CRISPR-cas9 试剂盒说明书

Datasheet for RKO/Cas9-hyg Stable Cell LineCatalog number:SL539Product:RKO cell line stably expressing CRISPR Cas9nucleaseDescription:This product is a cell line stably expressing the CRISPR Cas9nuclease.This cell line also expresses the hygromycin resistance gene.In combination withseparately transfected or transduced single guide RNAs(sgRNAs),this cell linewill sustain double-strand DNA breaks(DSBs)at targeted genome sites.Wefound the basal Cas9activity by T7Endonuclease I assay when sgRNA expressionat high levels using lentivirus transduction.This cell line can be used in vitro forgene knockout,transgene knockin,mutagenesis,transgene integration,or othergenome editing-related applications.Quantity:1vial of2x106cells;frozenShipping conditions:Dry iceStorage conditions:Liquid nitrogen vapor phase.Remove the item from the dry ice packaging and check all items for damage and leakage.Place immediately into storage at orbelow-1400C,preferably into the liquid nitrogen vapor phase,until use. Transgene integration:Source of parental line:RKOOrganism:Homo sapiens,humanTissue:colonCell type:epithelialQuality control:>95%viability before freezing.All cells were tested and found to be free ofmycoplasma,bacteria,viruses,and other toxins.Safety instructions:To ensure safety,protective gloves,clothing,and a face mask should be wornwhen handling frozen vials.Some leakage may occur into the vial during storage.The liquid nitrogen will be converted to gas upon thawing.Due to the nature ofnitrogen gas,pressure may build within the vial and possibly result in the vialexploding or losing its cap.This may cause flying debris.Thawing procedure:The vial of cells should be thawed in a370C water bath with gentle agitation.For optimal performance,the vial should be thawed in under two minutes.Ensurethat the cap of the vial did not loosen upon thawing,and re-tighten as needed.Spray the vial with70%EtOH and wipe ing aseptic technique,addthe contents of the vial to9ml of complete growth medium(without selection).Centrifuge for5min.at250x g.Aspirate the medium,being careful not todisturb the pellet.Resuspend in10mL of complete growth medium,and placeinto a culture vessel of your choice.Only add selection to the medium after24hours in culture.Culture conditions:Complete Growth MediumThe base medium for this cell line is RPMI1640.For optimal growth andmaintenance of selection,add the following components to the base medium:fetal bovine serum to a final concentration of10%.SelectionHygromycin to a final concentration of400µg/mLCulture temperature:370C with5%CO2Subculture:Rinse the cells with PBS without cations,digest cells with0.25%(w/v)Trypsin-EDTA(0.53mM)solution and split at1:3to1:12ratio.Cryopreservation:Freeze slowly in complete growth medium supplemented with 5%(v/v)DMSO.Mycoplasma:Negative (MycoAllert Mycoplasma Detection Kit from Lonza)Product QC:T7Endonuclease I (T7E1)AssayHUWE Site T7E1AssaysgRNA targeting to HUWE gene was transduced into RKO/Cas9-hyg Stable Cell Line by transduction.HUWE gene was cut by CAS9expressed inside the cells and repaired through NHEJ with mutation.A 525bp HUWE gene fragment from PCR was then tested by T7Endonuclease I (T7E1)Assay.The T7E1cleavage will results in two additional bands:one ~192bp and the other ~333bp.HUWE-F:AAGGGTGGGACGTGAACTTGTC HUWE-R:AGAATCTTCCCATCAACCCT100bp--200bp--300bp--400bp--500bp--600bp--700bp--M PCR T7E1Citation of product:If use of this item results in a publication,please use this information:CRISPR Cas9RKO/Cas9-hyg Stable cell line(SL539,GeneCopoeia,Inc.,Rockville,MD).Limited Use LicenseA limited use license is granted to the Buyer of the Product.The Product shall be used by the Buyer for internalresearch purposes only.The Product is expressly not designed,intended,or warranted for use in humans or for therapeutic or diagnostic use.The Product must not be resold,repackaged or modified for resale,or used tomanufacture commercial products without prior written consent from GeneCopoeia.This Product should be used in accordance with NIH guidelines developed for recombinant DNA and genetic e of any part of the Product constitutes acceptance of the above terms.Copyright©2018GeneCopoeia,Inc.C9SCL-DS-052218GeneCopoeia,Inc.9620Medical Center Drive,#101Rockville,MD20850USATel:301-762-0888;Fax:301-762-3888Email:***********************Web:。

慢病毒Cas9表达系统使用说明本说明书用于：¾构建Cas9蛋白表达细胞系¾在Cas9蛋白表达细胞系中敲除目的基因适用于以下产品货号货号pLV‐Cas9载体系列 CR2001，CR2002pLV‐Cas9‐Nick载体系列 CR2003，CR2004pGR载体系列 CR2011~CR2013pGR‐EGFP载体系列 CR2014~CR2016北京英茂盛业生物科技有限公司北京市昌平区沙河镇青年创业大厦B‐916Tel：010‐62495135Emai：order@Web site：目录1、产品简介 (2)1.1CRISPR/gRNA基因敲除原理 (2)1.2慢病毒Cas9表达系统特点 (2)2、Cas9表达慢病毒制备 (3)2.1试剂准备 (3)2.2简要实验流程 (4)2.3实验前准备 (4)2.4病毒制备步骤 (5)2.4 PEG纯化慢病毒 (6)3、筛选Cas9表达稳定细胞株 (7)3.1 试剂 (7)3.2 实验前准备 (7)3.3 筛选细胞系实验步骤 (8)3.4 Cas9表达细胞系检测 (9)4、用pTYNE载体对Cas9表达细胞系进行验证 (10)4.1验证Cas9蛋白表达细胞系 (10)4.2验证Cas9Nicknase蛋白表达细胞系 (11)5、pGR和pGR‐EGFP载体构建 (13)5.1 pGR和pGR‐EGFP载体图谱 (13)5.2 靶点设计 (13)5.3 pGR载体构建步骤 (14)附录1 用到的产品 (17)附录2 引物列表 (17)11、产品简介1.1CRISPR/gRNA基因敲除原理CRISPR (clustered, regularly interspaced, short palindromic repeats)是一种来自细菌降解入侵的病毒DNA或其他外源DNA的免疫机制。

在该机制中，Cas蛋白（CRISP‐associated protein）含有两个核酸酶结构域，可以分别切割两条DNA链。

Datasheet for A549/Cas9AAVS1Cell LineCatalog number:SL504(SCL-76321-G2)Product:A549cell line stably expressing CRISPR Cas9nucleaseDescription:This product is a cell line stably expressing the CRISPR Cas9nuclease.Cas9is integrated at the human AAVS1Safe Harbor locus(also known as PPP1R2C).Thiscell line also expresses copGFP and the hygromycin resistance gene.Incombination with separately transfected or transduced single guide RNAs(sgRNAs),this cell line will sustain double-strand DNA breaks(DSBs)at targetedgenome sites.This cell line can be used in vitro for gene knockout,transgeneknockin,mutagenesis,transgene integration,or other genome editing-relatedapplications.Quantity:1vial of2x106cells;frozenShipping conditions:Dry iceStorage conditions:Liquid nitrogen vapor phase.Remove the item from the dry ice packaging and check all items for damage and leakage.Place immediately into storage at orbelow-1400C,preferably into the liquid nitrogen vapor phase,until use. Transgene integration:Source of parental line:A549Organism:Homo sapiens,humanTissue:Lung carcinomaCell type:EpithelialQuality control:>95%viability before freezing.All cells were tested and found to be free ofmycoplasma,bacterial,viruses,and other toxins.Safety instructions:To ensure safety,protective gloves,clothing,and a face mask should be wornwhen handling frozen vials.Some leakage may occur into the vial during storage.The liquid nitrogen will be converted to gas upon thawing.Due to the nature ofnitrogen gas,pressure may build within the vial and possibly result in the vialexploding or losing its cap.This may cause flying debris.Thawing procedure:The vial of cells should be thawed in a370C water bath with gentle agitation.For optimal performance,the vial should be thawed in under two minutes.Ensurethat the cap of the vial did not loosen upon thawing,and re-tighten as needed.Spray the vial with70%EtOH and wipe ing aseptic technique,addthe contents of the vial to9ml of complete growth medium(without selection).Centrifuge for5min.at125x g.Aspirate the medium,being careful not todisturb the pellet.Resuspend in10mL of complete growth medium,and placeinto a culture vessel of your choice.Only add selection to the medium after24hours in culture.Culture conditions:Complete Growth MediumThe base medium for this cell line is F12Medium.For optimal growth andmaintenance of selection,add the following components to the base medium:fetal bovine serum to a final concentration of10%.SelectionHygromycin to a final concentration of800µg/mLCulture temperature:370C with5%CO2Subculture:Rinse the cells with PBS without cations,digest cells with0.25%(w/v)Trypsin-EDTA(0.53mM)solution and split at1:3to1:10ratio.Cryopreservation:Freeze slowly in complete growth medium supplemented with5%(v/v)DMSO. Mycoplasma:Negative(MycoAllert Mycoplasma Detection Kit from Lonza)1.T7Endonuclease I(T7E1)AssaysgRNA targeting to B2M gene was transduced into A549/CAS9AAVS1cell line by lenti-particals LPP-HCP000395-b-LvSG02.B2M gene was cut by CAS9expressed inside the cells and repaired through NHEJ with mutation.A669bp B2M gene fragment from PCR was then tested by T7E1Assay.The T7E1cleavage will results in two additional bands:one~291bp and the other~378bp.Mutation was generated for B2M gene by CAS9/CRISPR and NHEJ2.Junctional PCR(to confirm the Cas9gene integration into AAVS1site)(1)5'Junctional PCR(one primer from chromosomal outside of the5'homology arm region,the other primer from the Cas9-plasmid region)Junction-PCR5'F:ccggaactctgccctctaacJunction-PCR5'R:gcgtacttggcatatgatPredict product length：1100bp(2)3'Junctional PCR(one primer from chromosomal outside of the3'homology arm region,the other primer from the Cas9-plasmid region)Junction-PCR3'F:agctatctggtctcccttccJunction-PCR3'R:tcctgggataccccgaagagPredict product length：1250bpCitation of product:If use of this item results in a publication,please use this information: CRISPR Cas9stable A549cell line(SL504;GeneCopoeia,Inc.,Rockville,MD).Limited Use LicenseA limited use license is granted to the Buyer of the Product.The Product shall be used by the Buyer for internalresearch purposes only.The Product is expressly not designed,intended,or warranted for use in humans or for therapeutic or diagnostic use.The Product must not be resold,repackaged or modified for resale,or used tomanufacture commercial products without prior written consent from GeneCopoeia.This Product should be used in accordance with NIH guidelines developed for recombinant DNA and genetic e of any part of the Product constitutes acceptance of the above terms.Copyright©2018GeneCopoeia,Inc.C9SCL-DS-032018GeneCopoeia,Inc.9620Medical Center Drive,#101Rockville,MD20850USATel:301-762-0888;Fax:301-762-3888Email:***********************Web:。

RESULTS
Most available CRISPR/Cas9 editing methods relying on DNA and mRNA based tools do not allow efficient genome editing in hiPSC and results in the screening of hundreds of colonies to find a correctly edited hiPSC line. Common approaches to facilitate isolation of genome edited hiPSC clones is to introduce a positive selection marker with the desired edit into the genome to enrich for correctly edited hiPSCs, after which hiPSCs are seeded at limited dilutions to isolate potentially single cell derived clones. While the use of Cas9 protein now allows efficient editing in hiPSCs, its reliability is highly target and cell dependent. We thus developed a stably Cas9 expressing hiPSC line as a backbone to easily generate hiPSC-based disease models (Figure 2). Through lentiviral delivery we introduced Cas9 into hiPSCs (Cas9 hiPSCs) and after selection a stable cell line was obtained (Figure 2). The cell line generation process did not affect the biology of the hiPSCs as the cells maintained their hiPSC morphology, normal karyotype and expression of pluripotent markers (Figure 2). We furthermore evaluated the differentiation potential of the Cas9 hiPSCs both through spontaneous differentiation as embryoid bodies and directed differentiation to cardiomyocytes and dopaminergic neurons (Figure 3).