Genome-CRISP™ human sgRNA Library User Manual
Contents
1. Screen plates for response of phenotype of interest PCR sequence individual sgRNA ATTTAGCA CCGGGAA Figure 7 High thoroughput screening using Cas9 stable cell line and pooled sgRNA libraries Genome CRISP CRISPR AE sgRNA MJ Human stable cell line expressing Cas9 nuclease gene Cas9 integration site Quantity Application Cell line Promoter For convenient Human 1 tube of 2 SIUC transfection or H1299 cido x 106 cells transduction of sgRNAs oe especially in high Human 1 tube of 2 eGR HEK293T Eli x 108 cells throughout applications Cas9 knockin clones for human AAVS1 safe harbor site Cas9 ARE Selection Application Promoter integration marker site DC C9NU 03 To knock in Cas9 Puro CBh AAVS1 Cas9 AAVS1 nuclease to human DC CONU 04 knockin donor AAVS1 Safe Hygro CBh AAVS1 clone Harbor site using Human AAVS1 safe harbor gene knockin kits CatNo Product Description Genome TALER human AAVS1 safe Includes AAVS1 TALEN pair TN AAVS1 SH AVS K100 Bebo EO A AAVS1 donor cloning vector DC DON SH01 kit 9 AAVS1 positive control donor DC RFP SHO1 knock in verification primer pairs HQPAVSHR Genome TALER Includes SH AVS K000 human AAVS1 safe AAVS1 TALEN pair TN AAVS1 harbor gene knock in AAVS1 positive control DC RFP SHO1 kit without donor knock in verification primer pairs HQPAVSHR Includes Genome CRISP TM AAVS1 All in one sgRNA Cas9 expression clone hum2. GeneCopoeia pre made Cas9 lentiviral particles Human stable cell line expressing Cas9 nuclease gene For packaging lentiviral particles For packaging lentiviral particles For Cas9 nuclease packaging lentiviral particles or generating stable cell line with randomly integrated Cas9 nuclease gene For co transduction with sgRNA lentiviral particles or generating stable cell line with randomly integrated Cas9 nuclease gene For convenient transfection or transduction of sgRNAs especially in high throughout applications Genome CRISP CRISPR human sgRNA libraries lll Getting Started A Prepare library for use The GeneCopoeia Genome CRISP CRISPR human sgRNaA libraries are available as pools in 3 different delivery formats Bacterial stock transfection ready DNA and lentiviral particles Follow these guidelines for the preparation of each format for use 1 Bacterial stocks For isolation of transfection ready DNA and or lentiviral particles Prepare by using the protocol below Note To best maintain even representation of each sgRNA in the library amplification of library bacterial stocks in liquid culture is not recommended Instead library bacteria should be amplified by spreading on LB ampicillin agar plates followed by harvesting the bacterial colonies by scraping One 10 cm plate usually yields at least 30 ug of plasmid DNA Miniprep columns can be used for plasmid extraction If larger yields of plasmid DN
3. Science 343 80 Zhou et al 2014 High throughput screening of a CRISPR Cas9 library for functional genomics in human cells Nature 509 487 Genome CRISP CRISPR human sgRNA libraries VII Limited Use License and Warranty Limited Use License The following terms and conditions apply to use of the Genome CRISPR human sgRNa libraries the Product If the terms and conditions are not acceptable the Product in its entirety must be returned to GeneCopoeia within 5 calendar days A limited End User license is granted to the purchaser of the Product The Product shall be used by the purchaser for internal research 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 to manufacture commercial products or deliver information obtained in service without prior written consent from GeneCopoeia This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research Use of any part of the Product constitutes acceptance of the above terms Limited Warranty GeneCopoeia warrants that the Product meets the specifications described in the accompanying Product Datasheet If it is proven to the satisfaction of GeneCopoeia that the Product fails to meet these specifications GeneCopoeia will replace the Product In the event a replacement cannot be provi
4. cosider the following 1 Know the minimum concentration of puromycin needed to kill sensitive cells See previous section B Transfection of Target Cells With DNA 2 Know the optimal MOI for your cell line If you don t know this information already then perform a titration by infecting cells with serial dilutions of lentiviral particles expressing pCRISPR LvSGO3 carrying a scrambled control sgRNA GeneCopoeia catalog CCPCTRO1 LvSGO3 B or CCPCTRO1 LvSG03 10 for bacterial stock or transfection ready plasmid DNA respectively LPP CCPCTRO1 LvSGOS for pre made lentiviral particles Cells with serial dilutions of these lentiviral particles can be visualized for mCherry fluoresence under a microscope Genome CRISP CRISPR human sgRNA libraries Day 1 Plate cells Plate 2 10 x 104 of the target cells per well in a 24 well plate 24 hours prior to viral transduction Use 0 5 mL of cell specific medium supplemented with 5 heat inactivated fetal bovine serum and penicillin streptomycin optional for each well Incubate the cells at 37 C with 5 CO overnight Note Make sure the cells reach 70 80 confluence at the time of transduction Actual cell number to be plated depends on the cell types Day 2 Transduce target cells For each well prepare 0 5 mL of virus suspension diluted in complete medium with Polybrene at a final concentration of 5 8 ug mL Note Use several dilutions of pseudoviral stock 0 1 uL to 100 uL We recom
5. Purified Neomycin Lentifect Lentiviral Particles LPP CP LvC9NU 02 100 100 ul x 1 vial gt 1x107TU mL CMV eGFP Neomycin Genome CRISP Cas9 nuclease lentiviral expression clones Cat No Promoter Reporter gene Selection marker CP LvC9NU 01 Cas9 nuclease lentiviral expression clone Neomycin CP LvC9NU 02 Cas9 nuclease lentiviral expression clone CMV eGFP Neomycin IndelCheck CRISPR TALEN insertion or deletion detection system E oo O besonpton O IndelCheck CRISPR TALEN ICPE 050 insertion or deletion detection system 50 rxns IndelCheck ICPE 200 CRISPR TALEN insertion or deletion detection system 200 rxns Includes target site PCR kit TPCR 050 and T7 endonuclease assay kit TENI 050 Includes target site PCR kit TPCR 200 and T7 endonuclease assay kit TENI 200 TPCR 050 Target site PCR kit 50 rxns Target site PCR kit 50 rxns TPCR 200 Target site PCR kit 200 rxns Target site PCR kit 20 rxns T7 endonuclease assay kit 50 Cleave mismatched PCR products using T7 TENI 050 rxns endonuclease to detect indel mutations TENI 200 T7 endonuclease assay kit 200 Cleave mismatched PCR products using T7 rxns endonuclease to detect indel mutations VI Genome CRISP CRISPR human sgRNA libraries References Shalem et al 2014 Genome scale CRISPR Cas9 knockout screening in human cells Science 343 84 Wang et al 2014 Genetic screens in human cells using the CRISPR Cas9 system
6. A are required the procedure can be scaled up using larger plates and or increased numbers of plates a For each tube of library bacteria pre warm one 10 cm LB plate containing ampicillin inverted at 37 C for 1 2 hours b Fully thaw the library bacterial stocks at room temperature then place on ice c Mix the library bacterial stocks thoroughly by tapping and inverting the tubes several times Do not vortex Note If multiple preparations of plasmid DNA are needed aliquot the library stocks to multiple tubes to avoid repeated freeze thaw cycles Store each aliquot at 80 C d Pipette 1 uL of each library stock into 1 mL of liquid LB medium Mix thoroughly e Spread 200 uL of each diluted library stock on each pre warmed LB ampicillin plate Ensure that the bacteria is spread over the plate as evenly as possible f Incubate the plates inverted at 37 C for 16 18 hours Note Each plate should contain 0 5 2 x 10 colonies Individual colonies not a lawn should be visible g Pipette 5 mL of LB medium onto each plate Scrape the colonies off with a cell spreader or scraper h Pipette liquid suspension containing the scraped bacterial cells into a 15mL tube i Repeat step g amp h j Centrifuge tubes at 4 000 rpm for 10 minutes k Proceed with plasmid DNA extraction according to the manufacturer s instructions for the plasmid preparation kit you are using Genome CRISP CRISPR human sgRNA libraries 2 Tr
7. Gen Cor 5 pia Expressway to Discovery Genome CRISPR CRISPR human SQRNA libraries User Manual GeneCopoeia Inc 9620 Medical Center Drive 101 Rockville MD 20850 USA 301 762 0888 866 360 9531 inquiry genecopoeia com www genecopoeia com 2015 GeneCopoeia Inc USER MANUAL Genome CRISP CRISPR human sgRNA libraries Table of Contents l IF OQUCH ORAS raia ea bd o DS A 3 Es SSO PING ANd STO RAGS aa inss sina sds eia a O SOL ESA DD Sa DS DG pi aa 5 WN GONG Started ias ias cada Dai UTC Candida when teu dl aaa ad rede il ea la 7 A Prepare lIDrary TOF USC ais 7 I s Bacienal STOCKS Poole sanirane SE O DDS SAD DS 7 2 Transfection ready DNA pooled cccccccccecccceeceeeeeeeeeeeeeeeueeeeeeeeeeeeeeaaeeees 8 3 Lentiviral particles pooled cc ecccceeseeeeeseuseseuseeeueeeueeeeueueeeeuseeueneuss 8 B Analyze sgRNA representation in the lIDrary ce eccccccceccseeeaeeeeeeeeeeeeeeeeeeeeeeeneeeeaees 8 C Start with a Cas9 expressing stable Cell LINC cece ceccceeeeceeeeueeesaneeeaeeeeaeeeeneees 9 D Consider your screening readout reter rea rre arena 10 We JPROTOCOIS sissies orate daa rd asda ida DS Ih acre eek A UA E N 12 A Transfection of cells with DNA eee eeerere ear erne aaa rereanaado 12 B Transduction of cells with lentiviral particles cccccecccsececeeceseeeeeeeeesaeeeeeeeeeaeeeens 12 O SORCCIING a Re
8. P human sgRNA libraries A few examples of screening applications include 1 Drug target discovery Knockout of a subset of genes in a library could lead to lethality Performing serial dilutions in order to obtain single clones in 96 well plates would reveal that most wells contain healthy dividing cells while other wells would contain either cells with decreased growth rates or no living cells at all Screening for which sgRNAs are not found among surviving clones suggests drug target discovery candidates 2 Drug resistance A particular drug might be lethal to wild type cells Knockout of a subset of genes could make cells resistant to the drug and could provide insights into the drug s mechanism of action Determination of which genes are modified in surviving cells suggests candidates for targets for that drug 3 Change in visible cellular phenotype One example readout of this is growth of cells on soft agar a classical readout for cancer cell metastasis Knockout of a subset of genes in non invasive cells could cause cells to grow on soft agar Identification of the modified genes in the invasive cells suggests candidate genes important for metastasis under defined experimental conditions Genome CRISP CRISPR human sgRNA libraries 4 Protein trafficking Knockout of a subset of genes could cause a change in subcellular localization of a protein carrying an in frame fusion tag of a fluorescent protein such as GFP 5 Transcri
9. Shipped with ice pack Stored at 20 C Genome CRISP M human sgRNA libraries e Pooled lentiviral particles Each tube contains 2 5 x 108 TU lentiviral particles in 25 uL Catalog Shipping and Storage Innate kinases amp LO1 LS03 P1 a es ubiquitin ligases 102 LS03 P1 Nuclear hormone receptors L03 LS03 P1 Tumor metastasis genes LO4 LS03 P1 Oncogenes L05 LS03 p4 Tumor suppressor genes L06 LS03 P1 Protein kinases Lo7 1803 p1 ey genes in 50 pathways 4 tubes 118 119 SQRNAs each 2 tubes 118 sgRNAs each 2 tubes 57 sgRNAs each 4 tubes 144 sgRNAs each 4 tubes 115 116 SQRNAs each 10 tubes 131 132 SQRNAs each 2 tubes 139 sgRNAs each Shipped with dry ice Stored at 80 C Shipped with dry ice Stored at 80 C Shipped with dry ice Stored at 80 C Shipped with dry ice Stored at 80 C Shipped with dry ice Stored at 80 C Shipped with dry ice Stored at 80 C Shipped with dry ice Stored at 80 C Additional materials the following materials may be required but not supplied depending on your experiments a Eat No RR Product Application Clv PK 01 GeneCopoeia 293Ta Lentiviral packaging cell line HPK LvTR 20 HPK LvTR 40 HPK LvTR 50 HPK LvTR 100 CP LvC9NU 01 CP LvC9NU 02 clone LPP CP LvC9NU 01 100 LPP CP LvC9NU 02 100 SCL 01 CA1 SCL 02 CA2 GeneCopoeia LentiPac HIV Expression Packaging kit Genome CRISP Cas9 nuclease lentiviral expressing
10. an AAVS1 safe HCP AAVS1 CG02 oO ao gene knock in AAVS1 donor cloning vector DC DON SH01 kit AAVS1 positive control donor DC RFP SH01 knock in verification primer pairs HQPAVSHR Includes A TM ae e aie AAVS1 All in one sgRNA Cas9 expression clone SH AVS K002 HCP AAVS1 CG02 harbor gene knock in kit without donor AAVS1 positive control donor DC RFP SHO1 knock in verification primer pairs HQPAVSHR Genome CRISP CRISPR human sgRNA libraries Custom CRISPR sgRNA libraries In addition to our pre made CRISPR sgRNA libraries GeneCopoeia will upon request construct custom sgRNA libraries that cover a set of genes that you choose To request such custom libraries contact sales genecopoeia com Custom stable cell line services GeneCopoeia offers the generation of stable cell lines carrying TALEN or CRISPR Cas9 mediated site specific genomic modifications The services include project consultation design and generation of TALEN CRISPR Cas9 and knockin donor if needed constructs screening and isolation of monoclonal lines of correctly modified cells as well as generation of master cell banks Our stable cell line services can also be used with our human and mouse Safe Harbor Integration system For more information about our genome editing stable cell line services contact sales genecopoeia com Pre made Cas9 lentiviral particles Cat No Promoter Reporter gene Seletion marker LPP CP LvC9NU 01 100 Cas9 Nuclease
11. ansfection ready DNA For packaging into lentiviral particles use GeneCopoeia s Lenti Pac packaging products http www genecopoeia com product entiviral packaging kit cells Refer to the Lenti Pac documentation for packaging instructions For transfection into your cell line use GeneCopoeia s Endofectin transfection reagents http www genecopoeia com product endofectin 3 Lentiviral particles The lentiviral particles are ready to use For lentiviral transduction protocol refer to section IV B Analyze sgRNA representation in the library If you are amplifying library bacteria yourself for preparation of DNA it is important to ensure that the individual sgRNAs are as equally represented in the pools as possible We recommend analyzing a subset of sgRNAs approximately 10 of the total number of sgRNAs in the library which can be PCR amplified using one non sgRNA specific primer and one primer specific for each individual sgRNA You can order these primers for each member of the library from GeneCopoeia 1 Make a master PCR mix containing 5X PCR Buffer o uL Purified library plasmid DNA 10 ng primers Spmol uL 2 uL 25mM dNTP 0 2 uL PCR polymerase 5U uL 0 25 uL 20mM Mg2 2 5 pL ddH20 to25 pL Final 25 pL 2 Dispense equal volumes of the master mix into individual plate wells or tubes 3 Dispense equal volumes of a 5pmol yuL solution of each sgRNA specific primer 2 uL per sample inti each well 4 M
12. ded GeneCopoeia will provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to GeneCopoeia within 30 days of receipt of the Product GeneCopoeia s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price GeneCopoeia s liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty GeneCopoeia does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose GeneCopoeia is committed to providing our customers with high quality products If you should have any questions or concerns about any GeneCopoeia products please contact us at 301 762 0888 2015 GeneCopoeia Inc For Research Use Only 2015 GeneCopoeia Inc Trademark Genome CRISP GeneCopoeia CL 030315 GeneCopoeia Inc 21
13. e particles expressing Target cells Targeted cells Cas9 nuclease Transduction cl leeee zse 078F 00000000 Figure 1 Illustration of large scale screening with sgRNA library Advantages Applications Genome CRISP CRISPR human sgRNA libraries The Genome CRISP human sgRNA libraries are constructed as pools of 2 sgRNAs for each gene At the time of manufacture each sgRNA expressing clone is contained in individual bacterial stocks From there individual bacterial stocks are pooled and used either for direct shipment of pooled bacterial stock isolation of transfection ready DNA or further packaging of the DNA into lentiviral particles Figure 2 Figure 2 Pooling method of Genome CRISP human sgRNA libraries GeneCopoeia has determined a specific composition of each sgRNA pool Figure 3 Each gene is represented by 2 sgRNAs An A sgRNA and a B sgRNA The A sgRNAs are contained in separate pools from the B sgRNAs Further each pool carries up to 150 sgRNAs sgRNA A sgRNA A RNAB O 6 rms Ox 8 B Riscos 5 sgRNA B O Pool A Pool B Figure 3 Sub pooling principle of Genome CRISPTM human sgRNA libraries Genome CRISP CRISPR human sgRNA libraries ll Shipping and Storage e Pooled bacterial stock Each tube contains 200 ul of glycerol stock Catalog Shipping and Storage Innate kinases amp 4 tubes 118 119 Shipped with dry ice AE ub
14. identified by the mismatch cleavage assay are considered candidates that must be verified To do so transfect cells with clones or transduce cells with lentiviral particles expressing the individual candidate sgRNA s Once you have transfected or transduced your cells with the individual sgRNAs observe whether or not you are able to reproduce change in phenotype or assay you observed in the initial screen If you ordered your libraries as pools then you can purchase the individual sgRNA clones or lentiviral particle stocks directly from GeneCopoeia Genome CRISP CRISPR A sgRNA MPE V Related Products and Services Genome CRISP CRISPR Cas9 stable cell lines GeneCopoeia offers stable cell lines constitutively expressing the CRISPR Cas9 nuclease These cell lines provide you with a convenient means to carry out CRISPR genome editing applications with high efficiency such as high thoroughput loss of function screening using sgRNA libraries The Genome CRISP Cas9 stable cell lines are available pre made in human and mouse cell lines such as H1299 and HEK293T cells with the CRISPR Cas9 nuclease stably integrated into the human AAVS1 Safe Harbor site In addition we offer services to stably integrate the CRISPR Cas9 nuclease into your cell line of choice High Thoroughput Selection Screen Using sgRNA Library o Pe a Cen 200 ee E _ a SE Infect Cas9 stable cell line with lentiviral paticles of sgRNA library
15. iquitin ligases SQRNAs each Stored at 80 C L02 LS03 B1 Nuclear hormone 2 tubes 118 sgRNAs Shipped with dry ice receptors each Stored at 80 C l hi th E LO3 LS03 B1 Tumor metastasis 2 tubes 57 sgRNAs Shipped with dry ice genes each Stored at 80 C 4 tubes 144 sgRNAs Shipped with dry ice L04 LS03 B1 O i ER AE each Stored at 80 C Tumor suppressor 4 tubes 115 116 Shipped with dry ice oe aa genes SGRNAs each Stored at 80 C e 10 tubes 131 132 Shipped with dry ice LO6 LS03 B1 Protein kinases sgRNAs each Sorrel ath ONE LO7 LS03 B1 Key genes in 50 2 tubes 139 sgRNAs Shipped with dry ice pathways each Stored at 80 C e Pooled transfection ready plasmid DNA Each tube contains 20 30 mg of transfection ready plasmid DNA Catalog Shipping and Storage E SOR ubiquitin ligases SgRNAs each Stored at 20 C L02 LS03 F1 Nuclear hormone 2 tubes 118 sgRNAs Shipped with ice pack receptors each Stored at 20 C i hi ith i k LO3 LS03 F1 Tumor metastasis 2 tubes 57 sgRNAs Shipped with ice pac genes each Stored at 20 C 4 tubes 144 sgRNAs Shipped with ice pack L04 LS03 F1 O DE POE each Stored at 20 C Tumor suppressor 4 tubes 115 116 Shipped with ice pack RUAS Deal mes sgRNAs each Stored at 20 C ore 10 tubes 131 132 Shipped with ice pack LO6 LSO3 F1 Protein kinases sgRNAs each Sai i ANTS L07 LS03 F1 Key genes in 50 2 tubes 139 sgRNAs Shipped with ice pack Innate kinases amp pathways 4 tubes 118 119 each
16. ix well by gently pipetting up and down 5 Seal the plate or tubes and use the following PCR program 94 C 5 min 1 cycle 94 C 30s 58 C 30 s 25 cycles Ze 30s 72 C 5 min 1 cycle C Start with a Cas9 nuclease expressing stable cell line For the best result of all sgRNAs in a in individual cells it is important to obtain a derivative of your cell line that is stably or ind expressing the Cas9 nuclease gene integrated into the genome using one of the following approaches 1 Transduce your cells with Cas9 nuclease lentiviral particles Figure 4A Cas9 nuclease expressing plasmid DNA GeneCopoeia catalog numbers CP LvC9NU 01 CP LvC9NU 02 can be purchased for do it yourself packaging into lentiviral particles using the GeneCopoeia Lenti Pac viral packaging system http www genecopoeia com product lentiviral packaging kit cells Alternatively you can purchase pre made Cas9 lentiviral particles GeneCopoeia catalog numbers LPP CP LvC9NU 01 100 LPP CP LvC9NU 02 100 Transduction of cells with Cas9 expressing lentiviral particles followed by G418 selection leads to stable random integration of the Cas9 expressing plasmid into the genome 2 Knock in the Cas9 nuclease gene by using the GeneCopoeia human AAVS1 Safe Harbor Cas9 donor clones catalog numbers DC C9NU 04 DC C9NU 05 figure 4B and the GeneCopoeia human AAVS1 Safe Harbor knockin kits http www genecopoeia com product aavs1 safe harbor Integration of the Ca
17. mend gradient dilution of 0 1 uL 0 3 uL 3 uL 10 uL 30 uL for standard particles and 0 1 uL 0 3 uL 0 5 uL 0 7 uL 0 9 uL for purified particles Mix the virus with the medium gently by inverting the tubes several times Do not vortex 1 Infect the target cells by removing the old culture medium and replacing it with 0 5 ml of diluted viral supernatant For one well mock well control add 0 5 mL of complete medium with Polybrene Place the plates in a 37 C incubator with 5 CO and incubate cells overnight Optional Place the plates for 2 hours at 4 8 C then transfer the plates to a 37 C incubator with 5 CO and incubate cells overnight Note Incubating cells with lentivirus for 2 hours at low temperatures can significantly increase the transduction efficiency But this step should be omitted if the cells cannot tolerate low temperatures Day 3 Replace medium Split cell culture Replace the old medium with 0 5 mL of fresh complete medium without Polybrene Alternatively split the cells 1 5 to 1 25 depending on the cell types by trypsinizing and re seeding the cells onto 6 well plates or 10cm culture dishes Continue incubating for 48 hours in cell specific medium Day 5 Analyze transduced cells or start drug selection of stably transduced cells The infected target cells can be analyzed for transient expression of transgenes using an appropriate biological assay To select stably transduced cells replace old medium wi
18. ptional activation Knockout of a subset of genes could lead to expression of a fluorescent reporter such as GFP that is under the control of a promoter sequence Genome CRISP CRISPR human sgRNA libraries IV Protocols A Transfection of Cells With DNA Note Know the minimum concentration of puromycin needed to kill drug sensitive cells The Genome CRISP CRISPR human sgRNA library plasmids carry the puromycin resistance gene for stable selection of clones with integrated sgRNA expressing plasmids Figure 5 If you don t know the minimum concentration of puromycin needed to kill drug sensitive cells then perform a killing curve using serial dilutions of puromycin This is important because for lentiviral transduction puromycin selection is applied shortly after transduction and used for selection of cells with stable plasmid integration For DNA transfection we recommend using GeneCopoeia Endofectin transfection kits catalog numbers EFL1001 01 EFL1001 02 EFL1003 01 EFL1003 02 Refer to the kit documentation for detailed DNA transfection protocols SV40 polyA Figure 5 Lentiviral plasmid backbone used for the Genome CRISP CRISPR human sgRNA libraries Vector backbone contains lentiviral packaging elements Stable selection is provided by the puromycin resistance gene mCherry is used as a fluorescent reporter B Transduction of Cells With Lentiviruses Before transducing your cell line with sgRNA lentivirus particles
19. s9 nuclease gene using CRISPR or TALEN and either DC C9NU 04 or DC C9NU 05 occurs specifically at the human AAVS1 locus and provides consistent stable expression of Cas9 without deleterious effects on the cells 3 Order a custom Cas9 expressing stable cell line through GeneCopoeia s custom stable cell line service GeneCopoeia will stably integrate Cas9 into virtually any cell line of your choice For more information on our custom stable cell line services please visit http www genecopoeia com product stable cell line Genome CRISP CRISPR human sgRNA libraries A ULTE B 3X FLAG NLS A Figure 4 Cas9 nuclease expressing plasmids A The Cas9 nuclease lentiviral expression clones CP LvC9NU 01 left w o fluorescent reporter amp CP LvC9NU 02 right w eGFP is used as a fluorescent reporter B Example of Cas9 nuclease expression cassette knockin human AAVS1 locus D Consider your screening readout When using the Genome CRISP human sgRNA libraries it is essential to know how you are going to screen your cells after transfection or transduction The sgRNAs in each library are designed to knock genes out via NHEJ mediated frameshift alleles near the initiator ATG of the MRNA A readily observable phenotype or a convenient assay are needed in order to effectively use the sgRNA libraries for screening Your screening readout depends on the specific questions you addressing when using the Genome CRIS
20. sstehtrecdeetacacedtescseAadent sve sane maes E acao Su cada Si aa e apa pau cold do a 14 V Related products and services ss srerreeserea rea rea seen re arte ree rear era areas rea arado 15 Nils IROIOTONCOS silo os psp ns dennis date SRD T utara cn ada aces 18 VII Limited Use License and Warranty serena sera eresree rea ree sera rea rea arado 19 Genome CRISP CRISPR human sgRNA libraries Introduction Loss of function screening by gene knockout is a powerful tool for systematic genetic analysis in mammalian cells facilitating gene discovery genome scale functional interrogation e g signal transduction pathways and drug discovery e g target identification and drug mechanism studies Recently CRISPR sgRNA libraries have become available for gene knockout studies In the CRISPR Cas9 system a complex of the Cas9 nuclease with a single guide RNA sgRNA generates a double strand break DSB in the target DNA causing frameshiftmutations resulting from nonhomologous end joining NHEJ The Genome CRISP human single guide RNA sgRNA libraries are cloned into lentiviral vectors for dual use transfection or transduction delivery methods designed for large scale screens of interested genes For each targeted gene a minimum of 2 barcoded sgRNAs targeting different regions are optimized designed individually cloned and sequence verified to ensure efficient gene knockout sgRNA Stable cell lin
21. th fresh complete medium containing the appropriate selection drug every 3 4 days until drug resistant colonies become visible generally 7 14 days after selection Genome CRISP CRISPR human sgRNA libraries C Screening The screening strategy will be influenced by your readout i e the phenotype or assay you are using However there will be some common elements as illustrated in figure 6 Cas9 stable cell line y Transfect transduce with SQRNA pools Havest cells in Dilute cells to bulk individual clones l Screen for phenotype or assay PCR amp sequence SQRNA targets Figure 6 Workflow for Genome CRISP CRISPR human sgRNA library usage and screening Regardless of the phenotype or assay you are using as your library screening readout you will need to identify which gene s were knocked out to cause a change in that readout We recommend the following steps 1 Use the mismatch cleavage assay to analyze the genotype of each gene in the library The goal of the mismatch cleavage assay is to determine which gene s sustained CRISPR mediated knockout mutations Use the GeneCopoeia IndelCheck CRISPR TALEN insertion or deletion detection system catalog numbers ICPE 050 ICPE 200 TPCR 050 TPCR 200 TENI 050 and TENI 200 This assay can be performed on either pools of cells or isolated clones Refer to the IndelCheck system documentation for instructions 2 Genes that are
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