Gene Editing Tool with

Transcription

1 Gene Editing Tool with CRISPR-Cas9 System NFLS NANJING

2 INTRODUCTION & INSPIRATION

3 EASSILY Assembled Reassembled Replaced

4 CONNECTORS

5 REMOVE REPLACE REPAIR

6 CRISPR-Cas9 System

7 ACRISPR-Cas9 offers greater flexibility with custom designed Guide RNAs.

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9 TOOL KIT Recognition site grna Upstream homologous arms Downstream homologous arms

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12 grna, CRISPR and new sequence are transfected in nucleus as plasmid grna transcripts, CRISPR transcripts Cas9 transcripts, CRISPR transcripts and translates

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17 THE

18 grna-a grna-b grna-c thaf thbf thcf thar thbr thcr ENZYME

19 Upstream Homologous Arm with Target Gene Identifying sequence A Identifying sequence C Identifying sequence B

20 Downstream Homologous Arm with Target Gene Identifying sequence A Identifying sequence B Identifying sequence C

21 grna-a grna-b grna-c thaf thbf thcf thar thbr thcr ENZYME

22 grna-a (BBa_K ) Identifying sequence A TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACC AAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATAC GATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGAT ATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTT TAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTC GATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTAGAATTGATCAG AGGAACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAA CTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTAC AAAGTTGGCATTA

23 grna-b (BBa_K ) Identifying sequence B TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACCA AGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGA TACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATT AGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAA AATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATT TCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGAGGAACCGGATCTTATA ATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTT GGCATTA

24 grna-c (BBa_K ) Identifying sequence C TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACC AAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATAC GATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGAT ATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTT TTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTT CGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTACGCACCAC GTGTGATTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTAT CAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT TCTAGACCCAGCTTTCTT GTACAAAGTTGGCATTA

25 thaf (BBa_K ) TGAGTTTTAAAAAGAATAATTTAATCGATGATGATTCGGAGGCTACTGTCGCCGAAT CGGATTCGTTTTAAATATGTCTTACAGTATCACTACTCCATCTCAGTTCGTGTTCTTG TCATCAGCGTGGGCCGACCCAATAGAGTTAATTAATTTATGTACTAATGCCTTAGGA AATCAGTGTAGAATTGATCAGAGGAACCGG thbf (BBa_K ) TGAGTTTTAAAAAGAATAATTTAATCGATGATGATTCGGAGGCTACTGTCGCCGAAT CGGATTCGTTTTAAATATGTCTTACAGTATCACTACTCCATCTCAGTTCGTGTTCTTG TCATCAGCGTGGGCCGACCCAATAGAGTTAATTAATTTATGTACTAATGCCTTAGGA AATCAGTGAGGAACCGGATCTTATAATCGG thcf (BBa_K ) TGAGTTTTAAAAAGAATAATTTAATCGATGATGATTCGGAGGCTACTGTCGCCGAAT CGGATTCGTTTTAAATATGTCTTACAGTATCACTACTCCATCTCAGTTCGTGTTCTTG TCATCAGCGTGGGCCGACCCAATAGAGTTAATTAATTTATGTACTAATGCCTTAGGA AATCAGTGTACGCACCACGTGTGATTACGG

26 thar (BBa_K ) GTAGAATTGATCAGAGGAACCGGTTCAAACACAACAAGCTCGAACTGTCGTTC AAAGACAATTCAGTGAGGTGTGGAAACCTTCACCACAAGTAACTGTTAGGTTCC CTGACAGTGACTTTAAGGTGTACAGGTACAATGCGGTATTAGACCCGCTAGTCA CAGCACTGTTAGGTGCATTCGACACTAGAAATAGAAT thbr (BBa_K ) GAGGAACCGGATCTTATAATCGGTTCAAACACAACAAGCTCGAACTGTCGTTC AAAGACAATTCAGTGAGGTGTGGAAACCTTCACCACAAGTAACTGTTAGGTTCC CTGACAGTGACTTTAAGGTGTACAGGTACAATGCGGTATTAGACCCGCTAGTCA CAGCACTGTTAGGTGCATTCGACACTAGAAATAGAAT thcr (BBa_K ) GTACGCACCACGTGTGATTACGGTTCAAACACAACAAGCTCGAACTGTCGTTC AAAGACAATTCAGTGAGGTGTGGAAACCTTCACCACAAGTAACTGTTAGGTTCC CTGACAGTGACTTTAAGGTGTACAGGTACAATGCGGTATTAGACCCGCTAGTCA CAGCACTGTTAGGTGCATTCGACACTAGAAATAGAAT

27 TOOL KIT Recycle And Reuse Unlimited Times Of Replacement Unlimited Sets Of Components

28 Basic Plasmid Backbone Psb1c3 Add Prefix And Suffix

29 BLAST Identify REPETITION Specificity & Accuracy

30 ADVANTAGES

31 Flexibility Precisely And Accurately Targeting Genetic Diseases Pro. Daniel Anderson

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33 WHY NOT RESTRICTION ENZYMES? Limitations Inside The Nuclei ToPerform Post-translational Modifications Recognitions Site

34 WHY FROM GFP TO RFP? Replace a mutated or mis-inserted part without the need to reconstruct the entire sequence. Inserting a blank part to delete a certain malfunctioning part.

35 FUTURE PROSPECT

36 Inserting acertain for certain condition Inserting the arms on both side of the pathogen genome Replace or silence it in vivo Multiple sets of components Multiple replace at one time or multiple replaces at multiple times.

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38 感謝聆聽 THANK YOU

39 Guide-RNA grna Specific Recognition Sequence Transcription Vectors BioBrick cloning site prefix(ecor I,Xba I)and suffix(spe I,Pst I) Basic plasmid backbone psb1c3(bba_j04450)

40 thdna Target Recombinant Sequences of Vectors Upstream and Downstream Homologous Arm with Target Gene BioBrick cloning site prefix(ecor I,Xba I)and suffix(spe I,Pst I) basic plasmid backbone psb1c3(bba_j04450)

41 Greater flexibility in silencing Enhancing or repairing gene expression The ability to introduce larger gene fragments

42 Compatibility With igem database in mind, the kit follows a modular design and aims to facilitate future igem projects with greater flexibility and compatibility in gene editing. BioBrick cloning site prefix(ecor I,Xba I)and suffix (Spe I,Pst I) Basic plasmid backbone psb1c3(bba_j04450)

43 Promoter Start editing spontaneously

44 Build-up of the Parts

45 Proof of Principle Experiments

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