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the application of CRISPR/Cas9 system in genome editing

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CRISPR-cas9 system is a new robust genome editing tool and also this is a really easy to use system.
کاربرد سیستم کریسپر در ویرایش ژنوم.ppt

Published in: Health & Medicine
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  • DOWNLOAD FULL BOOKS, INTO AVAILABLE FORMAT ......................................................................................................................... ......................................................................................................................... 1.DOWNLOAD FULL. PDF EBOOK here { https://tinyurl.com/yxufevpm } ......................................................................................................................... 1.DOWNLOAD FULL. EPUB Ebook here { https://tinyurl.com/yxufevpm } ......................................................................................................................... 1.DOWNLOAD FULL. doc Ebook here { https://tinyurl.com/yxufevpm } ......................................................................................................................... 1.DOWNLOAD FULL. PDF EBOOK here { https://tinyurl.com/yxufevpm } ......................................................................................................................... 1.DOWNLOAD FULL. EPUB Ebook here { https://tinyurl.com/yxufevpm } ......................................................................................................................... 1.DOWNLOAD FULL. doc Ebook here { https://tinyurl.com/yxufevpm } ......................................................................................................................... ......................................................................................................................... ......................................................................................................................... .............. Browse by Genre Available eBooks ......................................................................................................................... Art, Biography, Business, Chick Lit, Children's, Christian, Classics, Comics, Contemporary, Cookbooks, Crime, Ebooks, Fantasy, Fiction, Graphic Novels, Historical Fiction, History, Horror, Humor And Comedy, Manga, Memoir, Music, Mystery, Non Fiction, Paranormal, Philosophy, Poetry, Psychology, Religion, Romance, Science, Science Fiction, Self Help, Suspense, Spirituality, Sports, Thriller, Travel, Young Adult,
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the application of CRISPR/Cas9 system in genome editing

  1. 1. Introduction
  2. 2. CRISPR C: Clustered R: regularly I: interspaced S: short P: palindromic R: repeat
  3. 3. The CRISPR system - based on bacterial immune system - normally occurring in bacterial processes - first reported in 1987 for the Ecoli But at the same time Their function was unknown 1
  4. 4. The CRISPR system -In 2000 similar repeats were identified in other bacteria -In 2002 similar repeats were named CRISPR and a set of proteins was found to be associated with CRISPR repeats (Cas: CRISPR associated gene) 2
  5. 5. The CRISPR system -In 2005 research showed that some CRISPR spacers are derived from bacteriophage -In 2007 researcher could use spacer DNA to alter the resistance of s.thermophilus to phage attack 3
  6. 6. The CRISPR system and finally in 2015 : Doudna and Charpentier discovered that how bacteria use spacers in it’s immune system Jennifer Doudna Emmanuelle Charpentier4
  7. 7. 5
  8. 8. 6
  9. 9. 7
  10. 10. Stage 1: CRISPR spacer acquisition 8
  11. 11. Stage 2: CRISPR expression 9
  12. 12. Stage 3: interference Interference system rely on : -association of Cas protein with sgRNAs and making ribonucleoprotein Complex (RNP) 10
  13. 13. Stage 3: interference 11 RNP Complex
  14. 14. Stage 3: interference Interference system rely on : - PAM: protospacer adjacent motifs 12
  15. 15. Stage 3: interference 5'-NGG-3' 13
  16. 16. Stage 3: interference -Cas is an endonuclease enzyme which have two cleavage domain (DSB) - Viral target DNA will cleaving at 3 bp on PAM upstream - Multiple viral spacer acquisition = Multiple cleavage 14
  17. 17. 15
  18. 18. Application of CRISPR/Cas9 in genome editing - Endonucleases are usually 4-8 bp cutter - Animal and plant DNA length is around 1000-3000 Mb - So it’s will make a lot of unwanted band (digestion produces) 16
  19. 19. - Cas-sgRNA complex is a smart and programmable Nuclease As stated before : - RNP recognition sites is 17-20 nt (spacer lenght) 17
  20. 20. Cas-sgRNA complex can: - Make a DSB in any desired target location -So CRISPR system have a great potential to become a genome editing tool 18
  21. 21. First : we have to designing spacer 19
  22. 22. - tracrRNA : trans-activating CRISPR RNA - crRNA : CRISPR RNA dsRNA 20
  23. 23. 21
  24. 24. 22
  25. 25. 23
  26. 26. second : Cas9 principles 24
  27. 27. Streptococcus pyogenes Cas9 : -the standard cas9 used in researches - PAM seq : 5’ NGG 3’ Staphylococcus aureus Cas9 : -Smaller than s.pyogenes Cas9 - PAM seq : 5’ NNGRRT 3’ 25
  28. 28. In term of cleavage : -There is no different between cds or non-cds locations and this is one of CRISPR/Cas9 advantages 26
  29. 29. third : what is our purpose to making a DSB in target DNA 27
  30. 30. Knock in HDR : homology directed repair Knock out NHEJ : non homologous end joining or 28
  31. 31. Gene of Interest Protospacer Adjacent Motif (PAM) Target Sequence 29
  32. 32. Non-Homologous End Joining (NHEJ) and DNA repair pathway 30
  33. 33. Transferring crRNA:tracrRNA-Cas9 complex to the cell nucleus 31
  34. 34. Non – viral based gene delivery : -Lipid-Mediated Transfection - Calcium phosphate transfection - Electroporation ghfgh 32
  35. 35. viral based gene delivery : -Lentivirus -Adenovirus -Adeno-Associated Virus (AAV) - crispr plasmid (pCRISPR) 33
  36. 36. 34
  37. 37. 35
  38. 38. Correction of Mutations in Zygote stages of Human? We have more knowledge and techniques on Human Embryo than Monkey’s36
  39. 39. 37
  40. 40. pXK7-AtCas9-2 14488 bp Egfp complementary comple 2 NLS-At NLS-At Cas9 attB1 attB2 Kan Sm/SpR p35S T35S LB RB 3X FLAG 38
  41. 41. conclusion
  42. 42. Application of CRISPR/Cas9 - Knockout/Knockin Mouse generation : Traditional methods: at least 6~12 months CRISPR/Cas9 : 2 Months with ~90% of efficiency 39 - with cas9 nickase ability off-target effects will reducing to 0% - there is no need to backcrossing F1 lines with parent lines - Site-directed mutagenesis: Disease Model Generation -Curing genetic diseases like HIV and Cancer -Gene Activation / Repression by dCas9 - It’s changes is inheritable - low price
  43. 43. In Here, donor strand is a ssDNA 40

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