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Review of CRISPR/Cas9

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A quick review of the CRISPR/Cas 9 I did for a Journal Club for a bioinformatician audience.

Published in: Science

Review of CRISPR/Cas9

  1. 1. CRISPR/Cas9 Thenewfrontierofgenome engineering 03.06.2015 JenniferA. Doudna & EmmanuelleCharpentier, Science346, 2014 DOI: 10.1126/science.1258096
  2. 2. Wikipedia GenomeeditingisatechniquewhereDNAis inserted,replacedorremovedfromagenome usingartificiallyengineered nucleases
  3. 3. 3 GenomeEditing
  4. 4. GenomeEditing Targetgenemutation Knockoutgene Studygenefunction 3
  5. 5. GenomeEditing Targetgenemutation Knockoutgene Studygenefunction Createtransgenicorganism Syntheticbiology 3
  6. 6. GenomeEditing Targetgenemutation Knockoutgene Studygenefunction Createtransgenicorganism Syntheticbiology Genetherapy 3
  7. 7. 2phases 4
  8. 8. 2phases CreateaDouble-StrandedBreak (DSB) 4
  9. 9. 2phases CreateaDouble-StrandedBreak (DSB) Meganucleases ZFNs TALENS 4
  10. 10. 2phases CreateaDouble-StrandedBreak (DSB) Meganucleases ZFNs TALENS CRISPR/Cas9 4
  11. 11. 2phases CreateaDouble-StrandedBreak (DSB) Letthecellrepairmechanisms fixit Meganucleases ZFNs TALENS CRISPR/Cas9 4
  12. 12. 2phases CreateaDouble-StrandedBreak (DSB) Letthecellrepairmechanisms fixit Meganucleases ZFNs TALENS CRISPR/Cas9 Non-HomogolousEndJoining(NHEJ) HomologyDirectRepair(HDR) 4
  13. 13. DSBrepairmechanisms 5
  14. 14. DSBrepairmechanisms 5
  15. 15. DSBrepairmechanisms 5 JoindirectlytheDNAends Pronetoerrors
  16. 16. DSBrepairmechanisms 5 DNAtemplates UseatemplateDNA Errorfree
  17. 17. DSBrepairmechanisms Mechanismsofwhichpathwayistakenisnotfullyunderstood DNAtemplates 5
  18. 18. DSBrepairmechanisms Mechanismsofwhichpathwayistakenisnotfullyunderstood Techniquesexiststoinduceoneoranother DNAtemplates 5
  19. 19. CRISPR/Cas9 Acleverimmunesystem
  20. 20. HowbacteriapreventDNAinvasionfromviruses 7
  21. 21. HowbacteriapreventDNAinvasionfromviruses CRISPR Protospacer 7Sander et al, Nature Biotechnology, 32, 347-355, 2014
  22. 22. HowbacteriapreventDNAinvasionfromviruses CRISPR=ClusteredRegularly InterspacedShortPalindromic RepeatsCRISPR Protospacer Protospacer: InvadingDNAfrom viruses,phages,… 7Sander et al, Nature Biotechnology, 32, 347-355, 2014
  23. 23. HowbacteriapreventDNAinvasionfromviruses CRISPR=ClusteredRegularly InterspacedShortPalindromic RepeatsCRISPR Protospacer Protospacer: InvadingDNAfrom viruses,phages,… 7Sander et al, Nature Biotechnology, 32, 347-355, 2014
  24. 24. HowbacteriapreventDNAinvasionfromviruses tracRNA:transactivating CRISPRRNA tracRNA tracRNA 8Sander et al, Nature Biotechnology, 32, 347-355, 2014
  25. 25. HowbacteriapreventDNAinvasionfromviruses tracRNA:transactivating CRISPRRNA tracRNA tracRNA 8Sander et al, Nature Biotechnology, 32, 347-355, 2014
  26. 26. HowbacteriapreventDNAinvasionfromviruses tracRNA:transactivating CRISPRRNA tracRNA tracRNA 8Sander et al, Nature Biotechnology, 32, 347-355, 2014
  27. 27. HowbacteriapreventDNAinvasionfromviruses 9Sander et al, Nature Biotechnology, 32, 347-355, 2014
  28. 28. HowbacteriapreventDNAinvasionfromviruses 9Sander et al, Nature Biotechnology, 32, 347-355, 2014
  29. 29. HowbacteriapreventDNAinvasionfromviruses tracRNAwill activatenuclease Cas9 9Sander et al, Nature Biotechnology, 32, 347-355, 2014
  30. 30. HowbacteriapreventDNAinvasionfromviruses Cas9willsearchthe matchingforeignDNA tocreateDSBand promotedegradation tracRNAwill activatenuclease Cas9 9Sander et al, Nature Biotechnology, 32, 347-355, 2014
  31. 31. HowbacteriapreventDNAinvasionfromviruses 9Sander et al, Nature Biotechnology, 32, 347-355, 2014 NewforeignDNAisaddedtoCRISPRregions
  32. 32. Cas9mechanism 10
  33. 33. Cas9mechanism PAM PAMmotif(‘NGG’) mandatorytocleaveDNA 10
  34. 34. Cas9mechanism PAM PAMmotif(‘NGG’) mandatorytocleaveDNA 2cleavagedomains: HNHandRuvC-like RuvC HNH 10
  35. 35. Fromanimmunesystemto anengineeredtechnique
  36. 36. Asimplifiedsystem 12
  37. 37. Asimplifiedsystem FusionofcrRNAandtracrRNAtoasinglegRNA (20 bp) 12Sander et al, Nature Biotechnology, 32, 347-355, 2014
  38. 38. Asimplifiedsystem FusionofcrRNAandtracrRNAtoasinglegRNA (20 bp) 12Sander et al, Nature Biotechnology, 32, 347-355, 2014
  39. 39. Asimplifiedsystem FusionofcrRNAandtracrRNAtoasinglegRNA 2-componentsystem (20 bp) 12Sander et al, Nature Biotechnology, 32, 347-355, 2014
  40. 40. CRISPR/Cas9engineeringtool 13
  41. 41. CRISPR/Cas9engineeringtool DNAcleavageisbasedonRNA/DNApatternandnot anymoreonProtein/DNA 13
  42. 42. CRISPR/Cas9engineeringtool DNAcleavageisbasedonRNA/DNApatternandnot anymoreonProtein/DNA Changerequireonlyinthe20’firstnucleotidesofthe gRNA(formercrRNA) 13
  43. 43. CRISPR/Cas9engineeringtool DNAcleavageisbasedonRNA/DNApatternandnot anymoreonProtein/DNA Changerequireonlyinthe20’firstnucleotidesofthe gRNA(formercrRNA) PossibilityoftargetingmultipleDNAsequencesatonce 13
  44. 44. CRISPR/Cas9engineeringtool DNAcleavageisbasedonRNA/DNApatternandnot anymoreonProtein/DNA Changerequireonlyinthe20’firstnucleotidesofthe gRNA(formercrRNA) MuchmoreeasiertotargetDNAsequence PossibilityoftargetingmultipleDNAsequencesatonce 13
  45. 45. Somelimitations:off-target 14
  46. 46. Somelimitations:off-target Off-target:toleranceofCas9tomismatchesintheRNA guidesequence. 14
  47. 47. Somelimitations:off-target LimitedbyPAMmotif Off-target:toleranceofCas9tomismatchesintheRNA guidesequence. 14
  48. 48. Somelimitations:off-target LimitedbyPAMmotif Dependofmismatchslocations,lengths,compositions Off-target:toleranceofCas9tomismatchesintheRNA guidesequence. 14
  49. 49. Somelimitations:off-target LimitedbyPAMmotif Dependofmismatchslocations,lengths,compositions Off-target:toleranceofCas9tomismatchesintheRNA guidesequence. Difficulttopredict 14
  50. 50. VariantsoftheCas9systems 15
  51. 51. VariantsoftheCas9systems:nickase 15
  52. 52. VariantsoftheCas9systems:nickase OnlyonestrandoftheDNAwillbecut 15
  53. 53. VariantsoftheCas9systems:CRISPRi 16
  54. 54. VariantsoftheCas9systems:CRISPRi dCas9 16 Nocleavage domain
  55. 55. VariantsoftheCas9systems:CRISPRi Repressmultipletargetgeneswithreversibility dCas9 16 Nocleavage domain
  56. 56. VariantsoftheCas9systems:CRISPRi Repressmultipletargetgeneswithreversibility dCas9 FuseCas9withactivator/repressor/fluorescent domains 16 Nocleavage domain
  57. 57. TheRevolution
  58. 58. CRISPR/Cas9isthenew‘graphene’hype Jineketal,Science337,2012 18
  59. 59. CRISPR/Cas9isthenew‘graphene’hype Jineketal,Science337,2012 Now 18
  60. 60. CRISPR/Cas9isthenew‘graphene’hype Jineketal,Science337,2012 Now >1000publications 18
  61. 61. CRISPR/Cas9isthenew‘graphene’hype Jineketal,Science337,2012 Now >1000publications Dozensoforganisms tested 18
  62. 62. CRISPR/Cas9isthenew‘graphene’hype Jineketal,Science337,2012 Now >1000publications PatentWar Dozensoforganisms tested 18
  63. 63. CRISPR/Cas9isthenew‘graphene’hype Jineketal,Science337,2012 Now >1000publications Severalstart-ups created PatentWar Dozensoforganisms tested 18
  64. 64. SomeApplications
  65. 65. Examplesofcelltypesandorganismsmodified 20
  66. 66. DynamicImagingofgenomicloci Chen et al., Cell, 2013, Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system 21
  67. 67. DynamicImagingofgenomicloci Chen et al., Cell, 2013, Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system AttachedaGFPtoanuclease-deficient Cas9(dCas9) 21
  68. 68. DynamicImagingofgenomicloci Chen et al., Cell, 2013, Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system AttachedaGFPtoanuclease-deficient Cas9(dCas9) 21
  69. 69. DynamicImagingofgenomicloci Chen et al., Cell, 2013, Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system AttachedaGFPtoanuclease-deficient Cas9(dCas9) 21
  70. 70. Firstmonkeyswithcustomizedmutationsborn 22
  71. 71. Firstmonkeyswithcustomizedmutationsborn Niu et al., Cell, 2014, doi: 10.1016/j.cell.2014.01.027 22
  72. 72. Firstmonkeyswithcustomizedmutationsborn Niu et al., Cell, 2014, doi: 10.1016/j.cell.2014.01.027 22
  73. 73. Firstmonkeyswithcustomizedmutationsborn CRISPR/Cas9targeting ofmultiple genesinmonkeyembryos Ppar-gandRag1double mutationin monkeysinonestep Niu et al., Cell, 2014, doi: 10.1016/j.cell.2014.01.027 22
  74. 74. 23
  75. 75. Geneticallymodifyhumanembryos 23
  76. 76. Geneticallymodifyhumanembryos Liang et al., Protein Cell, 2015, CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes 23
  77. 77. Geneticallymodifyhumanembryos Liang et al., Protein Cell, 2015, CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes Triedtomutatethehumanβ-globin(HBB)genein‘non-viable’ embryos(β-thalassaemia) 23
  78. 78. Geneticallymodifyhumanembryos Liang et al., Protein Cell, 2015, CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes Triedtomutatethehumanβ-globin(HBB)genein‘non-viable’ embryos(β-thalassaemia) 7of86embryosweresuccessfullymutated Muchmorehigherratesofoff-targeting 23
  79. 79. Geneticallymodifyhumanembryos Liang et al., Protein Cell, 2015, CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes Triedtomutatethehumanβ-globin(HBB)genein‘non-viable’ embryos(β-thalassaemia) 7of86embryosweresuccessfullymutated Muchmorehigherratesofoff-targeting Raisehugeethicalconcerns… 23
  80. 80. Conclusion
  81. 81. Mostpowerful&easiesttooltogenomeediting
  82. 82. Mostpowerful&easiesttooltogenomeediting Limitationsduetooff-targeting
  83. 83. Mostpowerful&easiesttooltogenomeediting Limitationsduetooff-targeting WorksonanyDNA(bacteria,mouse,rise,humans…)
  84. 84. Mostpowerful&easiesttooltogenomeediting Limitationsduetooff-targeting WorksonanyDNA(bacteria,mouse,rise,humans…) Manyapplicationswiththedifferentvariants
  85. 85. Raiseethicalquestions:“Howcanweusethispowerfultool insuchawayastoensuremaximumbenefitwhile minimizing risks?”
  86. 86. Raiseethicalquestions:“Howcanweusethispowerfultool insuchawayastoensuremaximumbenefitwhile minimizing risks?”
  87. 87. Thankyou

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