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CRISPR Cas System concept
- 1. Hossein Pourghadamyari Department ofClinical Biochemistry, Afzalipour School of Medicine, Kerman University of Medical Sciences Kerman University of Medical Sciences
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- 3. CRISPR Clustered Regularly InterspacedShort Palindromic Repeats. Hossein Pourghadamyari
- 4. In general, theprecise editing or regulation of genomic information at the DNA level requires the action of a molecular machine composed of two major parts: 1: DNA-binding domain that mediates sequence-specific DNA recognition and binding 2: An effector domain that enables DNA cleavage or regulates transcription near the binding site. Genome Editing Annu. Rev. Biochem. 2016. 85:227–64 Meganucleases Transcription activator-like effector nucleases (TALENs) zinc-finger nucleases (ZFNs)
- 5. CRISPR/CAS9: A GIFTFROM MOTHER NATURE Hossein Pourghadamyari
- 6. spacersspacers spacers spacers shor palindro repea short palindromic repeats short palindromic repeats short palindromic repeats short palindromic repeats JBacteriol. 1987 Dec;169(12):5429-33. CRISPR/CAS9: A GIFT FROM MOTHER NATURE
- 8. 2005–2006 CRISPRs havespacers of extrachromosomal origin viral, Phage Sequence and….. viral, Phage Sequence viral, Phage Sequence viral, Phage Sequence viral, phage Sequence spacersspacers spacers spacers shor palindro repea short palindromic repeats short palindromic repeats short palindromic repeats short palindromic repeats Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements Hypothesis CRISPR mediated by bacterial immunity
- 9. CRISPR mediated bybacterial immunity
- 10. (1) Adaptation When acomplex of Cas proteins excises a segment of the target DNA (known as a protospacer) and inserts it into the CRISPR array (where this sequence becomes a spacer) (2) Expression and processing Expression and processing of the precursor CRISPR (pre-cr) RNA resulting in the formation of mature crRNAs (3) interference When the effector module—either another Cas protein complex or a single large protein is guided by a crRNA to recognize and cleave target DNA (or in some cases, RNA) The defense activity of the CRISPR-Cas systems includes three stages: Dr. Hossein Pourghadamyari
- 11. CRISPR ArrayCas Genes Transcription Cas2 Translation Dr.Hossein Pourghadamyari
- 12. CRISPR ArrayCas Genes Expression (2)Expression and processing Processing RNase III Transcription Cas9 Cas9 Cas9 Cas9 Tracer RNA Cas9 mRNA Translation Dr. Hossein Pourghadamyari
- 13. CRISPR ArrayCas Genes Cas9 Dr.Hossein Pourghadamyari
- 14. • Protospacer adjacentmotif (PAM) CRISPR/Cas9 Components • CRISPR associated protein 9 (Cas9) • trans-activating crRNA (tracrRNA) • Single guide RNA (sgRNA)
- 15. • CRISPR associatedprotein 9 (Cas9)
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- 17. sgRNA • Single guideRNA (sgRNA)
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- 22. Double Strands Break(DSB) Repair Dr. Hossein Pourghadamyari
- 23. PAM-interacting (PI) domain NGGNGG NGG NGGNGG NGG NGG NGG PI Cas9 RuvCHNH PI Cas9Cas9 RuvCHNH sgRNA complement sequence Dr. Hossein Pourghadamyari
- 24. Engineer Cas9 Nuclease(Nickase) Dr. Hossein Pourghadamyari
- 25. PAM-interacting (PI) domain Dr.Hossein Pourghadamyari
- 26. Engineer Cas9 Nuclease(Dead cas9) Dr. Hossein Pourghadamyari
- 27. PAM-interacting (PI) domain GNGG NGGsgRNA complement sequence PI dCas9 + + + + + + Gene Activator ++ + Transcription CDSNGG Dead Cas9 (dCas9) Gene Activation Dr. Hossein Pourghadamyari
- 28. PAM-interacting (PI) domain GNGG NGGsgRNA complement sequence - -- CDSNGG Dead Cas9 (dCas9) Gene Inactivation -PI dCas9 - - - - - - Gene inhibitor Dr. Hossein Pourghadamyari
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- 33. Target RNA Design sgRNADesigning Dr. Hossein Pourghadamyari
- 34. Target RNA Design sgRNADesign Dr. Hossein Pourghadamyari
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- 38. • Surveyor nuclease •T7 Endonuclease I Methods for assessing on target cleveage Dr. Hossein Pourghadamyari
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- 41. More Slides Dr. HosseinPourghadamyari
- 42. 7 Endonuclease I(T7EI) mismatch assays Dr. Hossein Pourghadamyari
- 43. CRISPR ArrayCas Genes Transcription Cas2 Translation Dr.Hossein Pourghadamyari
- 44. CRISPR ArrayCas Genes Expression (2)Expression and processing Processing RNase III Transcription Cas9 Cas9 Cas9 Cas9 Tracer RNA Cas9 mRNA Translation Dr. Hossein Pourghadamyari
- 45. CRISPR ArrayCas Genes Cas9 Dr.Hossein Pourghadamyari
- 46. PAM-interacting (PI) domain NGGNGG NGG NGGNGG NGG NGG NGG PI Cas9 RuvCHNH PI Cas9Cas9 RuvCHNH sgRNA complement sequence Dr. Hossein Pourghadamyari
- 47. PAM-interacting (PI) domain GNGG NGGsgRNA complement sequence PI dCas9 + + + + + + Gene Activator ++ + Transcription CDSNGG Dead Cas9 (dCas9) Gene Activation Dr. Hossein Pourghadamyari
- 48. PAM-interacting (PI) domain GNGG NGGsgRNA complement sequence - -- CDSNGG Dead Cas9 (dCas9) Gene Inactivation -PI dCas9 - - - - - - Gene inhibitor Dr. Hossein Pourghadamyari
- 49. PAM-interacting (PI) domain Dr.Hossein Pourghadamyari
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- 51. CRISPR ArrayCas Genes Transcription Cas2 Translation Dr.Hossein Pourghadamyari
- 52. CRISPR ArrayCas Genes Expression (2)Expression and processing Processing RNase III Transcription Cas9 Cas9 Cas9 Cas9 Tracer RNA Cas9 mRNA Translation Dr. Hossein Pourghadamyari
- 53. CRISPR ArrayCas Genes Cas9 Dr.Hossein Pourghadamyari
- 54. PAM-interacting (PI) domain NGGNGG NGG NGGNGG NGG NGG NGG PI Cas9 RuvCHNH PI Cas9Cas9 RuvCHNH sgRNA complement sequence Dr. Hossein Pourghadamyari
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