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Genome-CRISP™ CRISPR-Cas9 Products and Services User Manual

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1. Cas9 Nuclease 5 DNA Site specific dsDNA break Figure 1 Illustration of CRISPR Cas9 mediated genome editing gt Targeted locus gt Targeted locus Chr Chr sgRNA Cas9 nuclease sgRNA Cas9 nuclease DSB DSB Chr Chr NHEJ Target gene Donor Clone u Ono Chr l Targeted site knockout Chr amp selection marker knockin B D R Figure 4 sgRNA guided gene engineering Left DSB created by sgRNA guided Cas9 nuclease is repaired by NHEJ Right DSB is repaired by the insertion of GOI amp selection markers or other genetic elements from a donor plasmid via HR Genome CRISP CRISPR Cas9 Products and Services Il Related Product and Services Services Description Validation Donor clone services see appendix Stable cell line services see appendix Transgenic mouse services Mismatch cleavage assay Donor clone design and construction Monoclonal colony Cell bank Transgenic mouse Chromosomal level functional validation Detects the presence of indels created by CRISPR Cas9 mediated NHEJ repair at the specific target site of the chromosome Customized plasmids designed to specifically transfer your gene of interest selection marker or other genetic elements into targeted sites through homologous recombination HR induced by our CRISPR Cas9 We offer various donor vector choices with different selection markers and genetic elements built in for your experime
2. cells and transfer 100 uL of cells from column 1 into column 2 Mix by gently pipetting Avoid bubbles Repeat these 1 2 dilutions through the entire plate Bring the final volume to 200 uL by adding 100 uL of medium to all but the last column of wells 4 Incubate plates undisturbed at 37 C 5 Cells will be observable via microscopy over 3 days and be ready to score in 5 8 days depending on the growth rate of cells Mark each well on the cover of the plate indicating which well contains a single colony These colonies can later be subcultured from the well into larger vessels Tech Note 1 Adding 4000 cells in well A1 2 X 104 cells mL is a good starting concentration Increase the concentration for more difficult to grow cell lines 2 If the reporter gene is fluorescent determine which of these colonies express it If the reporter gene is not observable you will have to wait until later in the culture process 3 Label each well with a single colony using a unique identification number and record this number on the plate and in your notebook E Validation of CRISPR Cas9 modified and HR recombinant cells 1 To confirm donor vector integration specifically at a target site junction PCR can be performed using PCR primer pairs that flank the 5 homology arm and 3 homology arm 2 Protocol for Junction PCR 1 Primers should be diluted to 10UM before use Validation of either the 5 or 3 homology arms for donor integration is
3. optimized Cas9 nuclease from Streptococcus pyogenes A Cas9 nickase clone expresses a mutant of Cas9 carrying an aspartate to alanine substitution at amino acid position 10 D10A In concert with a single guide RNA sgRNA the Cas9 nuclease creates a site specific double strand break DSB at a 20 nucleotide target site that is immediately followed by an N G G protospacer adjacent motif PAM DSBs are repaired by either nonhomologous end joining NHEJ which is error prone and leads to frameshift mutations or by homologous recombination HR in the presence of a repair template The ability of CRISPR to greatly stimulate DSBs has been exploited to knock genes out or create knockins of point mutations reporters or other modifications in many experimental systems The Cas9 D10A nickase uses the same recognition system as the wild type Cas9 except that it can create only single strand nicks which are not usually repaired by NHEJ or HR However when two correctly oriented sgRNAs are targeted to opposite strands flanking a desired site a DSB is generated and then repaired efficiently by NHEJ or HR This double nicking strategy is often used for applications demanding lower off target modification Catalog promoter Reporter gene Selection marker CP C9ONU 01 Cas9 nuclease expression clone mCherry Neomycin CP LvC9NU 01 Cas9 nuclease lentiviral expression clone CMV Neomycin CP LvC9NU 02 Cas9 nuclease lentiviral expression clone CM
4. A is sufficient to guide the Cas9 endonuclease to a specific genomic sequence to execute gene editing functions such as gene knockout knockin with donor plasmid modification and more This system can be simplified by fusing the crRNA and tracrRNA sequences to produce a synthetic chimeric single guided RNA SgRNA The selected target consists of a 20bp DNA sequence in the sgRNA followed by a trinucleotide 5 NGG 3 protospacer adjacent motif PAM which is recognized by Cas9 and is essential for gene editing functions The sgRNA hybridizes to the strand complementary to the PAM site Subsequently the Cas9 nuclease cleaves both strands of the DNA Figure 1 This RNA guided DNA recognition mechanism of CRISPR Cas9 provides a simple but powerful tool for precise genome engineering One of the most important advantages of CRISPR Cas9 systems is that the Cas9 protein can be guided by individual sgRNAs to modify multiple genomic target loci simultaneously Genome CRISP CRISPR Cas9 Products and Services Advantages e RNA guided sequence specific genome editing e Simple and fast design process No need to reengineer the nuclease for each new target e Efficient RNA guided DNA recognition regardless of the methylation state of the target site e Flexible and robust multiplexing edit multiple genome sites simultaneously sg Genome tr RNA acrRNA crRNA specific chimera sgRNA sequence Genomic DNA PAM 5 NGG 3
5. D02 CMV copGFP Neomycin N A pDonor D03 CMV N A Neomycin N A pDonor D04 CMV N A Puromycin N A pDonor D05 EFa1 N A Neomycin N A pDonor D07 EFa1 copGFP Puromycin TK Loxp pDonor D08 CMV copGFP Neomycin TK Loxp pDonor D09 EFa1 N A Puromycin TK Loxp pDonor D10 CMV N A Neomycin TK Loxp pDonor D11 PGK copGFP Puromycin TK Loxp pDonor D12 EF1a copGFP Hygromycin TK Loxp pDonor D13 PGK copGFP Neomycin TK Loxp pDonor D14 PGK N A Puromycin TK Loxp Sv40 PA Sv40 PA pDonor D01 Recombination arm L y we uoneuiqwovay Recombination arm L y wue uoneuiqwovsy Recombination arm L Recombination arm L y we uopeujqwovay y we uoneuiqwoovay Figure 8 Maps of some of the donor vectors Genome CRISP CRISPR Cas9 Products and Services Stable cell line services GeneCopoeia offers monoclonal stable cell line service with customized TALEN mediated genome modifications Cell banking service is also available TALEN CRISPR Stable Cell Line Development Services _DNAIRNA Standard cell line or Custom cell ine _ kba e Standard transfection l _ Selection antibiotics or eGFP L_ Pick single colonies q2 q e Select clones with single or double allele modifications to make cell bank e Select best clone with single or double modifications to make master cell bank e PCR verification for one vial of MCB 4 Genome CRISP CRISPR Cas9 Products and Services Transgenic mouse services Transgenic mouse models
6. Gen eCop a ajg Expressway to Discovery Genome CRISP CRISPR Cas9 Products and Services 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 Genome CRISP CRISPR Cas9 Products and Services USER MANUAL Genome CRISP CRISPR Cas9 Products and Services Introduction Il Related Services Ill Overview of Genome Editing Using CRISPR Cas9 IV Critical Steps V References VI Appendix A Clone Products and Services B Other Products and Services Vil Licensing and Warranty Statement I Introduction Background of CRISPR Cas9 system The clustered regularly interspaced short palindromic repeats CRISPR associated protein Cas systems are adaptive mechanisms evolved by bacteria and archaea to destroy invading viruses and plasmids Recently efficient genome editing by the CRISPR Cas9 system has been shown in multiple organisms including zebrafish mice rats C elegans plants and bacteria Several groups have demonstrated that compared with zinc finger nucleases ZFNs and transcription activator like effector nucleases TALENs CRISPR Cas9 mediated gene targeting has similar or greater genome editing efficiencies in multiple systems Efficient and flexible targeting In the CRISPR Cas9 system the complex of a CRISPR RNA crRNA annealed to a trans activating crRNA tracrRN
7. V eGFP Neomycin CP C9NI 01 Cas9 D10A nickase expression clone CBh N A CP C9NI 02 Cas9 D10A nickase expression clone CMV mCherry Neomycin JO JOWO1d OPAS CP C9ONU 01 Sv40 PolyA Figure 4 Map of Cas9 nuclease expression clone Genome CRISP CRISPR Cas9 Products and Services Figure 5 Maps of Cas9 nuclease lentiviral expression clone NLS GL Caras CP CONI 02 Figure 6 Maps of Cas9 Nickase Expression Clone we Targeting site sgRNA1 Cas9 Nickase 3 Genomic i i DNA 5 Site specific ssDNA nick Genomic 5P a 0 T 7 DNA 3 7 Site specific ssDNA nick DNA 3 5 Targeted site knockout GOI amp selection marker knockin Genomic 5 3 DNA 3 5 Analysis of GENE Knocked In Figure 7 Illustration of the double nicking strategy using the Cas9 D10 nickase mutant YLT 10 OWOJg OAS Genome CRISP CRISPR Cas9 Products and Services Genome CRISP sgRNA Design and Cloning Services GeneCopoeia offers single guide RNA SgRNA design and cloning services for the customer s target gene of interest sgRNA clones express a single stranded chimeric sgRNA consisting of crRNA and tracrRNA In the presence of the co transfected Cas9 endonuclease sgRNA can recognize the targeted DNA sequence and guide Cas9 nuclease to create DSBs and facilitate genome editing function for gene knockout knockin mutagenesis and more Multiple sgRNA clones can be constructed and co trans
8. are now considered ideal tools to delineate the molecular mechanisms of the gene products and their interactions with one another that influence all cellular processes that form the basis of physiological systems GeneCopoeia offers transgenic mice with customized TALEN or CRISPR Cas9 mediated genome modifications TALEN and CRISPR Cas9 Mediated Genome Modifications Overview of genome editing by TALEN and CRISPR Cas9 TALEN CRISPR Cas9 Ti moa NHEJ HR lt lt lt e o One step generation of mice with genome modifications TALEN or amp a ny sgRNA donor DNA R NHEJ or HR w gt Embryo transfer Blastocyst Mutant Genome CRISP CRISPR Cas9 Products and Services Vil Licensing and Warranty Statement Limited Use License Following terms and conditions apply to use of the Genome TALERTM Human AAVS1 Safe Harbor Gene Targeting Kit 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 p
9. ate 100 000 to 300 000 cells well in a 6 well plate following the recommended Tech Notes conditions for the cell type s being transfected Scale up and down the culture if needed On the day before transfection trypsinize and count the cells The number of cells plated in each well should be determined so that they are 70 80 confluent at the time of transfection 2 The next day prepare transfection complexes using suitable transfection reagents according to the manufacturer s instructions Leave the transfection complexes on the cells to react for gt 6 hours 1 Since transfection efficiencies vary across different cell lines we recommend optimizing the input for best results 2 For optimal results we recommend complexing DNA with transfection reagent in serum and antibiotic free media and cells growing in complete media e g DMEM F12 10 FBS w o antibiotics 3 For hard to transfect cells e g primary stem hematopoietic it may be advisable to utilize a non passive transfection method Please follow recommended guidelines provided by the manufacturer for the specific cell type s being transfected Genome CRISP CRISPR Cas9 Products and Services 3 24 hours post transfection remove transfection media and split the cells 1 10 and 1 20 in complete growth media w antibiotics Plate cells into 6 well plates and save a set of plate s for characterization Allow cells to recover for 24 hours 4 Begin antibiotic selecti
10. ease digestion 4 DNA heteroduplex formation At this point the amplified PCR product includes a mixture of both CRISPR modified and unmodified genomic DNA Place 300 ng of the PCR product in a thermocycler tube and perform the cross hybridization 5 T7 endonuclease assay Treat the cross hybridized homo and heteroduplexes with T7 endonuclease assay kit to determine CRISPR cleavage efficiency A gt Genome CRISP CRISPR Cas9 Products and Services HUWE sgRNA B 1000bp 900bp 800bp 700bp 600bp 500 517bp 400bp 300bp 200bp 100bp HUWE length 520bp sgRNAsite 330bp 190bp 1 HUWE sgRNA 2 sgRNA Ctrl Figure 3 Mutagenesis of the human HUWE gene using CRISPR Cas39 A HUWEI sgRNA design Expected bands of T7 endonuclease T7 ENI digestion are 330bp 190bp B The HEK293T cells were transfected with HUWE sgRNA Cas9 clone Lane 1 and scrambled sgRNA Cas9 control clone Lane 2 in a 6 well plate The cells were harvested 40h post transfection The genomic DNA was extracted and PCR amplified using HUWE specific PCR primer The PCR products were purified by gel purification 8uL purified PCR products were mixed with buffer denatured and reannealed and then digested with T7 ENI at 37 C for 60mins The expected HUWE PCR product size is 520bp and the expected T7 ENI digestion product sizes are 330bp and 190bp C Co transfection or transfection into target cells Pl
11. fected with one Cas9 clone to enable simultaneous editing of several sites within the genome offering greater efficiency and flexibility for the experiment design Vector Promoter sgRNA and Cas9 Selection Marker Reporter Gene PCRISPR SG01 sgRNA only Hygromycin PCRISPR LvSG03 U6 SgRNA only Puromycin mCherry oCRISPR CG01 U6 SUN Maveeliel Cui liam ete USE etiam type in the same all in one vector oCRISPR CG02 U6 sgRNA and CBh driven Cas9 wild N A type in the same all in one vector Compatile Cas9 Nuclease Cat No CP C9NU 01 CP LvC9NU 01 CP LvC9N U 02 or Cas9 D10A Nickase Cat No CP C9NI 01 Cat No CP C9NI 02 are available as separate expression clones pCRISPR SG01 Sv40 PolyA Sv40 Promotor JOJOWOJd OPAS pCRISPR CG01 pCRISPR CG02 Sv40 PolyA Figure 7 Maps of sgRNA clones Genome CRISP CRISPR Cas9 Products and Services A Other Products and Services Donor services GeneCopoeia offers customized donor clone design and construction services Donor clones are customized plasmids designed to specifically transfer your gene of interest selection marker or other genetic elements into a target site via HR mediated repair of DSBs induced by site specific genome editing tools Donor vectors are available with several options for selection markers and genetic elements to meet your experimental needs Donor vector types Reporter Gene Selection Marker LoxP Site pDonor D01 EFa1 copGFP Puromycin N A pDonor
12. lified plasmids we recommend restriction digestion analysis or direct sequencing B CRISPR chromosomal validation CRISPR modified DNA will have a few bases of sequence inserted or deleted near the Cas9 cut site due to NHEJ exonuclease activity We recommend using the GeneCopoeia s IndelCheck CRISPR TALEN insertion or deletion detection system ICPE 050 ICPE 200 Alternatives include the Cel1 mung bean and S1 nucleases The T7 endonuclease assay is carried out according to the user manual of the IndelCheck detection system We provide brief details here 1 24 or 48 hr post transfection collect cells for lysis recommended or for genomic DNA extraction 2 Use target PCR kit TPCR 050 TPCR 200 to amplify the region surrounding the SgRNA target site 3 Check the PCR result by running 5 uL of PCR product on a 2 agarose gel For all templates it is important to make sure that there is only a single band corresponding to the intended product for the primer pair The size of this band should be the same as calculated from the distance between the two primer annealing sites in the genome CRITICAL STEP If multiple amplicons are generated from the PCR redesign the primers and reoptimize the PCR conditions to avoid off target amplification In difficult cases in which a single band product cannot be achieved it is acceptable to gel extract the correct length band before proceeding with heteroduplex reannealing and Surveyor nucl
13. nt purpose Monoclonal stable cell line with CRISPR Cas9 mediated genome modifications Creation of a cell bank of monoclonal stable cell line with CRISPR Cas9 mediated genome modifications Transgenic mice with CRISPR Cas9 mediated genome modifications Genome CRISP CRISPR Cas9 Products and Services lll Overview of Genome Editing Using CRISPR Cas9 Plasmid Propagation Functional validation Knockout w o donor Transfection or transduction Seletion or sorting optional Isolate single clones Mismatch cleavage assay to identify knockout Sequence targets to confirm mutation Isolate more clones or transfect again to identify all allele knockouts optional Genome CRISP CRISPR Cas9 Products and Services IV Critical Steps A Plasmid propagation We recommend propagating the plasmids provided before your gene targeting experiment Plasmids can be transformed using standard conditions suitable in any RecA and EndA E coli competent cells For transformation of CRISPR Cas9 product plasmids we suggest plating 50 200 of transformed cells on fresh LB Ampicillin plates 50 ug ml Incubate the plates at 37 C overnight Inoculate colonies from the transformation and grow them at 37 C overnight in 200ml of LB media containing 50ug ml Ampicillin Use an endotoxin free plasmid DNA maxiprep kit to extract plasmid DNA after overnight growth To confirm the integrity of the amp
14. on 48 hours post transfection We recommend optimizing concentration of antibiotic for best results 5 For HR based knockin another round of antibiotic selection to enrich clones with donor integration is recommended Tech Note Establishing a kill curve on untransfected cells can determine the effective working antibiotic concentration for a target cell line The concentration of antibiotic that kills gt 90 of cells after 48hours of selection is the correct dose for the cells being selected D Clonal isolation of cell lines Serial dilution is widely used to isolate single clones with desired modifications followed by an expansion period to establish a new clonal cell line Like most clonal isolation methods there is no guarantee that the colonies arose from single cells A second round is advised to increase the likelihood of clonal isolation Also it is worth noting that cell types can vary substantially in their responses to single cell isolation so literature specific to the cell type of interest should be consulted 1 Fill each well of a sterile 96 well plate with 100 uL of medium except for well A1 which should remain empty NSS G 2 Add 200uL cell suspension io well A1 Mix 100 uL from A1 with the medium in well B1 Avoid bubbles Continue this 1 2 dilution through column 1 Add 100 uL of medium back to column 1 so that wells A1 through H1 contain 200 uL Genome CRISP CRISPR Cas9 Products and Services 3 Mix
15. random integration Genome CRISP CRISPR Cas9 Products and Services V References 1 Horvath P Barrangou R January 2010 CRISPR Cas the immune system of bacteria and archaea Science 327 5962 167 70 2 Marraffini LA Sontheimer EJ February 2010 CRISPR interference RNA directed adaptive immunity in bacteria and archaea Nat Rev Genet 11 3 181 190 3 Hale CR Zhao P Olson S et al November 2009 RNA Guided RNA Cleavage by a CRISPR RNA Cas Protein Complex Cell 139 5 945 56 4 van der Oost J Brouns SJ November 2009 RNAi prokaryotes get in on the act Cell 139 5 863 5 doi 10 1016 j cell 2009 11 018 5 Jinek M Chylinski K Fonfara l Hauer M Doudna J A and Charpentie E 2012 A programmable dual RNA guided DNA endonuclease in adaptiv bacterial immunity Science 337 816 821 6 Jiang W Bikard D Cox D Zhang F and Marraffini L A 2013 RNA guided editing of bacterial genomes using CRISPR Cas systems Nat Biotechnol 31 233 239 7 Hsu P D Scott D A Weinstein J A Ran F A Konermann S Agarwala V Li Y Fine E J Wu X Shalem O et al 2013 DNA targeting specificity of RNA guided Cas9 nucleases Nat Biotechnol Published online July 21 2013 Genome CRISP CRISPR Cas9 Products and Services VI Appendix A Clone Products and Services Genome CRISP Cas9 Nuclease and D10A Nickase Expression Clones A Cas9 clone expresses human codon
16. rior 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 provided 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 p
17. roducts please contact us at 301 762 0888 2015 GeneCopoeia Inc For Research Use Only 2015 GeneCopoeia Inc Trademark Genome CRISP EndoFectin GeneCopoeia CP 091215 GeneCopoeia Inc
18. usually sufficient however both arms can be done for additional confirmation 2 Protocol details for junction PCR assay a Isolate genomic DNA from positive control cells or test sample cells using a suitable genomic DNA miniprep kit Please follow the protocol recommended by the manufacturer b Perform junction PCR PCR reaction below Genome CRISP CRISPR Cas9 Products and Services CRISPR Cas9 cut Positive control donor Reagent e positive control donor only Genomic DNA 60 100ng ul 1uL 1uL 10u M 5 or 3 PCR Primer Mix 1uL 1uL 5X UltraPFTM Buffer Mg2 free SuL SuL 10 mM dNTPs 0 5uL 0 5uL 20mM MgSO4 2 5uL 2 5uL UltraPF 5U x 1 0 25uL 0 25uL PCR grade distilled water 14 75uL 14 75uL Total 25uL 25uL 98 C Smin 98 C 20sec 55 C 30sec 35 cycles 72 C 1min 72 C Tmin Hold at 4 16 C Run the PCR reaction out on the 1 Agarose EtBr gel in 1X TAE buffer to confirm the junction PCR result Sample results for 5 and 3 junction PCR assay depend on design Tech Note 1 The 3 junction PCR band and 5 junction PCR band may differ in brightness because the amplification efficiency may be different due to the nature of the chromosomal structure modification and sequence around that region 2 One positive in junction PCR is sufficient to confirm the integration 3 It is possible that random integration can coexist with site specific integration Negative selection can be used to detect coexisting

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