CRISPR/Cas9 system CRISPR/Cas9 system, DNA, Type II CRISPR/Cas9 system, Cas9 ( ) guide RNA ( RNA ), guide RNA DNA, Cas9 DNA ( protospacer adaptor moti

Transcription

1 CRISPR/Cas9 system U TEL (02) TEL (05) TEL (03) TEL (06) TEL (04) TEL (07) 免付費客服專線

2 CRISPR/Cas9 system CRISPR/Cas9 system, DNA, Type II CRISPR/Cas9 system, Cas9 ( ) guide RNA ( RNA ), guide RNA DNA, Cas9 DNA ( protospacer adaptor motif ( PAM ) ), gene knock-out gene knock-in Streptococcus Pyogenes native type CRISPR Locus Cas9 guide RNA, target DNA PAM 3 4 bp, double-strand break, non-homologous end joining (NHEJ), knock out, donor vectors, homologous recombination, 1

3 CRISPR/Cas9 system PAM 5' NNNNNNNNNNNNNNNNNNNNNGG 3' DNA, PAM (5'-NGG-3'), PAM DNA oligonucleotides (sense antisense) 20 nt 5' TGTATGAGACCACNNNNNNNNNNNNNNNNNNNN 3' 3' ACTCTGGTGNNNNNNNNNNNNNNNNNNNNCAAA 5' DNA oligonucleotides duplex 5' TGTATGAGACCACNNNNNNNNNNNNNNNNNNNN 3' 3' ACTCTGGTGNNNNNNNNNNNNNNNNNNNNCAAA 5' grna screening system grna,clone linearized Cas9 vector Transform competent cells, (LB/ Kanamycin plate 50 ug/ml) positive clones Transfect all-in-one construct Surveyor assay PCR HR donor vector, homology recombination, 2

4 CRISPR/Cas9 system Single guide RNA PAM (NGG site) 20bp sgrna target site CRISPR grna Design Online Tools : guide RNA grna, Clontech grna screening system, grnas Guide-it Complete sgrna Screening System Guide it sgrna In Vitro Transcription Kit (< 3 hours) grna in vitro reaction >4 µg grna grna in vitro screening / transfection : ( Scaffold Template, In Vitro Transcription Buffer, T7 Polymerase Mix, RNase Free Water, NucleoSpin Gel and PCR Clean Up Kit, High Yield PCR EcoDry Premix) 3

5 1. Design/purchase a 69 nt forward primer 2. Greate template for in vitro transcription of sgrna 3. Production of sgrna by in vitro transcription Guide it sgrna Screening Kit in vitro cleavage assay, grna cas9 Kit Recombinant Cas9 Nuclease 1. Use PCR to generate a target for cleavage 2. In Vitro cleavage of target sequence recombinant Cas9 and synthesized sgrna 3. Separate cleavage products on an agarose gel Guide-it sgrna In Vitro Transcription Kit 10 Rxns Guide-it sgrna Screening Kit 30 Rxns Guide-it Complete sgrna Screening System 1 System 4

6 CRISPR/Cas9 system CRISPR/Cas9 system, CRISPR/Cas9 system CRISPR/Cas9 All-in-one Plasmid Cas9 RNA Protein system Cas9 SmartNuclease system Cas9 SmartNickase system Cas9 SmartNuclease TM and SmartNickase TM RNA System Guide-it CRISPR/Cas9 Gesicle Production System Lentiviral CRISPR/Cas9 Systems AAV CRISPR/Cas9 Systems All-in-one Lenti-Cas9 All-in-one AAV-Cas9 vector Two-Vector Lenti-Cas9 Two-Vector AAV-Cas9 vector HR donor vector,,! 5

7 CRISPR/Cas9 basal plasmid All-in-One system: Cas9 sgrna GFP All-in-One tagged vector, transfection, T7 promoter in vitro transcription (IVT), Cas9 grna mrna, in vivo human codon-optimized Cas9 nuclease (hspcas9/mutant Cas9), nuclease domains, chimeric crrna-tracrrna transcript scaffold single guide RNA (grna) Cas9 protein N C Nuclear localization signals (NLS), Cas9 protein Woodchuck virus post-transciptional regulatory element (WPRE),, mrna transcript promoters Cas9, Myc tag, recombinant Cas9 protein, singel guide RNA DNA,, linearized all-in one vector : gene knock-out gene knock-in ( with HR donor vector) 6

8 CRISPR/Cas9 system Cas9 SmartNuclease All-in-One Vectors Wild type Cas9 Cas9 SmartNuclease All-in-One vector, sgrna Cas9, double-strand break, NHEJ, deletion/ insertion, knockout, Validation Data for Cas9 SmartNuclease Vectors All- in-one vector, transfection efficiency Cas9 SmartNuclease All-in-One system CAS900A-1 EF1-hspCas9-H1-gRNA linearized SmartNuclease vector 10 rxn CAS920A-1 CAG-hspCas9-H1-gRNA linearized SmartNuclease vector 10 rxn CAS940A-1 CMV-hspCas9-H1-gRNA linearized SmartNuclease vector 10 rxn CAS960A-1 MSCV-hspCas9-H1-gRNA linearized SmartNuclease vector 10 rxn CAS980A-1 PGK-hspCas9-H1-gRNA linearized SmartNuclease vector 10 rxn CAS700A-1 EF1-T7-hspCas9-H1-gRNA linearized SmartNuclease vector 10 rxn CAS720A-1 CAG-T7-hspCas9-H1-gRNA linearized SmartNuclease vector 10 rxn CAS740A-1 CMV-T7-hspCas9-H1-gRNA linearized SmartNuclease vector 10 rxn Cas9 SmartNuclease All-in-One tagged vector CAS700G-1 EF1-T7-hspCas9-T2A-GFP-H1-gRNA linearized SmartNuclease vector 10 rxn CAS701R-1 EF1-T7-hspCas9-T2A-RFP-H1-gRNA linearized SmartNuclease vector 10 rxn CAS720G-1 CAG-T7-hspCas9-T2A-GFP-H1-gRNA linearized SmartNuclease vector 10 rxn CAS721R-1 CAG-T7-hspCas9-T2A-RFP-H1-gRNA linearized SmartNuclease vector 10 rxn CAS740G-1 CMV-T7-hspCas9-T2A-GFP-H1-gRNA linearized SmartNuclease vector 10 rxn CAS741R-1 CMV-T7-hspCas9-T2A-RFP-H1-gRNA linearized SmartNuclease vector 10 rxn 7

9 Cas9 SmartNickase All-in-One Vectors One mutant (D10A) Cas9 SmartNickase All-in-One vector,d10a mutation Cas9 nuclease activity, single-strand break, grnas nickase sense antisense, 5' overhang single-strand break, 8 bp, off target activity, NHEJ deletion/insertion,, knockout Paired grna Nickase Approach 3 grna 1 TAGCCGTAACGAATGGCAAT -5 Targeting site 5 3 CCN GGN ATCGGCATTGCTTACCGTTA Cas9 (D10A) Nickase TAGCCGTAACGAATGGCAAT CGTAAGCTTACGCGATGCAC Cas9 (D10A) Nickase GCATTCGAATGCGCTACGTG NGG NCC CGTAAGCTTACGCGATGCAC grna overhang Choose your grna1 from the anti-sense strand upstream of your targeting site Choose your grna2 from the sense strand downstream of your targeting site Cas9 SmartNickase All-in-One system CAS800A-1 Cas9 Nickase : EF1-hspCas9-nickase-H1-gRNA linearized SmartNickase vector 10 rxn CAS820A-1 Cas9 Nickase : CAG-hspCas9-nickase-H1-gRNA linearized SmartNickase vector (10 rxn) 10 rxn CAS840A-1 Cas9 Nickase : CMV-hspCas9-nickase-H1-gRNA linearized SmartNickase vector (10 rxn) 10 rxn CAS750A-1 Cas9 Nickase : EF1-T7-hspCas9-nickase-H1-gRNA linearized SmartNickase vector 10 rxn CAS770A-1 Cas9 Nickase : CAG-T7-hspCas9-nickase-H1-gRNA linearized SmartNickase vector 10 rxn CAS790A-1 Cas9 Nickase : CMV-T7-hspCas9-nickase-H1-gRNA linearized SmartNickase vector 10 rxn Cas9 SmartNickase All-in-One tagged vector CAS750G-1 Cas9 Nickase: EF1-T7-hspCas9-nickase-T2A-GFP-H1-gRNA linearized SmartNickase vector 10 rxn CAS751R-1 Cas9 Nickase: EF1-T7-hspCas9-nickase-T2A-RFP-H1-gRNA linearized SmartNickase vector 10 rxn CAS770G-1 Cas9 Nickase: CAG-T7-hspCas9-nickase-T2A-GFP-H1-gRNA linearized SmartNickase vector 10 rxn CAS771R-1 Cas9 Nickase: CAG-T7-hspCas9-nickase-T2A-RFP-H1-gRNA linearized SmartNickase vector 10 rxn CAS790G-1 Cas9 Nickase: CMV-T7-hspCas9-nickase-T2A-GFP-H1-gRNA linearized SmartNickase vector 10 rxn CAS791R-1 Cas9 Nickase: CMV-T7-hspCas9-nickase-T2A-RFP-H1-gRNA linearized SmartNickase vector 10 rxn 8

10 CRISPR/Cas9 system Precision X multiplex grna cloning kit multiple grna kit, Cas9 SmartNickase All-in-One Vectors grnas, vectors H1 U6 promoter blocks multiple grna cassettes Primer grna overlapping scaffold-promoter block, PCR promoter multiple grnas cassettes multiple grnas cassettes clone linear expression vector grnas CAS9-GRNA-KIT PrecisionX Multiplex grna Cloning Kit 10 rxn CAS700A-KIT Multiplex grna Kit + EF1-T7-hspCas9-H1-gRNA linearized SmartNuclease vector 10 rxn CAS720A-KIT Multiplex grna Kit + CAG-T7-hspCas9-H1-gRNA linearized SmartNuclease vector 10 rxn CAS740A-KIT Multiplex grna Kit + CMV-T7-hspCas9-H1-gRNA linearized SmartNuclease vector 10 rxn CAS750A-KIT Multiplex grna Kit + Cas9 Nickase: EF1-T7-hspCas9-nickase-H1-gRNA linearized SmartNickase vector 10 rxn CAS770A-KIT Multiplex grna Kit + Cas9 Nickase: CAG-T7-hspCas9-nickase-H1-gRNA linearized SmartNickase vector 10 rxn CAS790A-KIT Multiplex grna Kit + Cas9 Nickase: CMV-T7-hspCas9-nickase-H1-gRNA linearized SmartNickase vector 10 rxn 9

11 Cas9 SmartNuclease Gene disruption/replacement Knockout of a luciferase gene and replacement with RFP HEK293 reporter cell line luciferase gene, Cas9 system luciferase gene, HR Donor Vector ( luciferase gene homologous sequences) homologous recombination, luciferase RFP EF1-hspCas9-H1-Luc-gRNA SmartNuclease Vector Luciferase reporter cell line Targeting data Luciferase Assay Surveyor Assay Luciferase gene replacement with RFP Luciferase, HR donor Luciferase gene RFP 10

12 CRISPR/Cas9 system Gene repair Removing a stop codon in an EGFP gene EGIP (Enhanced Green Fluorescent Inhibited Protein ) reporter cell line,aavs1 53 bp EGIP, stop codon EGIP, Cas9 System target AAVS1, EGFP HR Donor Vector homologous recombination, EGFP EF1-hspCas9-H1-Luc-gRNA SmartNuclease Vector EGIP reporter cell line EGFP, Targeting data Surveyor AAVS1 Assay EGFP Restoration Images 11

13 Lentiviral CRISPR/Cas9 Systems SBI Lentiviral CRISPR/Cas9 Systems All-in-One format: Cas9 grna Two vector format: Cas9, grna Lentivirus,5' LTR 3' LTR Human codon-optimized Cas9 nuclease (hspcas9/mutant Cas9), nuclease domains, chimeric crrna-tracrrna transcript scaffold guide RNA (grna) Cas9 protein N C Nuclear localization signals (NLS), Cas9 protein Woodchuck virus post-transciptional regulatory element (WPRE),, mrna transcript promoter Cas9, Myc tag, recombinant Cas9 protein, linearized vector transfection,, gene editing, stable Cas9 editing cell lines in vivo, 12

14 CRISPR/Cas9 system Two-Vector Cas9-Lentiviruses Efficiently Knockout Stable RFP Reporter Gene HEK293T Two Vector Cas9-Lentiviruses Cas9-Puro RFP grna, grna knock out RFP, MSCV-hspCas9-T2A-Puro(Cat #CASLV125VA-1) EF1-Blasticidin-H1-gRNA(Cat #CASLV500PA-B) RFP, Knockout CRISPR/Cas9 Lenti-system, stem cell cancer cell lines Transduce Stem Cells with Cas9-Lentivirus MSCV-hspCas9-EF1-copGFP (Cat #CASLV100VA-1) Transduce Cancer Cell Lines with Cas9-Lentivirus CMV-hspCas9-T2A-Puro (Cat #CASLV100VA-1) CMV-hspCas9-T2A-Puro (Cat #CASLV100VA-1) All-in-one Lentivector Two-Vector Lentivector(gRNA) grna Cloning and Expression Lentivectors Two-Vector Lentivector(Cas9/Nickase) WT Cas9/Nickase Dual Promoter EF1-GFP Format Lentivectors WT Cas9/Nickase T2A-Puro Format Lentivectors 13

15 All-in-one CRISPR/Cas9 Lentivectors CASLV300PA-1 CASLV320PA-1 CASLV400PA-1 CASLV420PA-1 CRISPR/Cas9 grna Lentivectors CMV-hspCas9-T2A-Puro-H1-gRNA linearized all-in-one SmartNuclease Lentivector Plasmid MSCV-hspCas9-T2A-Puro-H1-gRNA linearized all-in-one SmartNuclease Lentivector Plasmid Cas9 Nickase: CMV-hspCas9(D10A)-T2A-Puro-H1-gRNA linearized all-in-one SmartNuclease Lentivector Plasmid Cas9 Nickase: MSCV-hspCas9(D10A)-T2A-Puro-H1-gRNA linearized all-in-one SmartNuclease Lentivector Plasmid two vector CRISPR/Cas9 Lentivectors 10 rxn 10 rxn 10 rxn 10 rxn CASLV500PA-B EF1-Blasticidin-H1-gRNA linearized SmartNuclease Lentivector Plasmid 10 rxn CASLV501PA-G EF1-GFP-H1-gRNA linearized SmartNuclease Lentivector Plasmid 10 rxn CASLV502PA-R EF1-RFP-H1-gRNA linearized SmartNuclease Lentivector Plasmid 10 rxn CASLV510PA-B EF1-Blasticidin-U6-gRNA linearized SmartNuclease Lentivector Plasmid 10 rxn CASLV511PA-G EF1-GFP-U6-gRNA linearized SmartNuclease Lentivector Plasmid 10 rxn CASLV512PA-R EF1-RFP-U6-gRNA linearized SmartNuclease Lentivector Plasmid 10 rxn CRISPR/Cas9 Lentivector Plasmids & Kits CASLV100PA-1 CMV-hspCas9-T2A-Puro SmartNuclease Lentivector Plasmid CASLV100PA-KIT CMV-hspCas9-T2A-Puro SmartNuclease Lentivector Plasmid + LentiStarter Packaging Kit 1 Kit CASLV105PA-1 CMV-hspCas9-EF1-GFP SmartNuclease Lentivector Plasmid CASLV105PA-KIT CMV-hspCas9-EF1-GFP SmartNuclease Lentivector Plasmid + LentiStarter Packaging Kit CASLV120PA-1 MSCV-hspCas9-T2A-Puro SmartNuclease Lentivector Plasmid CASLV120PA-KIT MSCV-hspCas9-T2A-Puro SmartNuclease Lentivector Plasmid + LentiStarter Packaging Kit CASLV125PA-1 MSCV-hspCas9-EF1-GFP SmartNuclease Lentivector Plasmid CASLV125PA-KIT MSCV-hspCas9-EF1-GFP SmartNuclease Lentivector Plasmid + LentiStarter Packaging Kit CASLV200PA-1 Cas9 Nickase : CMV-hspCas9(D10A)-T2A-Puro SmartNickase Lentivector Plasmid CASLV200PA-KIT Cas9 Nickase : CMV-hspCas9(D10A)-T2A-Puro SmartNickase Lentivector Plasmid + LentiStarter Packaging Kit 1 Kit CASLV205PA-1 Cas9 Nickase : CMV-hspCas9(D10A)-EF1-GFP SmartNickase Lentivector Plasmid CASLV205PA-KIT Cas9 Nickase : CMV-hspCas9(D10A)-EF1-GFP SmartNickase Lentivector Plasmid + LentiStarter Packaging Kit 1 Kit CASLV220PA-1 Cas9 Nickase : MSCV-hspCas9(D10A)-T2A-Puro SmartNickase Lentivector Plasmid CASLV220PA-KIT Cas9 Nickase : MSCV-hspCas9(D10A)-T2A-Puro SmartNickase Lentivector Plasmid + LentiStarter Packaging Kit 1 Kit CASLV225PA-1 Cas9 Nickase : MSCV-hspCas9(D10A)-EF1-GFP SmartNickase Lentivector Plasmid CASLV225PA-KIT Cas9 Nickase : MSCV-hspCas9(D10A)-EF1-GFP SmartNickase Lentivector Plasmid + LentiStarter Packaging Kit 1 Kit 1 Kit 1 Kit 1 Kit 14

16 CRISPR/Cas9 system AAV CRISPR/Cas9 Systems SBI AAV Cas9 system, in vivo AAV cargo limit, Staphylococcus aureus sacas9 sacas9 spcas9, spcas9 1kb, AAV grna,sacas9 PAM sequences NNGRRT(R:A/G),gRNA 21 nt - 23 nt AAV CRISPR/Cas9 Systems All-in-One format: sacas9 grna Two vector format: sacas9, grna : AAV,L-ITR R-ILTR Human codon-optimized Cas9 nuclease (sacas9), nuclease domains, chimeric crrna-tracrrna transcript scaffold guide RNA ( grna ) EF1a promoter sacas9, U6 promoter grna, linearized vector transfection,, gene editting genome,,, in vivo, 15

17 Removing a stop codon in an EGFP gene Enhanced Green Fluorescent Inhibited Protein (EGIP) reporter cell line, stop codon GFP, AAV CRISPR/Cas9 systems HR donor stop codon, GFP EGIP reporter cell line GFP Cell pellet lysate Plasmid Supernatant precipitate Two-vector System All-in-one system AAV-Cas9 All-in-one system AAV-Cas9 Two-vector System All-in-one CRISPR/Cas9 AAV vectors CASAAV100PA-1 EF1a-hsaCas9-U6-gRNA(SA) linearized all-in-one SmartNuclease AAV Plasmid 10 rxn Two vector CRISPR/Cas9 AAV vectors CASAAV200PA-1 EF1a-hsaCas9 SmartNuclease AAV Plasmid CASAAV300PA-1 EF1-RFP-U6-gRNA(SA) linearized SmartNuclease AAV Plasmid 10 rxn 16

18 CRISPR/Cas9 system Cas9 RNA/Protein System Cas9 SmartNuclease and SmartNickase RNA System RNA-directed Cas9 System,SBI Cas9 mrna Cas9 protein, ready-to-use linearized T7 grna cloning vector, T7 promoter grna, in vitro transcription (IVT) RNA RNA,, in vivo, CRISPR-Cas9 RNA System All-in-One Vector System!! HR Integration at AAVS1 Images GFP Fluorescence Bright Field Phase CAS500A-1 Transfection-ready Cas9 SmartNuclease mrna (Eukaryotic version) 20 µg CAS502A-1 Transfection-ready Cas9 SmartNuclease mrna (Prokaryotic version) 20 µg CAS504A-1 Transfection-ready Cas9 SmartNickase mrna (Eukaryotic Nickase mutant version) 20 µg CAS510A-1 Linearized T7 grna SmartNuclease vector 10 rxn CAS510A-KIT T7 grna SmartNuclease Synthesis Kit (includes CAS510A-1 & T7 IVT synthesis reagents) 1 Kit CAS510A-PR Extra T7 grna PCR primer mix (5 um) 50 assays CAS530G-1 Transfection-ready hspcas9-t2a-gfp SmartNuclease mrna (wildtype Cas9 with GFP marker) CAS531R-1 Transfection-ready hspcas9-t2a-rfp SmartNuclease mrna (wildtype Cas9 with RFP marker) CAS534G-1 Transfection-ready hspcas9-nickase-t2a-gfp SmartNickase mrna (Cas9 Nickase mutant with GFP marker) CAS535R-1 Transfection-ready hspcas9-nickase-t2a-rfp SmartNickase mrna (Cas9 Nickase mutant with RFP marker) MR700A-2 mrnaexpress GFP transcript 20 ug 20 µg MR800A-2 mrnaexpress RFP transcript 20 ug 20 µg CAS400A-1 Purified Cas9 protein 50 µg CAS400A-KIT Cas9 protein and T7 grna SmartNuclease Synthesis Kit 50 µg 17

19 Delivery of Cas9-sgRNA to a Broad Range of Cell Types Guide-it CRISPR/Cas9 Gesicle Production System System Overview 1. gesicle packaging mix target-specific guide RNA plasmid glycoproteins 293T producer cells idimerize small ligand, Cas9-sgRNA ribonucleoprotein complex gesicle CherryPicker red fluorescent protein (RFP), Cas9-sgRNA ribonucleoprotein complex gesicle gesicle, (RFP) Cas9-sgRNA gesicles gesicles, Cas9-sgRNA complex (RFP) Cas9 protein nuclear localization signal (NLS) dimerizer ligand Cas9-sgRNA complex 18

20 CRISPR/Cas9 system Gesicles? Gesicles Cas9 protein single guide RNA (sgrna) cell-derived nanovesicles, Gesicles? Cas9 protein target-specific sgrna Gesicle plasmid-based delivery! Knockout of ZsGreen1 in Multiple Cell Types Gesicles containing Cas9-sgRNA protein complexes outperform plasmid transfection in harder-to-transfect cell types. Cell lines were created that contained an integrated ZsGreen1 fluorescent protein expression cassette. In this system, successful Cas9- mediated cleavage can be measured by loss of ZsGreen1 expression. These cell lines were treated with gesicles loaded with Cas9- sgrna protein complexes (with the sgrna generated against ZsGreen1), and then analyzed by flow cytometry. Cas9-sgRNA protein complex delivery and ZsGreen1 knockout via gesicles was efficient and comparable to plasmid-based delivery in easier-to-transfect cell types (left graph) and surpassed the results achieved via plasmid-based delivery in harderto-transfect cell types (right graph). 19

21 (genome integration) (off-target effects) plasmid viral delivery Cas9 protein, Gesicles delivery Cas9-sgRNA complex, EMX1 Off-target 4 Control Gesicles Plasmid Control Gesicles Plasmid Target sequence EMX1: GAGTCCGAGCAGAAGAAGAAGGG Potential off-target sequence: GAGTCTAAGCAGAAGAAGAAGAG PAM site 0% 14% 16% 0% 0% 26% Cleavage indicates Cas9-mediated editing EMX1 gene CCTGAGTCCGAGCAGAAGAA GAAGGGCTCCCAT GGACTCAGGCTCGTCTTCTT CTTCCCGAGGGTA EMX1 gene EMX1 sgrna1 Off-target 4 CATAGTAGACAAGAGTCTAAGCAGAAGAA GAAGAGAGCCAC GTATCATCTGTTCTCAGATTCGTCTTCTT CTTCTCTCGGTG OT4 EMX1 off target 4 Plasmid transfection Cas9 Gesicles CCTGAGTCCGAGCAGAAGAA GAAGGGCTCCCAT CCTGAGTCCGAGCAGAAGA- GAAGGGCTCCCAT CCTGAGTCCGAGCAGAAG GGCTCCCAT CCTGAG AAGGGCTCCCAT CCTGAGTCCGAGCAGAAG-- GAAGGGCTCCCAT CCTGAGTCCGAGCAGAAGAAAGAAGGGCTCCCAT CCTGAGTCCGAGCAGAAGAAAGAAGGGCTCCCAT CCTGAGTCCGAGCAGAAGAAAGAAGGGCTCCCAT CCTGAGTCCGAGCAGAAGAA GAAGGGCTCCCAT CCTGAGTCCGAGCAGAAG GGCTCCCAT CCTGAGTCCGAGCAGAAGAA -AAGGGCTCCCAT CCTGAGTCCGAGCAGAAGAA CAT CCTGAGTCCGAGCAGAAGAA G---GGCTCCCAT CCTGAGTCCGAGCAGAAGAAAGAAGGGCTCCCAT CCTGACTCCGAGCAGAAGAAAGAAGGGCTCCCAT CCTGAGTCCGAGCAGAAGAAAGAAGGGCTCCCAT CATAGTAGACAAGAGTCTAAGCAGAAGAA GAAGAGAGCCAC CCAC CATAGTAGACAAGAGTCTAAGCAGAAGA GAGCCAC CATAGTAGACAAGAGTCTAAGCAGAA--A GAAGAGAGCCAC CATAGTAGACAAGAGTCTA GAAGAGAGCCAC CATAGTAGACAAGAGTCTAAGCAGAAGAAAGAAGAGAGCCAC CATAGTAGACAAGAGTCTAAGCAGAAGAAAGAAGAGAGCCAC CATAGTAGACAAGAGTCTAAGCAGAAGAAAGAAGAGAGCCAC CATAGTAGACAAGAGTCTAAGCAGAAGAA GAAGAGAGCCAC CATAGTAGACAAGAGTCTAAGCAGAAGAA GAAGAGAGCCAC CATAGTAGACAAGAGTCTAAGCAGAAGAA GAAGAGAGCCAC CATAGTAGACAAGAGTCTAAGCAGAAGAA GAAGAGAGCCAC CATAGTAGACAAGAGTCTAAGCAGAAGAA GAAGAGAGCCAC CATAGTAGACAAGAGTCTAAGCAGAAGAA GAAGAGAGCCAC CATAGTAGACAAGAGTCTAAGCAGAAGAA GAAGAGAGCCAC CATAGTAGACAAGAGTCTAAGCAGAAGAA GAAGAGAGCCAC No off-target editing when using gesicles The use of gesicles decreases off-target effects. HEK 293T cells were either simultaneously cotransfected with plasmids encoding Cas9 DNA and a sgrna against EMX1, or treated with gesicles loaded with Cas9-sgRNA ribonucleoprotein complexes. After 72 hr, the EMX1 gene and a potential off-target locus (offtarget 4) were analyzed using the Guide-it Mutation Detection Kit (Cat. # ). With the gesicles, no off-target effect could be detected (top panel). Sequencing data for the different clones were aligned with the underlined wild-type sequence, revealing a range of deletions and insertions (indels; highlighted in red). Gesicles correctly edited the EMX1 gene with no off-target effects, whereas plasmid cotransfection resulted in indels in both the target site, EMX1, as well as off-target site 4 (bottom panel) Guide-it CRISPR/Cas9 Gesicle Production System( # #632616) 1 System pguide-it-sgrna1 Vector System 1 System Guide-it CRISPR/Cas9 Gesicle Packaging Set 10 Rxns Gesicle Producer 293T Cell Line 1 ml 20

22 CRISPR/Cas9 system Cas9 Cas9 polyclonal antibody CRISPR/ Cas9 Cas9 Protein, Cas9 antibody Cas9 RT-PCR primer Cas9 Protein wild-type Cas9, Nickase (D10A, H840A), Cas9 double mutant protein :Western blot analysis Immunofluorescence Cas9 ELISA assays Immunoprecipitation hspcas9 Cas9 RT-PCR primer set Cas9 nuclease Cas9 Nickase and Cas9 double mutant mrna primers,primer set 1:219 bp primer set 2 :122 bp CAS9-AB-1 Cas9 Polyclonal Antibody 25 µg CAS9-PR-1 Cas9 RT-PCR Primer Set (50ul at 5uM for each primer set) 50 reactions 21

23 HR Targeting Vectors CRISPR/Cas9, single guide RNA DNA, DNA, double-stand break (DSB) NHEJ,,, phenotype,, HR donor vectors homologous recombination (HR), HR donor vectors,, HR donor vector genome, knock-out knock-in Donor vector 1. marker (GFP RFP) luciferase 2. insulators, 3. LoxP sites, CRISPR/Cas9 DNA repair by NHEJ (Non-homologous End Joining) or HR (Homologous Recombination) Donor vector HR (homologous recombination) 1. :, gene knock-out knock-in 2., NHEJ mutation 3. donor vector marker knock-out/ knock-in, Cell with modification! Enrichment Genotyping Phenotype 22

24 CRISPR/Cas9 system Gene Knock-out positive selection ( Puromycin resistant gene ) negative selection (TK marker ), Puromycin HR donor vector, ganciclovir /fialuridine HR donor vector template HR110PA-1 donor vector, gene knock-out,hr RFP puromycin-resistent gene Gene Knock-in 23

25 Gene Tagging GFP fusion vectors Gene tagging with dual GFP and Luciferase markers IRES-GFP co-expression vectors 24

26 CRISPR/Cas9 system Example of Target using Cas9 tagging marker HR donor CRISPR/Cas9 system, homologous arms clone HR donor, tagging marker fusion gene knock out HR110PA-1 Gene Knock-Out HR Targeting Vector [MCS1-EF1α-RFP-T2A-Puro-pA-MCS2] HR210PA-1 Gene Knock-Out HR Targeting Vector with TK selection [MCS1-LoxP-EF1α-GFP-T2A- Puro-P2A-hsvTK-pA-LoxP-MCS2] HR410PA-1 Gene Knock-Out HR Targeting Vector [MCS1-EF1a-GFP-T2A-Puro-pA-MCS2] HR510PA-1 Gene Knock-Out HR Targeting Vector [MCS1-EF1a-RFP-T2A-Hygro-pA-MCS2] HR700PA-1 Gene Knock-Out HR Targeting Vector w/dual Selection Markers (GFP+Puro) and Negative Selection (TK) Against Random Integration HR710PA-1 Gene Knock-Out HR Targeting Vector w/dual Selection Markers (RFP+Hygro) and Negative Selection (TK) Against Random Integration HR720PA-1 Gene Knock-Out HR Targeting Vector w/single Selection Marker (Blasticidin) and Negative Selection (TK) Against Random Integration gene knock in HR100PA-1 Basic HR Targeting Vector [MCS1-LoxP-MCS2-MCS3-pA-LoxP-MCS4] for Gene Knock- In/Out gene tagging HR120PA-1 GFP-Fusion HR Targeting Vector [GFP-pA-LoxP-EF1α-RFP-T2A-Puro-pA-LoxP-MCS] HR130PA-1 T2A-GFP Co-Expression HR Targeting Vector [T2A-GFP-pA-loxP-EF1α-RFP-T2A-PuropA-LoxP-MCS] HR150PA-1 GFP-T2A-Luciferase Co-Expression HR Targeting Vector [GFP-T2A-Luc-pA-loxP- EF1α-RFP-T2A-Puro-pA-LoxP-MCS] HR180PA-1 IRES-GFP Co-Expression HR Targeting Vector [IRES-GFP-pA-loxP-MCS1-EF1α-RFP- T2A-Puro-pA-LoxP-MCS2] HR220PA-1 GFP-Fusion HR Targeting Vector [GFP-pA-LoxP-EF1a-RFP-T2A-Hygro-pA-LoxP-MCS] 25

27 CRISPR/Cas9 system CRISPR/Cas9 Cas9 SmartNuclease AAVS1 kit AAVS1 (PPP1R2C locus) 19, safe harbor genomic locus,,, AAVS1 SBI Cas9 SmartNuclease AAVS1 kit, AAVS1 safe harbor genomic locus, AAVS1 Donor Vectors, homologous recombination, : Cas9 SmartNuclease AAVS1-gRNA Targeting Vector, AAVS1,, : CRISPR/Cas9 AAVS1, AAVS1 copy number:, AAVS1 donor vector GFP Puro marker, AAVS1 SAFE HARBOR AAVS1-gRNA Cas9 AAVS1-gRNA Targeting Vector AAVS1 Donor Vector AAVS1-gRNA Targeting vector AAVS1- CAS601A-1 Cas9 SmartNuclease AAVS1-gRNA Targeting Vector [EF1a promoter] GE620A-1 AAVS1 Safe Harbor Targeting Vector All-Purpose Donor (AAVS1-SA-puro-MCS) GE622A-1 AAVS1 Safe Harbor Targeting Vector GOI Knock-in Donor (AAVS1-SA-puro-EF1-MCS) GE624A-1 AAVS1 Safe Harbor Targeting Vector Reporter Knock-in Donor (AAVS1-SA-puro-MCS-GFP) GE602A-1 AAVS1 Safe Harbor cdna/mirna Donor Vector [paavs1d-pgk.mcs-ef1a.copgfppuro] GE603A-1 AAVS1 Safe Harbor Positive Control Donor Vector [paavs1d-cmv.rfp-ef1a.copgfppuro] 26

28 Nucleofector TM Nucleofactor TM,,,, Easy :, Fas t :,, High efficiency :, protocol 4D 96 well shuttle NFIIb Nucleofector TM Family HT Nucleofector TM device 27

29 Large-Scale Transfection~ Nucleofector LV Unit :, 10 9 : 700 Ex-vivo modification for cell therapy application (EX:genome editing,generation of CAR-T cells) Transient production of potential therapeutic proteins Antibodies for construct screening format 1 ml 1 ml Nucleocuvette Cartridge 1 ml filling volume Up to 1x10 8 cells Manual filling via sterile injection port 20 ml Up to 20 ml processing volume (in 1 ml steps) Up to 1x10 9 cells Automatic filling via reservoirs or bags Efficiency(%) Viability(PI neg cells; %), Transferability from Small to Large Scale. Comparison of various exemplary cell types transfected with pmaxgfp Vector in small volume (100 µl Nucleocuvette Vessels) or larger volumes (1 ml Nucleocuvette Cartridge or LV Nucleocuvette Cartridge) using the same conditions.transfection efficiency and cell viability were analyzed 24 hours post Nucleofection. 28

30 CRISPR/Cas9 system TEL (02) TEL (05) TEL (03) TEL (06) TEL (04) TEL (07) 免付費客服專線