INTRODUCTION & INSPIRATION EASSIL Y Assemble d Rea ssemble d - - PowerPoint PPT Presentation

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INTRODUCTION & INSPIRATION EASSIL Y Assemble d Rea ssemble d - - PowerPoint PPT Presentation

Apr 01, 2023 549 likes •1.02k views

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

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Gene Editing Tool with

CRISPR-Ca s9 System

NFLS · NANJ ING

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INTRODUCTION & INSPIRATION

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EASSIL Y Assemble d Rea ssemble d Re pla ce d

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CONNECTORS

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REMOVE REPLACE REPAIR

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CRISPR-Ca s9 System

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

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TOOL K IT

gRNA

Upstrea m homologous a rms

Downstre a m homologous a rms Re cognition site

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  • 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
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THE

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

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Upstre a m Homologous Arm with Ta rget Gene

Ide ntifying seque nce A Ide ntifying seque nce B Ide ntifying se que nce C

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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

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

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gRNA-A (BBa _K 1 2 52 001 )

Ide ntifying se que nce A

TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACC AAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATAC GATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGAT ATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTT TAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTC GATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTAGAATTGATCAG AGGAACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAA CTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTAC AAAGTTGGCATTA

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gRNA-B (BBa _K 1 2 52 002 )

Ide ntifying se que nce B

TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACCA AGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGA TACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATT AGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAA AATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATT TCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGAGGAACCGGATCTTATA ATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTT GGCATTA

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gRNA-C (BBa _K 1 2 52 003)

Ide ntifying se que nce C

TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACC AAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATAC GATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGAT ATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTT TTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTT CGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTACGCACCAC GTGTGATTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTAT CAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT TCTAGACCCAGCTTTCTT GTACAAAGTTGGCATTA

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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

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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

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TOOL K IT

  • Re cycle And Re use
  • Unlimite d Time s Of

Re pla ceme nt

  • Unlimite d Sets Of

Compone nts

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

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  • BLAST
  • Ide ntify REPETITION
  • Spe cificity & Accura cy
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ADVANTAGES

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  • Flexibility
  • Precisely And Accurately Ta rgeting
  • Genetic Disea ses
  • Pro. Da niel Anderson
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WHY NOT RESTRICTION ENZYMES?

  • Limitations
  • Inside The Nuclei To Pe rform Post-tra nslationa l

Modifications

  • Recognitions Site
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WHY FROM GFP TO RFP?

  • Re pla ce

a mutate d

  • r

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.

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FUTURE PROSPECT

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  • 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.

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感謝聆聽 感謝聆聽

THANK YOU

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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）

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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）

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  • Greater flexibility in silencing
  • Enha ncing or repa iring gene

expression

  • The a bility to introduce la rger gene

fra gments

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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）

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  • Promoter
  • Sta rt editing sponta neously
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Build-up of the Pa rts

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Proof of Principle Experiments

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