Genome-CRISP™ CRISPR-Cas9 Products and
Contents
1. Gen eCop 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 2014 GeneCopoeia Inc Genome CRISP CRISPR Cas9 Products and Services USER MANUAL Genome CRISP CRISPR Cas9 Products and Services Introduction ll Products and Services lll Related Services IV Overview of Genome Editing Using CRISPR Cas9 V Critical Steps VI References VII Appendix VIII Licensing and Warranty Statement 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 tracrRNA is sufficient to gui2. D08 CMV copGFP Neomycin TK Loxp pDonor D09 EFa1 N A Puromycin TK Loxp pDonor D10 CMV N A Neomycin TK Loxp 15 Genome CRISP CRISPR Cas9 Products and Services Sv40 PA J E 7 S C S S E E 2 S 2 T E s 3 2 F pDonor D02 z 2 o 2 E 3 E 8 2 8 2 3 o 3 ow D a a will g e z p pa O Ki 4 o c 3 c 3 ES 2 E gt 5 Ei E A E 2 E 2 S se S E 3 s z G x a E EA E a 5 E S S 2 E 5 t S S o w oO o 3 x E Sv40 PA Sv40 PA E E R e rs p e 3 oO Le o ki O c R Ki S S E gt E 5 S S 5 E e v o O 3 E 5 ZS 3 8 3 a e 3 x E E E A 4 KA c 3 E 2 E e 8 5 c ZS S 5 2 2 S S A OO w D E 3 Ka bi Ei 3 E Figure 9 Maps of donor vectors 16 Genome CRISP CRISPR Cas9 Products and Services Stable cell line services GeneCopoeia offers monoclonal stable cell line service with customized CRISPR Cas9 mediated genome modifications Cell banking service is also available TALEN CRISPR Stable Cell Line Development Services e Clone Standard cell line or Custom cell line e Standard transfection e Selection antibiotics or eGFP e PCR screening of stable clones e Pick single colonies Safe Harbor clone only optional e Southern blot to rule out RI e Select clones with single or double allele modifications to make cell bank e Select best clone with singl
3. Figure 7 Maps of sgRNA clones Genome CRISP CRISPR Cas9 Products and Services lll Related Product and Services Services Validation Donor clone services see appendix Stable cell line services see appendix Transgenic mouse services Surrogate reporter assay T7 endonuclease assay Donor clone design and construction Monoclonal colony Cell bank Transgenic mouse Description Plasmid level functional validation Detects activities of genome editing tools by observing the expression level of a surrogate reporter gene 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 experiment 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 IV Overview of Genome Editing Using CRISPR Cas9 JT UU to confirm mutatio
4. Mutagenesis of the human HUWE gene using CRISPR Cas9 A HUWEI sgRNA design Expected bands of T7 endonuclease T7 ENI digestion are 330bp 190bp B HUWEI sgRNA Cas9 clones were transfected into HEK293T cells in a 6 well plate The medium was changed at 16h post transfection Cells were harvested at 40h post transfection Genomic DNA was then extracted for PCR PCR products were gel purified 8uL purified product was mixed with 1uL 10x NEB buffer 2 followed by annealing in a PCR machine programmed as follows 94 C10min 93 C 25s 92 C 25s x35 20 C5min 4 C After annealing the products were cut with 2U T7 Endonuclease for 60mins at 37 C and analyzed by agarose gel electrophoresis C Co transfection or transfection into target cells 1 Plate 100 000 to 300 000 cells well in a 6 well plate following the recommended 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 Tech Notes 1 2 11 Since transfection efficiencies vary across different cell lines we recommend optimizing t
5. PolyA Figure 5 Maps of Cas9 Nickase Expression Clone Genome CRISP CRISPR Cas9 Products and Services ye Targeting site sgRNA1 Cas9 Nickase TET TEE 3 Genomic TET TTT TL 5 DNA 3 4 LELLEL ELL Cas9 Nickase Site specific sgRNA2 ssDNA nick Genomic es t rs 3 DNA 3 5 Site specific ssDNA nick DNA KEN 5 Targeted site knockout GOI amp selection marker knockin Genomic 5 3 DNA 3 CN Analysis of GENE Knocked In Figure 6 Illustration of the double nicking strategy using the Cas9 D10 nickase mutant Genome CRISP sgRNA Design and Cloning Services SQRNA clones express a single stranded chimeric sgRNA In the presence of the Cas9 endonuclease an sgRNA guides the Cas9 nuclease to create a DSB at the target site Multiple SgRNA clones can be co expressed with one Cas9 clone to enable simultaneous editing of several sites in the genome offering great efficiency and flexibility Vector types Selection Marker Vector Promoter sgRNA Cas9 Nuclease Reporter Gene Host pCRISPR SG01 U6 1 Sold separately Hygromycin Mammalian pCRISPR CG01 CMV driven Cas9 in Neomycin mCherry Mammalian the same vector pCRISPR CG02 CBh driven Cas9 in N A Mammalian the same vector pCRISPR LvSG02 U6 Sold separately Puromycin Mammalian mCherry Lentiviral Genome CRISP CRISPR Cas9 Products and Services A JOJOWOJd OPAS pCRISPR SG01 Sv40 PolyA Sv40 Promotor
6. 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 A bk 2 Reagent Genomic DNA 60 100ng ul 10uM 5 or 3 PCR Primer Mix 5X UltraPF Buffer Mg free 5ul 10 mM dNTPs 20mM MgSO z UltraPF 5U ul PCR grade distilled water Total 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 Protocol for Junction PCR Primers should be diluted to 10uM before use Validation of either the 5 or A homology arms for donor integration is usually sufficient however both arms can be done for additional confirmation 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 CRISPR Cas9 cut positive control donor 1 ul Positive control donor only 1u
7. 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 14 Genome CRISP CRISPR Cas9 Products and Services VII Appendix 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 Selection Reporter Vector Promoter LoxP Site Marker Gene pDonor D01 EFa1 copGFP Puromycin N A pDonor 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
8. c elements from a donor plasmid via HR Genome CRISP Cas9 nuclease lentiviral expression clone A Cas9 nuclease lentiviral expression clone is a premade clone containing the sequence of engineered CRISPR associated Cas gene 9 constructed in a lentiviral backbone A lentiviral system is very effective at delivering genetic material to whole model organisms and almost all mammalian cells including non dividing inactive or growing and difficult to transfect cells including neuron primary and stem cells ALT Genome CRISP CRISPR Cas9 Products and Services T7 SV40 Figure 4 Maps of Cas9 nuclease lentiviral expression clone Genome CRISP Cas9 Nickase Expression Clone A Cas9 nickase expression clone is a premade clone containing the sequence of engineered Cas9 nickase Figure 4 Wildtyoe Cas9 has two catalytic domains one cuts the binding strand and the other cuts the complementary strand The Cas9 D10A nickase contains an Aspartate to Alanine amino acid substitution at position 10 This mutation makes the Caen nuclease able to cut only the binding strand thereby creating a single strand nick at the targeted site In a carefully designed double nicking strategy two sgRNAs targeted to opposite strands of the DNA will enable the Cas9 D10A nickase to create a staggered end DSB which can be repaired by NHEJ or HR NLS amer G CP CONI 01 Cas9 nickase JOJOWOJd OPAS CP C9ONI 02 Sv40
9. de 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 Advantages RNA guided sequence specific genome editing Simple and fast design process No need to reengineer the nuclease for each new target Genome CRISP CRISPR Cas9 Products and Services Efficient RNA guided DNA recognition regardless of the methylation state of the target site Flexible and robust multiplexing edit multiple genome sites simultaneously sgRNA tracrRNA crRNA chimera Genome specific sgRNA sequence Genomic DNA PAM 5 NGG 3 Cas9 Nuclease Genom
10. e or double modifications to make master cell bank e PCR verification for one vial of MCB 17 Genome CRISP CRISPR Cas9 Products and Services VIII Limited Use License and Warranty Limited Use License Following terms and conditions apply to use of the Genome CRISP Product and services 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 provided GeneCopoeia will provide the purchaser with a ref
11. he input for best results For optimal results we recommend complexing DNA with transfection reagent in serum and antibiotic free media and cells growing in complete media e g Genome CRISP CRISPR Cas9 Products and Services 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 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 selection 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 c
12. ic SOT LTT ee Site specific dsDNA break Figure 1 Illustration of CRISPR Cas9 mediated genome editing ll Products and Services Genome CRISP Cas9 Nuclease Expression Clone Cat CP C9NU 01 Figure 2 A Cas9 nuclease expression clone is a premade clone containing the sequence of a mammalian codon optimized CRISPR associated Cas 9 gene from Streptococcus pyogenes Co expression of Cas9 with an sgRNA guides the Cas9 nuclease to create a site specific double strand break DSB in the host genome DSB repair mediated by non homologous end joining NHEJ can then reconnect the DNA and induce insertion or deletion mutations at the site of the break Alternatively specific sequences genes or base pair changes can be introduced into the DSB site by homologous recombination with an exogenous double stranded donor DNA fragment Genome CRISP CRISPR Cas9 Products and Services 17 JOJOW OI OPAS CP CONU 01 Sv40 PolyA Figure 2 Map of Cas9 Nuclease Expression Clone gt Targeted locus sgRNA Cas9 nuclease DSB Chr NHEJ Target gene knock out Chr gt Targeted locus sgRNA Cas9 nuclease DSB Sonor A AS E A EIERE SR Il Targeted site knockout Chr amp selection marker knockin DR GEREENT M Figure 3 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 geneti
13. l 1 ul 1 ul Sul 0 5ul 0 5ul A Du A Du OU Zu OU Zu 14 75ul 14 75ul 25ul 25ul 13 Genome CRISP CRISPR Cas9 Products and Services 98 C Smin 98 C 20sec 55 C 30sec 35 cycles 72 C 1min 72 C 7min 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 3 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 Though rare it is possible that random integration can coexist with site specific integration Negative selection can be used to detect coexisting random integration VI 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
14. lonal 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 d Cf SR ES kend Si d 5 ad A 5 d C p 2 Add 200ul cell suspension to well AT Mix 100 ul from AT 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 A Mix 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 12 Genome CRISP CRISPR Cas9 Products and Services 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 2x104
15. ns identify all allele knockouts optional optional WO Genome CRISP CRISPR Cas9 Products and Services V 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 200ul of transformed cells on fresh LB Ampicillin plates 50ug 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 amplified 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 Surveyor mutation detection kit for standard gel electrophoresis Transgenomic cat no 706025 for this assay Alternatives include the Cel1 T7 mung bean and S1 nucleases For an example see Figure 5 The Surveyor procedure is carried out according to the manufacturer s instructions and is described in greater detail in the Surveyor manual We provide brief details here 1 24
16. or 48 hr post transfection collect cells to extract genomic DNA 2 PCR 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 nuclease digestion 1 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 2 Surveyor Nuclease S digestion Treat the cross hybridized homo and heteroduplexes with Surveyor Nuclease S to determine TALEN cleavage efficiency 10 1000bp 900bp 800bp 700bp 600bp 500 517bp 400bp 300bp 200bp 100bp Genome CRISP CRISPR Cas9 Products and Services HUWE length 520bp sgRNA site 330bp 190bp 1 HUWE sgRNA 2 sgRNA Ctrl HUWE sgRNA Figure 8
17. und 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 2014 GeneCopoeia Inc For Research Use Only 2014 GeneCopoeia Inc Trademark Genome CRISP EndoFectin GeneCopoeia GeneCopoeia Inc CP 010214 18
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