar, J., Alok, A. & Tuli, R. RNA guided genome editing for target gene mutations in wheat. G3 (Bethesda) 3, 2233 2238 (2013). Feng, Z. et al. Efficient genome editing in plants using a CRISPR/Cas system. Cell Res. 23, 12291232 (2013). Miao, J. et al. Targeted mutagenesis in rice using CRISPRCas system. Cell Res. 23, 12331236 (2013). Jiang, W. et al. Demonstration of CRISPR/Cas9/sgRNAmediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic acids Res. 41, e188 (2013).


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