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This presentation contain some fundamentals of CRISPR & its application.

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  1. 1. CRISPR - A New Tool inCRISPR - A New Tool in Molecular BiologyMolecular Biology Presented by- Musharraf Ali M.Tech- 2 sem Roll No.-15012006 School of BCE , IIT-BHU
  2. 2. CRISPR- • Clustered regularly interspaced short palindromic repeats • found in PK DNA as repeats followed by a short spacer DNA segment • spacer DNA segment could contain previously exposured bacterial plasmid or virus • contributed as an adaptive/acquired immunity in PK system. • common in archae bacteria
  3. 3. Kind of Repeats observed- • Regularly Spaced Repeats • Direct Repeats in Mycobacteria • TREPs in Haloarchaea • short repeat (SR) arrays Later, Jansen and Mojica proposed replacing SRSR with the more explicit, elegant and now widely adopted acronym CRISPR, for Clustered Regularly Interspaced Short Palindromic Repeats .
  4. 4. Distribution of CRISPR Arrays Amongst Archaea and Bacteria
  5. 5. CRISPR-Cas Function Revealed • Early Hypotheses- - modulating gene expression - binding sites for regulatory proteins - regulation of neighbouring genes - role in recombination - role in replicon partitioning - chromosomal rearrangement
  6. 6. • The Link to Invading Genetic Elements - Two reports in 2005 revealed the presence of spacer - analyses of isolates of Streptococcus pyogenes and Yersina pestis - BLAST searches s revealed that 2 % of analysed spacers showed close similarities to viral DNA or plasmid DNA - CRISPR-Cas system in immunity was demonstratedCRISPR-Cas system in immunity was demonstrated experimentally forexperimentally for S.thermophilus in 2007
  7. 7. Functional Components of CRISPR-Based Systems • composed of - - regularly alternating repeats and spacers - a leader sequence at one end of the array - a set of cas genes these components participate in different stages of the CRISPR-Cas pathway, generating CRISPR-RNAs (crRNAs) and Cas proteins
  8. 8. Fig- Schematic representation of CRISPR-Cas systems and the main steps of CRISPRmediated interference
  9. 9. Identification of CRISPR-associated Proteins • Four cas (CRISPR-associated) genes, cas1–cas4, were originally identified • Cas1 and Cas2, the Cas3 and Cas4 protein families were proposed to be homologous to DNA-helicases and exonucleases, respectively • CRISPR-Cas systems into three main types (I, II and III) on the presence of particular signature cas genes
  10. 10. APPLICATION 1- Resistance Against Viruses Dairy fermentations using Streptococcus thermophilus. 3 approaches are- a)-‘‘CRISPerization’’: Phage Resistance Improvement b)-Artificial Spacer Engineering c)-Transfer Between Microorganisms
  11. 11. 2- Immunity Against Non-Viral Nucleic Acids- -provides immunity against nucleic acids through base pairing between spacer-derived crRNAs and complementary target sequences Beside immunity it also confer- a)-Plasmid Interference- In Staphylococcus epidermidis where CRISPR-encoded spacers lowered efficiency of plasmid uptake. b)-Interference Against Other Mobile Elements – CRISPR-mediated interference against antibiotic resistance genes. ability of S. thermophilus to naturally acquire spacers that target an antibiotic resistance gene
  12. 12. 3- CRISPR-Based Gene Regulation – •potential to use CRISPR-Cas systems for the regulation, transcriptional control, or regulation of transcript levels within a cell (patent application WO/2010/075424) •ability of CRISPR spacers to lower transcript levels, showing that a spacer homologous to the histidyl-tRNA synthetase sequence lowers His-tRNA levels (Aklujkar and Lovley 2010) •Analogies between CRISPR-mediated interference and RNA interference •to harness the flexibility and modularity of CRISPR-Cas systems for RNA interference in bacteria and archaea(patent applications WO/ 2010/011961 and WO/2010/054108)
  13. 13. 4- CRISPR-Based Strain Typing – -CRISPR loci comprise of two main elements, the CRISPR spacer array and a group of CRISPR-associated (cas) genes -CRISPR applications existed long before their function was elucidated. The first use of spacer information for subtyping was in spacer- oligonucleotide typing, or “spoligotyping,” of Mycobacterium tuberculosis strains. -principle of spoligotyping is PCR amplification of the CRISPR array with labeled primers that recognize the DR sequences
  14. 14. Patent applications related to various uses of CRISPR-Cas systems Publication number Title Inventor Publication Date WO/2006/073445 Detection and typing of bacterial strains Russell et al. 13.07.2006 WO/2007/025097 Use of CRISPR- associated genes (cas) Horvath et al. 01.03.2007 WO/2007/136815 Tagged microorganisms and methods of tagging Barrangou et al. 29.11.2007 WO/2008/108989 Cultures with improved phage resistance Barrangou et al. 12.09.2008
  15. 15. CONCLUSION & FUTURE PERSPECTIVE • CRISPR-based typing techniques have been well established for some bacterial species such as Mycobacterium and are currently being developed and studied extensively, especially for human pathogens but also for some agriculturally important species • strain-specific portions of a CRISPR allele using spacer- specific primers or third-generation long-read sequencing • Harnessing CRISPR biology, specifically, identifying the presence/ absence of spacer sequences for subtyping purposes
  16. 16. REFRENCES • Deveau, H., Garneau, J. E. & Moineau, S. CRISPR/Cas system and its role in phage-bacteria interactions. Annu. Rev. Microbio. • Horvath, P. & Barrangou, R. CRISPR/Cas, the immune system of bacteria and archaea. Science. • Mojica, F. J., Diez-Villasenor, C., Soria, E. & Juez, G. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol. Microbiol. • Marraffini, L. A. & Sontheimer, E. J. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science • Garneau, J. E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature • Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR–Cas9 complex. Nature
  17. 17. FURTHER INFORMATION >Eugene V. Koonin’s homepage: COGs: >CRISPRdb: >NCBI CRISPR/Cas: CRISPRclass/index.html >PDB: TIGRFAMs: >CRISPRi protocol by Nature Protocols: http://www.nature. com/nprot/journal/v8/n11/full/nprot.2013.132.html