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CRISPR Cas9

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Introduction to CRISPR Cas9 technology. View in slide show after downloading for better viewing. Description is minimal, but it will be worth going through the slides that are full of pictures, if you have a minimal understanding of CRISPR.
Prepared in Oct 2015

Introduction to CRISPR Cas9 technology. View in slide show after downloading for better viewing. Description is minimal, but it will be worth going through the slides that are full of pictures, if you have a minimal understanding of CRISPR.
Prepared in Oct 2015

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CRISPR Cas9

  1. 1. Ashikh Seethy Dept of Biochemistry, MAMC- New Delhi CRISPR/Cas9
  2. 2. Gene Editing:  In vivo gene editing:  Zinc Finger Nucleases (ZFN)  TALENs  Homing Endonucleases (Meganucleases)  CRISPR/Cas9
  3. 3. Introduction
  4. 4. Zinc Finger Nucleases:
  5. 5. Introduction
  6. 6. TALENS: Transcription Activator-Like Effector Nucleases  DNA binding domain: 33-34 amino acid sequence •Secreted by Xanthomonas when they infect various plant species •Bind promoter sequences in the host plant •Activate the expression of plant genes that aid bacterial infection
  7. 7. Meganucleases or Homing Endonucleases:  5 families:  LAGLIDADG  GIY-YIG  HN  His-Cys box  PD-(D/E)XK  5 families:  LAGLIDADG  Recognizes 14-40 bp long sequences
  8. 8.  Protein based gene editing systems are difficult to engineer  Expensive  Time consuming  Off-targeting  Alternative: CRISPR/Cas9 System
  9. 9. CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats  1987
  10. 10. 1987 2000: CRISPR is present in > 90% archae and > 40% bacteria 2002: Identification of cas- CRISPR Associated
  11. 11. 2005: Insights into Origin of Spacers
  12. 12. 2007: CRISPR Adaptive Immunity in Bacteria
  13. 13. 2010: Cas9 Cleaves Target DNA via Double Stranded Breaks
  14. 14.  crRNA: CRISPR RNA  tracrRNA: trans-activating CRISPR RNA  PAM: Protospacer Adjacent Motifs NGG in S.pyogenes  sgRNA: single guide RNA aka crRNA-tracrRNA chimera
  15. 15. Introduction
  16. 16. Introduction
  17. 17. Types of CRISPR/Cas Systems:
  18. 18. Designing CRISPR/Cas9:
  19. 19. Designing CRISPR/Cas9:
  20. 20. Off Targeting by CRISPR/Cas9:
  21. 21. CRISPR Nickases: ↓Off-targeting D10A H840A
  22. 22. Gene manipulations with CRISPR/Cas9
  23. 23. Gene Regulation: D10A H840A
  24. 24. CRISPR/Cas9 Advantages  Based on Watson-Crick base-pairing of sgRNA-DNA and an NGG PAM motif straightforward and flexible  Multiplex gene targeting  Much larger targetable sequence space Limitations  The requirement of a protospacer adjacent motif (PAM) sequence limits the number of potential target sequences  Sequence specificity to target loci is only 14 nt long (12 nt of sgRNA and 2nt of the PAM), which can recur around ~11 times in a human genome  Endogenous chromatin states and modifications may prevent the sequence specific binding
  25. 25. Introduction
  26. 26. Hereditary tyrosinemia Type I  Fumaryl-acetoacetate hydrolase (FAH)  Homozygous GA mutation in the last exon  Liver failure and death in infancy
  27. 27. Introduction
  28. 28. Tool Against the World’s Deadliest Animal
  29. 29. Than k

Notas del editor

  • 12 patients studied: NEJM 2014
  • FOK1 cuts DNA once it is bound to the DNA. Generally, this binding is favoured by the palindromic sequence in the restriction site. In ZFN and TALENS, the binding is favoured by the DNA binding domains of ZFN and TALENS
    repeat-variable diresidue (RVD)
  • Whereas Type II restriction enzymes bind short, usually symmetric, recognition sequences of 4 to 8 bp, homing endonucleases bind very long and in many cases asymmetric recognition sequences spanning 12 to 40 bp
  • 2A Peptides:
    These peptides, first discovered in picornaviruses, are short (about 20 amino acids) and produce equimolar levels of mulitple genes from the same mRNA. The term "self-cleaving" is not entirely accurate, as these peptides are thought to function by making the ribosome skip the synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream.4 The "cleavage" occurs between the Glycine and Proline residues found on the C-terminus meaning the upstream cistron will have a few additional residues added to the end, while the downstream cistron will start with the Proline. 
  • The length of the repeat can vary from 21bp to 48 bp, whereas spacers are typically between 26 bp and 72 bp
  • HNH is a domain in the active site
    RuvC domain can be inactivated by a D10A mutation and the HNH domain can be inactivated by an H840A mutation.
  • The KRAB domain, which is found in the amino-terminal region of the proteins, behaves as a transcriptional repressor domain by binding to corepressor proteins, whereas the C2H2 zinc-finger motifs bind DNA.

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