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CRISPR/Cas9Gene Editing Tools
2
Guide-it™ Products for Successful CRISPR/Cas9 Gene Editing
Gene editing for all researchersThe CRISPR/Cas9 system has democratized genome modifi cation; targeted modifi cation can now be achieved at virtually any genomic locus in virtually any cell type. This powerful method is allowing researchers to ask questions that were previously unaskable, leading to new insights into the basis of fundamental biological processes and to new innovative therapeutic strategies for treating disease—and that’s good science!
Best-in-class tools for your gene editing project—from start to fi nishGuide-it products further improve the usability of the CRISPR/Cas9 system by providing straightforward, streamlined methods at every step of your genome editing workfl ow.
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Why choose Guide-it products?• Optimized methods
designed for speed and ease of use
• Complete kits that don’t require additional reagents
• Backed by our team of technical support scientists, ready to help at any point in your experiment Design sgRNAs
Transcribe and screensgRNAs in vitro
Deliver sgRNA and Cas9 to cells
Detect Cas9 protein and confi rm gene editing
Identify indels
Typical Gene Editing Workfl ow
www.takarabio.com/CRISPR3
What is CRISPR/Cas9 gene editing?CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 technology is based on an antiviral adaptive immune system in bacteria. The CRISPR/Cas9 system has been harnessed to create a simple, RNA-programmable method to mediate genome editing in mammalian cells. CRISPR/Cas9 editing is mediated by two components: a single-guide RNA (sgRNA) and Cas9 nuclease. The sgRNA directs the Cas9 nuclease to a specifi c genomic site where it introduces double-stranded DNA breaks. When this DNA damage is repaired by endog-enous cellular DNA repair mechanisms, mutant alleles can be introduced.
At the bench: Design the best sgRNAThe fi rst step in any CRISPR/Cas9 experiment is to choose a target sequence in the gene that you want to manipulate, and then design your guide RNA. In theory, selecting a target sequence is simple, but in practice, not all sgRNAs perform with equal effi ciency and specifi city. It is recommended that for any given target, multiple sgRNAs are designed and tested.
Learn more about best practices for choosing a target site at www.takarabio.com/sgRNA-design.
Design sgRNAs
sgRNA (single guide RNA)1 sgRNA + Cas9 protein2
Target-specificcrRNA sequence
tracrRNA
Your favourite gene
PAM sequence(5'-NGG-3')
Cas9
sgRNA
Target-specific cleavage3 Cellular repair4
4
Test sgRNA performance before cell delivery
At the bench: Effective sgRNAs are identifi ed in vitro
Identifying effective sgRNAs. Panel A. Cleavage effi ciency of four different sgRNAs (1–4) targeting CXCR4. Negative control (NC) lacked sgRNA. sgRNA3 was predicted to be the least effective. Panel B. HeLa cells were cotransfected with plasmids encoding Cas9 and one of the four different sgRNAs. Effi ciency of CXCR4 gene disruption was assessed by a FITC-labeled antibody against CXCR4. In agreement with the Screening Kit results, sgRNA3 resulted in the lowest percentage of knockout cells.
Guide-it sgRNA In Vitro Transcription Kit: Produce high yields of sgRNAs• Fast, effi cient method for sgRNA production
• Transcribe 10-20 ug of sgRNA per in vitro reaction
• sgRNAs can be used for in vitro screening and/or delivery into cells via transfection/electroporation
Guide-it sgRNA In Vitro Screening Kit: Test sgRNA effi cacy• Highly optimized in vitro cleavage assay that allows
estimation of cleavage effi ciency
• Includes high-quality, recombinant Cas9 protein
200
Co
un
ts
160
120
80
40
0100 101
Knockout71.26%
102 103
FL1-H
sgRNA1
104
200
Co
un
ts
160
120
80
40
0100 101
wtwt wtwt
Knockout73.62%
102 103
FL1-H
sgRNA2
104
200
Co
un
ts
160
120
80
40
0100 101
Knockout11.50%
102 103
FL1-H
sgRNA3
104
200
Co
un
ts
160
120
80
40
0100 101
Knockout74.90%
102 103
FL1-H
sgRNA4
104
Transcribe and screensgRNA in vitro
Guide-it sgRNA Screening Kit in vitro Cas9 cleavage
assay
M NC 1 2 3 4 0% ~55% ~42% ~8% ~71%
A B
1 Design/purchase a 56- to 58-nt forward primer
2 Create template for in vitro transcription of sgRNA
3 Production of sgRNA by in vitro transcription and purification with the provided Guide-it IVT RNA Clean-Up Kit (Cat. No. 632638)
T7 promoter(17 nt + 4 nt)
Target-specificcrRNA (20 nt)
tracrRNA
Homology to scaffoldtemplate (15 nt)
56- to 58-ntforwardprimer Reverse primer
PCR
Scaffold template(tracrRNA)
Target-specificcrRNA
T7promoter
sgRNA
1 Use PCR to generate a target for cleavage
2 In vitro cleavage of target sequence by recombinant Cas9 and synthesized sgRNA
3 Separate cleavage products on an agarose gel
Uncleaved
Cleaved
PAM sequence(5'-NGG-3')
Cas9
sgRNA
Your target gene
PAMsequence
www.takarabio.com/CRISPR5
At the bench: Tight control ofCas9 expression
Lenti-XTM CRISPR/Cas9 Systems: Lentiviral delivery• Optimized systems for producing lentiviral particles to
deliver sgRNA/Cas9 expression cassettes
• Express Cas9 either constitutively or with doxycycline induction under control of the Tet-On® 3G promoter
– +200
Co
un
ts
160
120
80
40
0100 101
– Dox
102 103
M1M2
1.39%knockout
200
Co
un
ts
160
120
80
40
0100 101 102 103
M1M2
58.61%knockout
+ Dox
A Guide-it Mutation Detection Kit
B FACS
Doxycycline-induced genome editing in cell lines transduced with our Lenti-X Tet-On 3G CRISPR/Cas9 System. This clonal population of cells was transduced with both dox-inducible Cas9 and sgRNA pro- ducing lentiviral particles. As seen in panel A, cleavage of the target is seen after adding doxycycline (+), while there is no background genome editing before adding doxycycline (-). Panel B demonstrates functional editing of a surface membrane protein, with 59% knockout achieved after adding doxycycline.
Deliver sgRNA/Cas9 expression cassettes to any target cell
pGuide-it vector simultaneously expresses Cas9, an sgRNA, and a fl uores-cent reporter (ZsGreen1 or TdTomato).
Guide-it CRISPR/Cas9 Systems: Plasmid-based delivery • Complete system for the cloning and expression of
sgRNAs and Cas9 in mammalian cells
• Pre-linearized expression plasmids allow for simple, single-step cloning of sgRNAs
• Vectors include bright fl uorescent reporters, ZsGreen1 or tdTomato (2.5x and 6x brighter than EGFP, respectively) for identifi cation of transfected cells
AAVpro® CRISPR/Cas9 Helper Free Systems: AAV-mediated delivery• Optimized systems for producing AAV2 particles to
deliver sgRNA/Cas9 expression cassettes. Packaging vectors for additional serotypes (AAV1, AAV5, AAV6) are available.
• Improved results in hard-to-transfect cells/suitable for in vivo use
• AAV delivery precludes genomic integration and persistent expression of Cas9, reducing potential off-target effects.
pAAV-Guide-it-Up Vector pAAV-Guide-it-Down Vector
Transfection+ pRC2-mi342 Vector+ pHelper Vector
HEK 293T Cells
AAV2-Up Particles AAV2-Down Particles
Cotransduction
Target cell
Cas9/sgRNAcomplex
Expression of full-length Cas9 & user-defined sgRNA
AAV Extraction Solution
HEK 293T Cells
Cas9
sgRNA
A two-vector system overcomes the size restrictions of the AAV genome. The SpCas9 gene is split between two vectors with a 1.6 kb region of homology that mediates recombination in target cells, producing full-length SpCas9.
Deliver sgRNA andCas9 to cells
• AAVpro CRISPR/Cas9 Helper Free System (with Streptococcus pyogenes Cas9): The SpCas9 gene is split between pAAV-Guide-it-Up and pAAV-Guide-it-Down plasmids with a 1.6-kb region of homology; the homologous region results in recombination in target cells, producing a full-length SpCas9 gene.
• AAVpro CRISPR/SaCas9 Helper Free System (with Staphylococcus aureus Cas9): The SaCas9 gene fi ts into one single plasmid that also contains the sgRNA expression cassette.
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At the bench: Functional knockout of the endogenous CD81 gene by sgRNA transfection
HT1080 cells stably expressing Cas9 (HT1080-Cas9) were transfected with 50 pmol of sgRNA targeting CD81, either once (1x) or twice (2x). Seven days later, cells were immunostained with a CD81 antibody (Ab) conjugated to an FITC fl uorophore and analyzed by fl ow cytometry. A control sample, comprised of HT1080-Cas9 cells, was analyzed by fl ow cytometry, either without (-Ab) or with (+Ab) the CD81 antibody. Both single and double transfection with sgRNA resulted in a shift in fl uorescence intensity, indicating successful CRISPR/Cas9-mediated knockout of CD81.
Xfect™ RNA Transfection Reagent: RNA delivery• Single reagent for transfection of cell lines and primary cells with mRNA and/or sgRNA• Very low cytotoxicity and high transfection effi ciency in primary cells• Simple, serum-compatible protocol
–Ab –sgRNA +Ab –sgRNA
99.89%
Co
un
ts
Co
un
ts
Co
un
ts
Co
un
ts
17.72%knockout
+Ab +sgRNA 2x+Ab +sgRNA 1x
19.43%knockout0.07%
Workfl ow of CRISPR/Cas9 Gesicle Production. The target-specifi c sgRNA is cloned into the provided plasmid, which is then mixed with the CRISPR/Cas9 Gesicle Packaging Mixes 1 and 2. Following a 10-minute incubation, the complete nanoparticle mix is applied to the Gesicle Producer 293T Cell Line in the presence of the provided A/C Heterodimerizer ligand. After 48–72 hours, gesicles containing active Cas9 protein complexed with your sgRNA can be collected and concentrated via centrifugation.
RNA and Protein delivery - minimize off-target effects
Guide-it CRISPR/Cas9 Gesicle Production System: Protein deliveryCRISPR/Cas9 gesicles are cell-derived nanovesicles containing active Cas9 protein complexed with a target-specifi c sgRNA. Gesicles are engineered with glycoproteins on their surface that mediate binding and fusion with the membranes of a wide range of target cells, allowing delivery of Cas9/sgRNA even to diffi cult-to-transfect and non-dividing cells. The CRISPR/Cas9 Gesicle Production System provides all components needed for cloning and expressing your target-specifi c guide RNA, and packaging reagents for producing CRISPR/Cas9 gesicles.
• Effi cient delivery of Cas9/sgRNA ribonucleoprotein (RNP) complexes to a broad range of cell types• Cas9 protein delivery eliminates genomic integration and reduces off-target effects• Tight control over dose and timing of delivery and editing
Add 10 μg ofplasmid encodingfor sgRNA (600 µl
final) to Mix 1
Vortex and addto Mix 2
Add A/C Heterodimerizer
Vortex andincubate 10 minutes
Apply to 293T cells
Incubate 48–72 hr
Collect media,centrifuge
Deliver sgRNA andCas9 to cells
www.takarabio.com/CRISPR7
Knock out of cell surface marker CD81 in induced pluripotent stem cells. hiPSC-18 cells were electroporated with each vendor’s complete Cas9 ribonucleoprotein (RNP) system (the guide RNAs were produced either synthetically or with their in vitro transcription system), with all RNPs targeting the same sequence in CD81. Ten days after electroporation, cells were stained with a fl uorescent anti-CD81 antibody and then run on a FACS machine to detect CD81. The positive and negative staining controls show the FACS data from untreated cells with and without antibody staining. The Takara RNP system demonstrated very effi cient editing of the target compared to its competitors.
Guide-it Recombinant Cas9 (Electroporation-Ready): Protein delivery• Recombinant wild-type Streptococcus pyogenes Cas9 nuclease (SpCas9) with C-terminal nuclear-localization signal• High concentration (3 ug/ul) and low glycerol (10%)• Target hard-to-edit cells, including hematopoietic stem cells (HSCs) and human iPS cells (hiPSCs)• Electroporation of mammalian cells with Cas9-sgRNA ribonucleoprotein (RNP) complexes minimizes off-target effects
Deliver sgRNA andCas9 to cells
Start-To-Finish hiPSC Editing and Single-Cell-Cloning SystemsTraditionally, the establishment of a clonal population from edited hiPS cells grown and passaged as colonies is ineffi cient, challenging, and time consuming and often results in cell death or premature differentiation. The Cellartis® gene editing and single-cell cloning systems contain a defi ned culture system for effi cient single-cell cloning and expansion of edited hiPSC clones, based on Cellartis DEF-CS™.
• Cellartis iPSC Single-Cell Cloning DEF-CS Culture Media Kit: A complete system for effi cient expansion and scale-up manufacturing of hiPSCs in a feeder-free and defi ned environment. Superior single-cell survival Maintenance of pluripotency and stable karyotype
• Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System: A complete system that allows effi cient gene editing of hiPSCs via electroporation followed by single-cell cloning and expansion into 48-well plates. Recombinant Cas9, sgRNA In Vitro Transcription Kit, and Cellartis iPSC Single-Cell Cloning DEF-CS Culture Media Kit are included.
• Cellartis iPSC CRISPR/Cas9 Gesicle and Single-Cell Cloning System: A complete system that allows effi cient gene editing of hiPSCs via gesicle delivery of Cas9/sgRNA complexes followed by single-cell cloning and expansion into 48-well plates. Guide-it CRISPR/Cas9 Gesicle Production System and Cellartis iPSC Single-Cell Cloning DEF-CS Culture Media Kit are included.
At the bench: Effi cient CD81 knockout in hiPS cells cultured in DEF-CSHuman induced pluripotent stem cells (hiPSCs) growing in DEF-CS medium were electroporated with Cas9 in a ribonucleoprotein (RNP) complex with sgRNA specifi c to CD81. FACS analysis identifi ed that over 86% of the hiPSCs were CD81(-), indicating a high effi ciency in gene editing.
At the bench: Higher effi ciency compared to other vendor systems
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Deliver sgRNA andCas9 to cells
Guide-it Long ssDNA Production System: Produce ssDNA donor templates for knockinsThis kit uses an effi cient and simple in vitro protocol for the production of long single-stranded DNA (ssDNA) strands for use as repair template in knockin experiments. The kit provides a simple method for converting a dsDNA PCR product into ssDNA by selectively digesting either the sense or the antisense strand. The kit contains suffi cient reagents to create 50 ssDNA strands (25 pairs of sense and antisense strands).
• Create long ssDNA donor templates up to 5 kb• ssDNA can be co-electroporat ed with Cas9/sgRNA ribonucleoprotein (RNP) complexes for delivery into target cells• Benefi ts of using ssDNA as a template over dsDNA:
Drastically reduced tendency to randomly integrate into the genome, resulting in improved gene editing efficiency and lower background Low cytotoxic response to ssDNA template delivery No expression from non-integrated templates, making identification of correctly edited clones significantly easier
At the bench: ssDNA donor template production up to 5 kb
Production of ssDNA from a dsDNA template starting material. Long ssDNA was produced for tagging GADPH and Tyr genes with AcGFP1. The ssDNA has a smaller molecular weight than the corresponding dsDNA (Panel A). Panel B shows the successful production of ssDNA ranging from 2–5 kb for the CCR5 locus.
www.takarabio.com/CRISPR9
A streamlined method for detecting mutationsGuide-it Mutation Detection Kit: Mismatch-based detection of indels• Complete system for confi rming the presence of indels
after genome editing
• Ultra-fast PCR-based method – direct amplifi cation from cells without genomic DNA extraction or sequencing
• Includes Guide-it Resolvase enzyme, a mismatch-specifi c nuclease that provides better specifi city than CEL1 nuclease
• Quick screen to determine the mutation frequency in your cell population, the entire protocol can be completed within 3-4 hrs
Guide-it Resolvase cleavesimperfectly matched DNA
Region of interest is amplified(mutation is marked in blue)
Amplify genomic DNA from cellsusing Terra™ PCR Direct Polymerase
1 Denature and reanneal2
Cleaved and uncleaved PCR fragments have different sizes
3 Separate cleaved and uncleavedPCR fragments using agarosegel electrophoresis
4Uncleaved
Cleavedfragment A
Cleavedfragment B
Uncleaved fragmentCleaved fragment ACleaved fragment B
At the bench: Improved mutation detection versus the CEL1 assay
HEK 293T cells were transfected with plasmids encoding Cas9 and a sgRNA targeting the AAVS1 locus. Mutations were easily discernible when using the Guide-it kit. In contrast, the CEL1 assay showed considerable smearing, making it diffi cult to determine cleavage effi ciency and reducing the ability to detect low levels of mutations.
Guide-it CEL1 nuclease assa y* M 1 2 3 4 5 6 M 1 2 3 4 5 6
bp
468 –
291– 177–
Uncleaved fragment
* Note: Smearing is due to non-specifi c cleavage
Cleavage productCleavage product
Sensitive detection of Cas9 expressionGuide-it Cas9 Monoclonal Antibody (Clone TG8C1)• Recognizes wild-type Cas9 from Streptococcus
pyogenes
• Suitable for western blotting and immunocytochemistry
• High sensitivity and specifi city: recognizes less than 1 ng Cas9 by Western Blot
At the bench: Wild-type and nickase mutant Cas9 are detected with high sensitivity
HEK 293 cells were transfected with plasmids encoding either wt or mutant Cas9 transcripts expressed from the CMV promoter; untransfected cells were used as a negative control. After 48 hours, the cells were lysed and Western blotting was performed. Blots were probed with primary anti-Cas9 polyclonal antibody (1:5,000 dilution). For detection, an HRP-conjugated goat anti-rabbit secondary antibody was used.
250 —
150 —
100 —
75 —
50 —
Unt
rans
fect
ed
wt C
as9
H840AD10A
H840A D10A
Cas9 mutants
H983A N863
Detect Cas9 protein and confi rmgene editing
Guide-it Cas9 Polyclonal Antibody• Recognizes wild-type Cas9, as well as nickase and
nuclease-defi cient versions
• Suitable for western blotting and immunocytochemistry
• High sensitivity and specifi city: recognizes as little as 0.15 ng of recombinant Cas9 by Western Blot
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Determine if one or both copies of your gene have indels
Characterize indels in four steps
Guide-it Genotype Confi rmation Kit: Simple detection of monallelic and biallelic mutations in single clones• Streamlined protocol that uses direct amplifi cation of
target genomic DNA from cells
• Includes highly purifi ed recombinant Cas9 nuclease for in vitro cleavage
Guide-it Indel Identifi cation Kit: Characterize indels at the sequence level• Complete kit contains all of the components needed to amplify, clone, and prepare modifi ed target sites for
DNA sequence analysis
• Ultra-fast protocol includes PCR amplifi cation directly from cells, and the In-Fusion® Cloning system for ligation-free cloning in 15 minutes
• Can be used to characterize the variety of indels in cell populations or to analyze single clones
INDELS
Cas9/sgRNAcleaves
both copies
wt MonoallelicMutation
BiallelicMutation
Cas9/sgRNAcleaves only
wt copy
No Cas9/sgRNA
cleavage
1 largefragment
1 large fragmentand
2 small fragments
2 small fragments
The Guide-it Genotype Confi rmation Kit includes a Cas9/sgRNA-mediated in vitro cleavage reaction for genotype determination after CRISPR/Cas9 gene editing. The same sgRNA that was used for gene editing must be used in the cleavage assay.
HEK 293 cells were treated with Cas9 and an sgRNA targeting the C4BPB gene to generate 15 single-cell clones. Panel A. The Guide-it Genotype Confi rmation Kit was used to determine the genotype, with wt, monoallelic (M), and biallelic (B) control reactions included in the analysis. Panel B. Sequencing results are shown for select clones from Panel A. Lowercase letters represent the wt sequence. As predicted, clone 2 is monoallelic and clones 7 and 9 are biallelic.
Identify indels
Controls
Clone #2
7
9
Individual clones after C4BPB editing
WT M B WT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Target sitePAM
Target site
PAM
Target sitePAM
Target sitePAM11
A
B
At the bench: Accurate genotype determination in HEK 293 cells
www.takarabio.com/CRISPR11
P R O D U C T SCat. # Product Size
Transcribe and screen sgRNAs in vitro
632636 Guide-it™ Complete sgRNA Screening System (includes Cat. # 632635, 632638, and 632639) 50 Rxns
632635 Guide-it™ sgRNA In Vitro Transcription Kit 50 Rxns
632638 Guide-it™ IVT RNA Clean-Up Kit 50 Rxns
632639 Guide-it™ sgRNA Screening Kit 50 Rxns
Deliver sgRNA and Cas9 into cells
632601 Guide-it™ CRISPR/Cas9 System (Green) 1 System
632602 Guide-it™ CRISPR/Cas9 System (Red) 1 System
632608 AAVpro® CRISPR/Cas9 Helper Free System (AAV2) 1 System
632609 AAVpro® CRISPR/Cas9 Vector System 1 System
632619 AAVpro® CRISPR/SaCas9 Helper Free System (AAV2) 1 System
632618 AAVpro® CRISPR/SaCas9 Vector System 1 System
632629 Lenti-X™ CRISPR/Cas9 System Each
632630 pLVX-hyg-sgRNA1 Vector System 10 Rxns
632631 pLVX-puro-Cas9 Vector 20 uL
632633 Lenti-X™ Tet-On® 3G CRISPR/Cas9 System 1 System
631450 Xfect™ RNA Transfection Reagent 1.2 mL
632613 Guide-it™ CRISPR/Cas9 Gesicle Production System (includes Cat. # 632612 and 632616) 1 System
632617 Gesicle Producer 293T Cell Line 1 mL
632612 pGuide-it-sgRNA1 Vector System 1 System
632616 Guide-it™ CRISPR/Cas9 Gesicle Packaging Set 10 Rxns
632642 Cellartis® iPSC CRISPR/Cas9 Gesicle and Single-Cell Cloning System 1 System
632640 Guide-it™ Recombinant Cas9 (Electroporation-Ready) 3 x 100 ug
632641 Guide-it™ Recombinant Cas9 (Electroporation-Ready) 100 ug
632643 Cellartis® iPSC rCas9 Electroporation and Single-Cell Cloning System 1 System
Y30021 Cellartis® iPSC Single-Cell Cloning DEF-CS™ Culture Media Kit 1 Kit
632644 Guide-it™ Long ssDNA Production System 25 Rxns
632645 Guide-it™ Long ssDNA Strandase Kit 25 Rxns
Confi rm Cas9 expression
632606 Guide-it™ Cas9 Polyclonal Antibody 3 x 100 uL
632607 Guide-it™ Cas9 Polyclonal Antibody 100 uL
632627 Guide-it™ Cas9 Monoclonal Antibody (Clone TG8C1) 3 x 100 ug
632628 Guide-it™ Cas9 Monoclonal Antibody (Clone TG8C1) 100 ug
Determine genome editing effi ciency in target cell population
631443 Guide-it™ Mutation Detection Kit 100 Rxns
631448 Guide-it™ Mutation Detection Kit 25 Rxns
Identify Indels
632611 Guide-it™ Genotype Confi rmation Kit 100 Rxns
631444 Guide-it™ Indel Identifi cation Kit 10 Rxns
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Gene Editing Resources
www.takarabio.com/CRISPR
Visit our website to explore our evolving portfolio of products, learning resources, and technical information for CRISPR/Cas9 gene editing.
• New products• Selection guides• Technical notes• Videos• Protocols• Learning resources
Takara Bio EuropeordersEU@takarabio.com • techEU@takarabio.com • infoEU@takarabio.com • +33 139 046 880For Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale. Takara and the Takara logo are trademarks of TAKARA HOLDINGS, Kyoto, Japan. AAVpro is a trademark of Takara Bio, Inc. Clontech, the Clontech logo, Guide-it, In-Fusion, that’s GOOD science!, Terra, and Xfect are trademarks of Takara Bio USA, Inc. All other marks are the property of their respective owners. Certain trademarks may not be registered in all jurisdictions.©2017 Takara Bio Europe
ZZGIBR0817www.takarabio.com
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