[PDF] ゲノム編集ツール Edit-R CRISPR-Cas9
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- おさむ さわなか
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1 imagination at work ゲノム編集 RNAi 遺伝子発現 ゲノム編集ツール Edit-R CRISPR-Cas9 テクニカル マニュアル Dharmacon ダーマコン
2 1 CRISPR-Cas CRISPR-Cas...3 CRISPR-Cas Edit-R CRISPR-Cas Edit-R SMARTCas9 Expression Plasmid Cas9...4 Edit-R trans-activating CRISPR RNA tracrrna...5 Edit-R CRISPR RNA crrna...5 crrna...6 Dharmacon CRISPR RNA Configurator crrna...6 crrna Edit-R CRISPR-Cas Edit-R CRISPR-Cas Edit-R CRISPR-Cas Appendix...12 Blast R Edit-R CRISPR-Cas9 Nuclease Expression Plasmid Dharmacon Edit-R SMARTCas9 Expression Plasmid Dharmacon Edit-R tracrrna crrna Dharmacon DharmaFECT Duo Transfection Reagent FAQ Evrogen...19
3 1 CRISPR-Cas9 CRISPR-Cas CRISPR clustered regularly interspaced palindromic repeats -Cas CRISPR-associated proteins DNA DNA CRISPR CRISPR CRISPR CRISPR RNA CRISPR RNA crrna crrna CRISPR RNA crrna RNA crrna DNA crrna CRISPR-associated Cas DNA protospacer-adjacent motif PAMBhaya, 2011 CRISPR-Cas9 CRISPR-Cas Cas Streptococcus pyogenes S. Pyogenes CRISPR-Cas9 S. pyogenes DNA PAM 3 Jinek, Cas9 2 crrna CRISPR CRISPR RNA 3 trans-activating CRISPR RNA CRISPR RNA crrna 1 tracrrna Deltcheva, 2011 DNA NHEJ HR NHEJ Mali, 2014; Sampson, 2014 DNA S. pyogenes CRISPR-Cas9 ゲノムDNA PAM crrna tracrrna 1 Cas9 tracrrna crrna crrna DNA PAM 5' 3
4 2 Edit-R CRISPR-Cas9 Edit-R CRISPR-Cas9 3 1 Cas tracrrna 3 crrna S.pyogenes CRISPR-Cas9 DharmaFECT Duo Transfection Reagent 3 2 ターゲットcrRNA の 設 計 crrnaの 配 列 を 1 遺 伝 子 あたり3~5 種 類 カスタム 設 計 します Webで 注 文 1. カスタム 設 計 したcrRNA 2. tracrrna 3. Cas9ヌクレアーゼを 発 現 用 プラスミド 4. DharmaFECT Duo トランスフェクション 試 薬 納 品 後 トランスフェクション Cas9ヌクレアーゼ 発 現 用 プラスミド tracrrna:crrna 2 Edit-R CRISPR-Cas9 培 養 2~3 日 間 ゲノムが 編 集 された 細 胞 を FACS 法 で 濃 縮 培 養 3~4 日 間 ゲノムが 編 集 された 細 胞 を 抗 生 物 質 選 択 で 濃 縮 dharmacon.gelifesciences.co.jp > SMARTCas9 Expression Plasmid (mkate2) 使 用 時 SMARTCas9 Expression Plasmid (Puro R ) 使 用 時 Edit-R SMARTCas9 Expression Plasmid Cas9 S. pyogenes Cas9 Csn1 mkate2 Evrogen Puro R Cas9 3A Cas9 3' 5' mkate2 Puro R C Cas9 3B SMARTCas9 Expression Plasmid DNA 4
5 mkate2 T2A Cas9 pa Edit-R SMARTCas9 SMARTCas9 (mkate2) Expression Plasmid SMARTchoice プロモーター hcmv mcmv hef1α mef1α PGK CAG SMARTchoice プロモーター hcmv mcmv hef1α mef1α PGK CAG mkate2 T2A Cas9 pa Puro R T2A Cas9 pa SMARTCas9 (mkate2) Expression Plasmid SMARTCas9 (Puro R ) Expression Plasmid SMARTchoice プロモーター Cas9 mkate2 T2A pa hcmv mcmv hef1α mef1α PGK CAG Puro R SMARTCas9 (Puro R ) Expression Plasmid T2A S. pyogenes Cas9 tracrrna crrna 588 nm 653 nm Cas9 pa mkate2 Cas9 BGH 3A Edit-R SMARTCas9 Expression Plasmid Cas9 Puro R T2A pa S. pyogenes Cas9 tracrrna crrna Puro R Cas9 BGH hcmv mcmv hef1α mef1α PGK CAG 1α 1α β 3B Edit-R SMARTCas9 Expression Plasmid hcmv Cas9 12 Appendix Edit-R trans-activating CRISPR RNA tracrrna S. pyogenes tracrrna Jinek, 2012 HPLC RNA Edit-R CRISPR RNA crrna DNA 20 tracrrna S. pyogenes RNA DNA DNA PAM S. pyogenes PAM NGG 4 5
6 crrna crrna DNA PAM S. pyogenes NGG 5' 20 DNA RNA U6 5' G DNA 4 PAM 5' DNA Fu, 2013; Hsu, 2013; Wang, 2013 PAM 3 DNA Cas9 Dharmacon CRISPR RNA Configurator crrna crrna Dharmacon CRISPR RNA Configurator dharmacon. gelifesciences.com/gene-editing/edit-r/custom-crrna/ "I want to design a crrna" crrna crrna PAM 5' crrna crrna 20 crrna dharmacon.gelifesciences.com/gene-editing/edit-r/custom-crrna/ "Use my own crrna sequence" crrna 20 Dharmacon CRISPR RNA Configurator S. pyogenes 3' 42 crrna 4 4 PPIB chr PAM 5' crrna 20 Dharmacon CRISPR RNA Configurator DNA RNA 22 S. pyogenes 3' 42 crrna crrna 6
7 3 Edit-R CRISPR-Cas9 Edit-R CRISPR-Cas9 3 Cas9 Expression Plasmid DNA tracrrna crrna DharmaFECT Duo Transfection Reagent Edit-R CRISPR-Cas9 Edit-R CRISPR-Cas9 Web dharmacon.gelifesciences.com Edit-R SMARTCas9 Expression Plasmid 120 µg SMARTCas9 Expression Plasmid mkate2 Edit-R hcmv_mkate2-cas9 Expression Plasmid DNA (cat #U ) Edit-R mcmv_mkate2-cas9 Expression Plasmid DNA (cat #U ) Edit-R hef1α_mkate2-cas9 Expression Plasmid DNA (cat #U ) Edit-R mef1α_mkate2-cas9 Expression Plasmid DNA (cat #U ) Edit-R PGK_mKate2-Cas9 Expression Plasmid DNA (cat #U ) Edit-R CAG_mKate2-Cas9 Expression Plasmid DNA (cat #U ) SMARTCas9 Expression Plasmid Puro R Edit-R hcmv_puro R -Cas9 Expression Plasmid DNA (cat #U ) Edit-R mcmv_puro R -Cas9 Expression Plasmid DNA (cat #U ) Edit-R hef1α_puro R -Cas9 Expression Plasmid DNA (cat #U ) Edit-R mef1α_puro R -Cas9 Expression Plasmid DNA (cat #U ) Edit-R PGK_Puro R -Cas9 Expression Plasmid DNA (cat #U ) Edit-R CAG_Puro R -Cas9 Expression Plasmid DNA (cat #U ) Edit-R CRISPR Cas9 Nuclease Expression Plasmid Blast R 120 µg Dharmacon U Appendix mkate2 Transfection Optimization Plasmid 120 g Dharmacon U Appendix tracrrna 5 nmol 120 µg Dharmacon U crrna 20 nmol Dharmacon CTM-XXXXXX-XXX dharmacon.gelifesciences.com/gene-editing/ edit-r/custom-crrna/ DharmaFECT Duo Transfection Reagent Dharmacon T ml T ml T ml T ml 5 7
8 CellTiter-Blue Cell Viability Assay Promega G8081 Fisher Scientific BP S Fisher Scientific BP mm Tris ph 7.4 Edit-R CRISPR-Cas9 DharmaFECT Duo Transfection Reagent Cas9 Expression Plasmid DNA tracrrna crrna HEK293T ,000 HEK293T 100, ml ml % CO 2 1 Cas9 Expression Plasmid DNA Cas9 Expression Plasmid DNA tracrrna crrna RNA Cas9 RNA Cas ph mm Tris 1.2 ml Cas9 Expression Plasmid DNA 120 µg 100 ng/ µl Cas9 260 nm DNA 100 ng/µl 7. ph mm Tris 500 µl tracrrna 5 nmol 10 µm tracrrna 260 nm RNA 10 µm 8
9 8. ph mm Tris 2 ml crrna 20 nmol 10 µm crrna 260 nm RNA 10 µm ml tracrrna crrna 50 nm Cas9 Expression Plasmid DNA 1 1 µg mg/ml DharmaFECT Duo Transfection Reagent 120 µl 2 ml 60 µg/ml DharmaFECT Duo tracrrna 10 µm crrna 10 µm Cas9 100 ng/µl DharmaFECT Duo 60 µg/ml 1 Cas9 Expression Plasmid DNA Cas9 Expression Plasmid DNA tracrrna crrna HEK293T µl DharmaFECT Duo 50 µl DharmaFECT Duo 1 3 µg DharmaFECT Duo 100 µl µl 500 µl µl % CO Cas9 2 SMARTCas9 Expression Plasmid a. mkate2-cas9 Expression Plasmid DNA FACS mkate2-cas9 Expression Plasmid DNA FACS mkate2 3 9
10 mkate2 588 nm 633 nm Omega Optical QMAX-Red XF102-2 Texas Red b. Puro R -Cas9 Expression Plasmid DNA Puro R -Cas9 Expression Plasmid DNA % SURVEYOR Mutation Detection Kit Integrated DNA Technologies T7 Endonuclease Guschin, 2010; Reyon, 2012; Cong, 2013 DNA Reyon, Edit-R CRISPR-Cas9 Edit-R mkate2-cas9 Expression Plasmid DNA 588 nm 633 nm 70% 96 DharmaFECT Duo Transfection Reagent RNA RNA % DharmaFECT Duo Transfection Reagent DharmaFECT Duo Transfection Reagent µg/ml µl ng
11 µg/well A 100 ng 200 ng 100 ng 200 ng 0.3 B 100 ng 200 ng 100 ng 200 ng 0.4 C 100 ng 200 ng 100 ng 200 ng 0.5 D 100 ng 200 ng 100 ng 200 ng 0.6 E 100 ng 200 ng 100 ng 200 ng 0.7 F 100 ng 200 ng 100 ng 200 ng 0.8 G 100 ng 200 ng 100 ng 200 ng H DharmaFECT Duo Transfection Reagent A G ng 200 ng ng ng H % ph mm Tris 1.2 ml 120 µg 100 ng/µl Cas9 260 nm DNA 100 ng/µl 3. 7 DharmaFECT Duo µg/ml 1 mg/ml DharmaFECT Duo Transfection Reagent 10 µl 15 µl 20 µl 25µl 30 µl 35 µl 40 µl 500 µl DharmaFECT Duo µg 5 4. Cas9 10 µl 4 DharmaFECT Duo 10 µl 20 µl DharmaFECT Duo 20 DharmaFECT Duo 4 96 Cas9 100 ng/lµl DharmaFECT Duo µg/ ml 1 Edit-R mkate2-cas9 Expression Plasmid DNA 100 ng Edit-R mkate2-cas9 Expression Plasmid DNA 200 ng µl µl 100 µl µl 11
12 7. mkate2 mkate2 a b % DharmaFECT Duo 70% 9 8. SMARTCas9 Nuclease Expression Plasmid tracrrna crrna tracrrna crrna nm 25 nm mkate2 Edit-R mkate2-cas9 Expression Plasmid DNA tracrrna crrna 5 Appendix Blast R Edit-R CRISPR-Cas9 Nuclease Expression Plasmid S. pyogenes Cas9 Csn1 hcmv 40 SV40 Blast R Cas9 5 DNA Vector elements of the Edit-R Cas9 Nuclease Expression plasmid hcmv Cas9 phcsvblast-cas9 Blast R SV40 hcmv Cas9 SV40 Blast R hcmv mkate2 S. pyogenes Cas9 phcsvblast-mkate tracrrna crrna 40 Blast R Blast R SV40 5 Edit-R BlastR-Cas9 Nuclease Expression Plasmid 12
13 Vector elements of the Edit-R Cas9 Nuclease Expression plasmid hcmv Cas9 Edit-R CRISPR-Cas9 Nuclease Expression Plasmid Cas9 mkate2 mkate2 Transfection Optimization Plasmid 6 phcsvblast-cas9 Blast R mkate2 Transfection Optimization Plasmid SV40 hcmv mkate2 phcsvblast-mkate Blast R SV40 hcmv mkate2 588 nm 653 nm SV40 40 Blast R Blast R 6 Edit-R mkate2 Transfection Optimization Plasmid Dharmacon Edit-R SMARTCas9 Expression Plasmid ph mm Tris Dharmacon Edit-R tracrrna crrna ph mm Tris Dharmacon DharmaFECT Duo Transfection Reagent Web dharmacon.gelifesciences.com Service & Support Documents & Downloads Technical Resources RNAi & RNA Accell Stability Product Bulletin
14 FAQ tracrrna crrna Edit-R tracrrna MALDI-TOF HPLC crrna RNA 260 nm Dharmacon 2 -ACE crrna Dharmacon 2 -ACE RNA crrna 2 crrna crrna RNA tracrrna crrna RNA Edit-R Plasmid tracrrna crrna 4 5 tracrrna crrna RNA ph 7.4 RNA 4 5 Edit-R Cas9 Nuclease Expression Plasmid mkate Transfection Optimization Plasmid tracrrna crrna 2 4 RNA DNA Edit-R Gene Engineering System Edit-R Cas9 Expression plasmid DNA mkate2 Transfection Optimization plasmid DNA tracrrna crrna RNA RNA 260 nm = cm crrna tracrrna nm cm 10 mm 1 2 mm
15 Edit-R Cas9 Expression Plasmid DNA DNA DharmaFECT Duo Transfection Reagent DharmaFECT Web dharmacon.gelifesciences.com Edit-R Cas9 Gene Engineering Cas9 Edit-R Cas9 Expression Plasmid 1) Cas9 CMV Blast R Cas9 U ) FACS SMARTCas9-mKate2 Expression Plasmid 6 3) FACS SMARTCas9-Puro R Expression Plasmid 6 Cas9 Cas9 Cas9 mkate2 Cas9 Edit-R Cas9 Expression Plasmid SMARTCas9-mKate2 Expression Plasmid mkate2 15
16 Edit-R mkate2 Transfection Optimization Plasmid Edit-R Cas9 Nuclease Expression Plasmid Cas9 mkate2 Edit-R mkate2 Trasfection Optimization Plasmid SMARTCas9 Expression Plasmid SMARTCas9-mKate2 Expression Plasmid Edit-R tracrrna crrna Cas9 tracrrna crrna Edit-R Cas9 Expression Plasmid Cas9 crrna tracrrna (Streptococcus pyogenes) CRISPR-Cas9 Cas9 Cas9 Cas9 DNA tracrrna crrna 1 RNA sgrna RNA tracrrna crrna Edit-R crrna Edit-R sgrna DNA DharmaFECT Duo Edit-R DharmaFECT Duo DNA RNA DharmaFECT Duo Edit-R DNA Surveyor(IDT) T7EI(NEB) DNA DNA PCR CRISPR-Cas9 1. Cas9 NHEJ DNA 2. DNA PCR 3. CRISPR-Cas9 16
17 Edit-R Edit-R Edit-R Cas9 Edit-R RNA crrna tracrrna Streptococcus pyogenes CRISPR-Cas9 Cas9 Cas9 Cas9 DNA tracrrna crrna Edit-R RNA Cas9 mrna Cas9 tracrrna crrna Edit-R Cas9 Expression Plasmid Cas9 Cas9 mrna crrna tracrrna Streptococcus pyogenes CRISPR-Cas9 Cas9 Cas9 Cas9 Cas9 mrna DNA tracrrna crrna CRISPR-Cas9 CRISPR-Cas9 SNP B crrna SNP 17
18 D. Bhaya, M. Davison, et al. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu. Rev. Genet. 45, (2011). L. Cong, F. A. Ran, et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science. 339(6121), (2013). E. Deltcheva, K. Chylinski, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor Nuclease III. Nature. 471(7340), (2011). Y. Fu, J. D. Sander, et al. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. (2014). D.Y. Guschin, A. J. Waite, et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol. Biol. 649, (2010). F. Heigwer, G. Kerr, et al. E-CRISP: fast CRISPR target site identification. Nat. Methods. 11(2), (2014). P.D. Hsu, D. A. Scott, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31(9), (2013). M. Jinek, K. Chylinski, et al. A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 337(6096), (2012). P. Mali, L. Yang, et al. RNA-guided human genome engineering via Cas9. Science. 339(6121), (2013). N. K. Pyzocha, F. A. Ran, et al. RNA-Guided Genome Editing of Mammalian Cells. Methods Mol. Biol. 1114, (2014) D. Reyon, C. Khayter, et al. Engineering designer transcription activator-like effector nucleases (TALENs) by REAL or REAL-Fast assembly. Curr. Protoc. Mol. Biol. 100, (2012). T. R. Sampson, D. S. Weiss. Exploiting CRISPR/Cas systems for biotechnology. Bioessays. 36(1), (2014). T. Wang, J. J. Wei, et al. Genetic screens in human cells using the CRISPR-Cas9 system. Science. 343(6166), (2014). 18
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