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Gene Editing Tool with CRISPR-Ca s9 System NFLS NANJ ING INTRODUCTION & INSPIRATION EASSIL Y Assemble d Rea ssemble d Re pla ce d CONNECTORS REMOVE REPLACE REPAIR CRISPR-Ca s9 System A CRISPR-Ca s9 offe rs gre ate r flexibility

  1. Gene Editing Tool with CRISPR-Ca s9 System NFLS · NANJ ING

  2. INTRODUCTION & INSPIRATION

  3. EASSIL Y Assemble d Rea ssemble d Re pla ce d

  4. CONNECTORS

  5. REMOVE REPLACE REPAIR

  6. CRISPR-Ca s9 System

  7. A CRISPR-Ca s9 offe rs gre ate r flexibility with custom de signe d Guide RNAs.

  9. TOOL K IT gRNA Re cognition site Upstrea m homologous a rms Downstre a m homologous a rms

  12. • gRNA, CRISPR a nd new se que nce a re tra nsfe cte d in nucle us a s pla smid • gRNA tra nscripts, CRISPR tra nscripts • Ca s9 tra nscripts, CRISPR tra nscripts a nd tra nslate s

  17. THE

  18. gRNA-B gRNA-C gRNA-A thCF thAF thBF thAR thCR thBR ENZYME

  19. Upstre a m Homologous Arm with Ta rget Gene Ide ntifying seque nce A Ide ntifying se que nce C Ide ntifying seque nce B

  20. Downstre a m Homologous Arm with Ta rget Ge ne Ide ntifying se que nce A Ide ntifying seque nce B Ide ntifying se que nce C

  21. gRNA-B gRNA-C gRNA-A thCF thAF thBF thAR thCR thBR ENZYME

  22. gRNA-A (BBa _K 1 2 52 001 ) Ide ntifying se que nce A TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACC AAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATAC GATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGAT ATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTT TAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTC GATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTAGAATTGATCAG AGGAACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAA CTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTAC AAAGTTGGCATTA

  23. gRNA-B (BBa _K 1 2 52 002 ) Ide ntifying se que nce B TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACCA AGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGA TACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATT AGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAA AATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATT TCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGAGGAACCGGATCTTATA ATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTT GGCATTA

  24. gRNA-C (BBa _K 1 2 52 003) Ide ntifying se que nce C TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACC AAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATAC GATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGAT ATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTT TTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTT CGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTACGCACCAC GTGTGATTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTAT CAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT TCTAGACCCAGCTTTCTT GTACAAAGTTGGCATTA

  25. thAF (BBa _K 1 2 52 004) TGAGTTTTAAAAAGAATAATTTAATCGATGATGATTCGGAGGCTACTGTCGCCGAAT CGGATTCGTTTTAAATATGTCTTACAGTATCACTACTCCATCTCAGTTCGTGTTCTTG TCATCAGCGTGGGCCGACCCAATAGAGTTAATTAATTTATGTACTAATGCCTTAGGA AATCAGTGTAGAATTGATCAGAGGAACCGG thBF (BBa _K 1 2 52 005) TGAGTTTTAAAAAGAATAATTTAATCGATGATGATTCGGAGGCTACTGTCGCCGAAT CGGATTCGTTTTAAATATGTCTTACAGTATCACTACTCCATCTCAGTTCGTGTTCTTG TCATCAGCGTGGGCCGACCCAATAGAGTTAATTAATTTATGTACTAATGCCTTAGGA AATCAGTGAGGAACCGGATCTTATAATCGG thCF (BBa _K 1 2 52 00 6 ) TGAGTTTTAAAAAGAATAATTTAATCGATGATGATTCGGAGGCTACTGTCGCCGAAT CGGATTCGTTTTAAATATGTCTTACAGTATCACTACTCCATCTCAGTTCGTGTTCTTG TCATCAGCGTGGGCCGACCCAATAGAGTTAATTAATTTATGTACTAATGCCTTAGGA AATCAGTGTACGCACCACGTGTGATTACGG

  26. thAR (BBa _K 1252007) GT AGAAT T GAT CAGAGGAACCGGT T CAAACACAACAAGCT CGAACT GT CGT T C AAAGACAAT T CAGT GAGGT GT GGAAACCT T CACCACAAGT AACT GT T AGGT T CC CT GACAGT GACT T T AAGGT GT ACAGGT ACAAT GCGGT AT T AGACCCGCT AGT CA CAGCACT GT T AGGT GCAT T CGACACT AGAAAT AGAAT thBR (BBa _K 1252008) GAGGAACCGGAT CT T AT AAT CGGT T CAAACACAACAAGCT CGAACT GT CGT T C AAAGACAAT T CAGT GAGGT GT GGAAACCT T CACCACAAGT AACT GT T AGGT T CC CT GACAGT GACT T T AAGGT GT ACAGGT ACAAT GCGGT AT T AGACCCGCT AGT CA CAGCACT GT T AGGT GCAT T CGACACT AGAAAT AGAAT thCR (BBa _K 1252008) GT ACGCACCACGT GT GAT T ACGGT T CAAACACAACAAGCT CGAACT GT CGT T C AAAGACAAT T CAGT GAGGT GT GGAAACCT T CACCACAAGT AACT GT T AGGT T CC CT GACAGT GACT T T AAGGT GT ACAGGT ACAAT GCGGT AT T AGACCCGCT AGT CA CAGCACT GT T AGGT GCAT T CGACACT AGAAAT AGAAT

  27. TOOL K IT • Re cycle And Re use • Unlimite d Time s Of Re pla ceme nt • Unlimite d Sets Of Compone nts

  28. Ba sic Pla smid Ba ckbone Psb1 c3 Add Prefix And Suffix

  29. • BLAST • Ide ntify REPETITION • Spe cificity & Accura cy

  30. ADVANTAGES

  31. • Flexibility • Precisely And Accurately Ta rgeting • Genetic Disea ses • Pro. Da niel Anderson

  33. WHY NOT RESTRICTION ENZYMES? • Limitations • Inside The Nuclei To Pe rform Post-tra nslationa l Modifications • Recognitions Site

  34. WHY FROM GFP TO RFP? • Re pla ce a mutate d or mis-inserted pa rt without the ne ed to reconstruct the e ntire se que nce. • Inserting a bla nk pa rt to de lete a ce rta in ma lfunctioning pa rt.

  35. FUTURE PROSPECT

  36. • Inse rting a ce rta in for ce rta in condition • Inserting the a rms on both side of the pathoge n ge nome Re pla ce or sile nce it in vivo Multiple sets of compone nts • Multiple re pla ce at one time or multiple re pla ce s at multiple time s.

  38. 感謝聆聽 感謝聆聽 THANK YOU

  39. Guide -RNA • gRNA Specific Recognition Sequence Tra nscription Vectors • BioBrick cloning site prefix （ EcoR I ， Xba I ） a nd suffix （ Spe I ， Pst I ） • Ba sic pla smid ba ckbone pSB1 C3 （ BBa _J 04450 ）

  40. thDNA Ta rget Re combina nt Se que nces of Ve ctors • Upstre a m a nd Downstre a m Homologous Arm with Ta rget Ge ne • BioBrick cloning site pre fix （ EcoR I ， Xba I ） a nd suffix （ Spe I ， Pst I ） • ba sic pla smid ba ckbone pSB1 C3 （ BBa _J 04450 ）

  41. • Greater flexibility in silencing • Enha ncing or repa iring gene expression • The a bility to introduce la rger gene fra gments

  42. Compatibility • With iGEM data ba se in mind, the kit follows a modula r de sign a nd a ims to fa cilitate future iGEM proje cts with gre ate r flexibility a nd compatibility in ge ne e diting. • BioBrick cloning site pre fix （ EcoR I ， Xba I ） a nd suffix （ Spe I ， Pst I ） • Ba sic pla smid ba ckbone pSB1 C3 （ BBa _J 04450 ）

  43. • Promoter • Sta rt editing sponta neously

  44. Build-up of the Pa rts

  45. Proof of Principle Experiments

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