Download - Genome Modulation
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Genome modulation and editingGain the confidence to imagine, innovate, and engineer
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2 Life Technologies | Genome editing
Our missionInquire, understand, break through to discovery
We believe genome editing will change the way we create energy, produce food, optimize industrial processing, and detect, prevent, and cure diseasesimproving the human condition and the world around us. We are committed to offering unparalleled technology and solutions to the research community.
Through design and engineering, this unique science enables researchers to study, alter, create, and re-create highly complex pathways, DNA sequences, genes, and natural biological systems to understand and answer some of lifes most challenging questions. Weve developed this interactive electronic flip book to give you an understanding of the technologies and tools available today, and to guide you in selecting the right tools and solutions to enable you to break through to discovery faster and with less effort.
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3Life Technologies | Genome editing
Contents
Introduction to genome modulation and editing 2
Genome modulation and editing workflow 4
Genome modulation and editing technologies 6
Genome editing technologies 8
What is genome editing? 8
Genome editing selection guide 10
CRISPR-Cas9 technology 12 GeneArt CRISPR-Cas9 tools
TAL effector technology 24 GeneArt TAL effector tools
Site-specific gene integration tools 32
Gene modulation RNAi technologies 36
Transfection tools 44
Superior Transfecton Reagents 44
Genome editing detection and analysis tools 48
Detect and analyze 48
GeneArt Genomic Cleavage Detection Kit 50
GeneArt Genomic Cleavage Selection Kit 54
Genome editing analysis tools 56
Custom cell line engineering services 60
Support 62
Ordering information 64
Des
ign
Det
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ana
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Del
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4 Life Technologies | Genome editing
Genome engineering combines molecular biology and engineering principles to generate modified genotypes, enabling researchers to study how the genome influences phenotype. We have developed tools and solutions for every step in the cell engineering workflow, and this combined tool set comprises a collection of optimized, validated technology systems designed to work together to help answer important scientific questions faster and with less effort.
From bioinformatics in silico design tools, to genome modification tools, to transfection, cell culture, and sample preparation, to detection and analysis of genetic variants, we have what you need. Our featured technologies include Flp-In and Jump-In protein expression systems, widely used RNAi, with siRNA and miRNA for gene modulation, and the newest technologies: GeneArt TALs and GeneArt CRISPR-Cas9. For delivery, we offer reagents for a variety of different transfection methods, and to complete the workflow, we have reagents for monitoring transfection and cleavage efficiency, and for analysis of knockdown/knockout events and off-target effects.
Our goal is a broad, integrated cell engineering workflow solution that brings together the power of cutting-edge science from multiple disciplines to create a single source of genome engineering tools and technology for the researcher of the future. Balancing performance and cost, our portfolio is built on 20 years of industry-leading innovation and can grow with your research needs.
Genome modulation and editing workflowDiscover the most comprehensive genome-engineering portfolio
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5Life Technologies | Genome editing
Design ( Identify targets sites and design)
Deliver (Transfection)
Detect and analyze(Screen and enrich)
Genome editing GeneArt CRISPR-Cas9
GeneArt TAL effectors
Gene modulation Ambion miRNA mimics
and inhibitors
Silencer Select siRNA
Homologus recombination Flp-In System
Jump-In Ccell engineering
platform
Lipofectamine 3000 reagent
Lipofectamine RNAiMAX reagent
Lipofectamine MessengerMAX reagent
Lipofectamine 2000 reagent
Neon Transfection System
GeneArt Genomic Cleavage Detection Kit
Real-time PCR assay
Ion Torrent next-generation sequencing
Cell sorting or bead-based enrichment
Complete workflow solution Complete set of tools and reagents for gene engineering workflow Custom services available for each step in the workflow or for the entire workflow Cell line generation services leveraging genome editing tools and cell culture reagents
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6 Life Technologies | Genome editing
Genome modulation and editing technologiesFind the right technology for your application
Technology class RNAi Site-specific gene integration CRISPR-Cas9 tools TAL effector toolsRequirement Transient knockdown of multiple transcripts Targeted integration of your gene of interest
into specific integration sitesRapid and efficient editing with multiplexing capabilities
Precise and flexible editing; targeting to any gene in any cell, with all-around freedom
Products Silencer Select siRNAAmbion miRNA mimics and inhibitors
Jump-In Targeted Integration KitsFlp-In System
GeneArt CRISPR Nuclease VectorGeneArt CRISPR Nuclease mRNA
GeneArt Precision TALsGeneArt PerfectMatch TALs
Benefits High-potency, transient knockdown of target transcript with lower off-target effects (siRNA)
Medium potency targeting of multiple transcripts with shared seed sequence (miRNA)
Rapid and highly efficient production of nonisogenic or isogenic stable cell lines
Easy-to-design genome engineering system Affordable, ready-to-use mRNA or cloning vectors Enrichment for hard-to-transfect or difficult-to-modify
cell lines (GeneArt CRISPR Nuclease Vector) Multiplexing and screening capable, with easy
construction and low cost
Precise technology that recognizes the DNA sequence you specify
Flexible design lets you choose the effectors and Gateway cloningcompatible vector that meet your needs
Reliable, because the final clone contains a verified, optimized sequence for improved expression
Fast service delivers your clone, typically within 2 weeks of your order confirmation
Independent of target sequence, with no restrictions for GeneArt PerfectMatch TALs
Modification options Downregulation (knockdown) Integration (knock-in) Gene deletion (knockout) Integration (knock-in)
Gene deletion (knockout) Downregulation (knockdown) Integration (knock-in) Gene activation
Ease of use Predesigned constructs available for transfection in cell culture
Easy vector construction, transfection, and clone identification
Simple and fast design process Difficult to design (except when using the GeneArt TALs online design tool)
Type of recognition RNARNA DNADNA RNADNA ProteinDNAIssues in recognition
Seed region could play a role Multiple integration sites may be targeted by PhiC31 in the Jump-In Fast System
FRT site (34 bp) in genome is needed for gene integration; this is provided in the Flp-In cell line
PAM site (NGG at the end of the 20-bp target sequence)
Active range of spacing needed for activity, no design constraints
Cell type Mammalian Mammalian
Areas of application Gene function Gene-based therapeutics Gene modulation Pathway analysis Systems biology
Protein production Antibody production
Gene function Gene modulation Gene tagging Pathway engineering Metabolic engineering Protein production
Gene function Gene modulation Gene tagging Pathway engineering Metabolic engineering Protein production
Limitations Only for mRNA cleavage; degradation of mRNA is transient
Only for transient inhibition of translation (miRNA)
Only for integrations Off-target effects Inhibited by chromatin structure Large size for delivery
Off-target effects Moderate to high Low Moderate LowMultiplexing Capable No Capable Rarely used
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7Life Technologies | Genome editing
With all the tools and technologies available today, which ones are right for your research? Wed like to help simplify your choice, based on your research needs. Once you determine which technology best suits your needs, you can learn more about the specific tools available in the following sections
Genome modulation and editing technologiesFind the right technology for your application
Technology class RNAi Site-specific gene integration CRISPR-Cas9 tools TAL effector toolsRequirement Transient knockdown of multiple transcripts Targeted integration of your gene of interest
into specific integration sitesRapid and efficient editing with multiplexing capabilities
Precise and flexible editing; targeting to any gene in any cell, with all-around freedom
Products Silencer Select siRNAAmbion miRNA mimics and inhibitors
Jump-In Targeted Integration KitsFlp-In System
GeneArt CRISPR Nuclease VectorGeneArt CRISPR Nuclease mRNA
GeneArt Precision TALsGeneArt PerfectMatch TALs
Benefits High-potency, transient knockdown of target transcript with lower off-target effects (siRNA)
Medium potency targeting of multiple transcripts with shared seed sequence (miRNA)
Rapid and highly efficient production of nonisogenic or isogenic stable cell lines
Easy-to-design genome engineering system Affordable, ready-to-use mRNA or cloning vectors Enrichment for hard-to-transfect or difficult-to-modify
cell lines (GeneArt CRISPR Nuclease Vector) Multiplexing and screening capable, with easy
construction and low cost
Precise technology that recognizes the DNA sequence you specify
Flexible design lets you choose the effectors and Gateway cloningcompatible vector that meet your needs
Reliable, because the final clone contains a verified, optimized sequence for improved expression
Fast service delivers your clone, typically within 2 weeks of your order confirmation
Independent of target sequence, with no restrictions for GeneArt PerfectMatch TALs
Modification options Downregulation (knockdown) Integration (knock-in) Gene deletion (knockout) Integration (knock-in)
Gene deletion (knockout) Downregulation (knockdown) Integration (knock-in) Gene activation
Ease of use Predesigned constructs available for transfection in cell culture
Easy vector construction, transfection, and clone identification
Simple and fast design process Difficult to design (except when using the GeneArt TALs online design tool)
Type of recognition RNARNA DNADNA RNADNA ProteinDNAIssues in recognition
Seed region could play a role Multiple integration sites may be targeted by PhiC31 in the Jump-In Fast System
FRT site (34 bp) in genome is needed for gene integration; this is provided in the Flp-In cell line
PAM site (NGG at the end of the 20-bp target sequence)
Active range of spacing needed for activity, no design constraints
Cell type Mammalian Mammalian
Areas of application Gene function Gene-based therapeutics Gene modulation Pathway analysis Systems biology
Protein production Antibody production
Gene function Gene modulation Gene tagging Pathway engineering Metabolic engineering Protein production
Gene function Gene modulation Gene tagging Pathway engineering Metabolic engineering Protein production
Limitations Only for mRNA cleavage; degradation of mRNA is transient
Only for transient inhibition of translation (miRNA)
Only for integrations Off-target effects Inhibited by chromatin structure Large size for delivery
Off-target effects Moderate to high Low Moderate LowMultiplexing Capable No Capable Rarely used
Mammalian Bacterial Yeast
Insect Stem cells Zebrafish
Mammalian Bacterial Yeast Plants
Insect Stem cells Zebrafish
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8 Life Technologies | Genome editing
Genome editing is precise, site-specific DNA modification in a live cell. Genome editing involves the use of engineered nucleases, in conjunction with endogenous repair mechanisms, to insert, delete, or replace DNA sequences from a specific location in genomic DNA (Figure 1). The ability to edit the genome in a precise and targeted manner can be used to provide a more comprehensive understanding of biology and disease mechanisms. Genome editing has a variety of applications, such as creating disease-resistant transgenic plants, stem cell engineering, and gene therapy, and is also widely used in creating tissue and animal disease models.
What is genome editing?Engineering made easy with engineered nucleases
GENEDISRUPTION
GENEINSERTION INVERSION
GENEDELETION
NON-HOMOLOGOUS END JOINING (NHEJ)Without added homologous DNA:
repairs with indels
GENECORRECTION
GENEADDITION
HOMOLOGY-DIRECTED REPAIR (HR)With added homologous DNA:
insert/replace DNA
Cells repair mechanism is harnessed to heal DNA breaks
CHROMOSOME & ENGINEERED NUCLEASE
DNA-SPECIFIC DOUBLE-STRANDED BREAK
Figure 1. Engineered nuclease-mediated genome editing. Engineered nucleases such as the CRISPR-Cas9 or TAL effectors can be designed to target specific sites in the genome, creating double-strand breaks (DSBs) at desired locations. The natural repair mechanisms of the cell repair the break by either homologous recombination (HR) or non-homologous end joining (NHEJ). HR is more precise, since it requires a template, allowing the introduction of foreign DNA into the target gene. Homologous DNA donor sequences can be used with homology-directed repair (HDR) to introduce a defined new DNA sequence. DSB repair by NHEJ is likely to introduce errors such as insertions or deletions (indels), leading to a nonfunctional gene.
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9Life Technologies | Genome editing
Get your free copy of the Evolution of Genome Engineering poster*
Find publications on genome editing here.
CO09619 0914
* Terms and conditions apply. For complete details, go to lifetechnologies.com/genomeeditposters
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10 Life Technologies | Genome editing
Recent advances in molecular and cell biology have equipped researchers with powerful genome editing tools, such as the clustered regularly interspaced short palindromic repeat (CRISPR) system and transcription activatorlike (TAL) effectors, thereby making it relatively easier to target and regulate user-defined endogenous genes in a sequence-specific manner. Here is a guide designed to help you select the right technology in the right format for your needs.
Genome editing selection guideFind the right genome editing technology for your application
Mammalian cells
Do you have target sequence constraints (i.e., lack of PAM sequence)?
Will you useedited cells for
commercial applications?
Multiplex? What is yourdownstream applicationor host cell type?
What is yourdownstream application
or host cell type?
GeneArt CRISPRnuclease mRNA/gRNA
In vivo applications (i.e., microinjection)
GeneArt CRISPR nuclease mRNA/gRNA
GeneArt CRISPR nuclease vector
In vivo applications(i.e., microinjection)Mammalian cells
GeneArt TAL mRNA service
GeneArt TAL mRNA service
GeneArt TAL vector
GeneArt CRISPRnuclease mRNA/gRNA
YesNo
YesNo
YesNo
YesNo
GeneArt TALmRNA service
GeneArt TALvector
GeneArt TALvector**
Are you working with plants?
Yourdesiredgene edit
Generepression(knockdown)
Customeffectordomain
Geneactivation
GeneArt TAL VP16 or VP64activator effector domain
Knockoutor
knock in*
GeneArt TAL KRABrepressor effector domain
GeneArt TAL
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11Life Technologies | Genome editing
Recent advances in molecular and cell biology have equipped researchers with powerful genome editing tools, such as the clustered regularly interspaced short palindromic repeat (CRISPR) system and transcription activatorlike (TAL) effectors, thereby making it relatively easier to target and regulate user-defined endogenous genes in a sequence-specific manner. Here is a guide designed to help you select the right technology in the right format for your needs.
Mammalian cells
Do you have target sequence constraints (i.e., lack of PAM sequence)?
Will you useedited cells for
commercial applications?
Multiplex? What is yourdownstream applicationor host cell type?
What is yourdownstream application
or host cell type?
GeneArt CRISPRnuclease mRNA/gRNA
In vivo applications (i.e., microinjection)
GeneArt CRISPR nuclease mRNA/gRNA
GeneArt CRISPR nuclease vector
In vivo applications(i.e., microinjection)Mammalian cells
GeneArt TAL mRNA service
GeneArt TAL mRNA service
GeneArt TAL vector
GeneArt CRISPRnuclease mRNA/gRNA
YesNo
YesNo
YesNo
YesNo
GeneArt TALmRNA service
GeneArt TALvector
GeneArt TALvector**
Are you working with plants?
Yourdesiredgene edit
Generepression(knockdown)
Customeffectordomain
Geneactivation
GeneArt TAL VP16 or VP64activator effector domain
Knockoutor
knock in*
GeneArt TAL KRABrepressor effector domain
GeneArt TAL
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12 Life Technologies | Genome editing
Technology overviewClustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins are derived from bacteria and archaea, where they are part of an adaptive immune system that protects the organism against invading DNA (Figure 2).
Three-component CRISPR-Cas9 editingGenome editing uses engineered nucleases in conjunction with endogenous repair mechanisms to alter the DNA in a cell. The CRISPR-Cas9 system takes advantage of a short guide RNA to target the bacterial Cas9 endonuclease to specific genomic loci. Because the guide RNA supplies the specificity, changing the target only requires a change in the design of the sequence that encodes the guide RNA. The CRISPR-Cas9 system used in gene editing consists of three components: the Cas nuclease Cas9 (a double-stranded DNA endonuclease), a target-complementary crRNA, and an auxiliary tracrRNA (see Figure 3).
CRISPR-Cas9 technologyRevolutionizing the field of genome editing
Figure 2. CRISPR-Cas9 mechanism. Small segments of plasmids or viral genomes from an earlier infection are incorporated into CRISPR loci. CRISPR loci are transcribed and processed into short crRNAs that are complementary to previously encountered foreign DNA. The mechanism of target recognition relies on a sequence within the crRNA and a conserved sequence adjacent to the crRNA binding region called the protospacer-adjacent motif (PAM). A trans-activating crRNA (tracrRNA) pairs with a complementary pre-crRNA to form a duplex. That RNA duplex is cut by RNase III to form the so-called guide RNA (gRNA), a crRNA-tracrRNA chimera that guides Cas9 endonuclease to cleave and inactivate a new intruder.
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13Life Technologies | Genome editing
Figure3. A CRISPR-Cas9 targeted double-strand break. Cleavage occurs on both strands, 3 bp upstream of the NGG protospacer- adjacent motif (PAM) sequence at the 3 end of the target sequence. The specificity is supplied by the guide RNA (gRNA), and changing the target only requires a change in the design of the sequence encoding the guide RNA. After the gRNA unit has guided the Cas9 nuclease to a specific genomic locus, the Cas9 protein induces a double-strand break (DSB) at the specific genomic target sequence.
Watch this video for a quick understanding of how the CRISRP-Cas9 system works. Bench Tip Video: CRISPR-Cas Genome Editing Technology
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14 Life Technologies | Genome editing
With their highly flexible but specific targeting, CRISPR-Cas9 systems can be manipulated and redirected to become powerful tools for genome editing. CRISPR-Cas9 technology permits targeted gene cleavage and gene editing in a variety of cells, and because the endonuclease cleavage specificity in CRISPR-Cas9 systems is guided by RNA sequences, editing can be directed to virtually any genomic locus by engineering the guide RNA sequence and delivering it along with the Cas endonuclease to your target cell. Based on your research needs, you can choose from our two different formats of CRISPR tools: CRISPR-Cas9 all-in-one expression plasmids, or CRISPR-Cas9 mRNA and gRNA. You can find more detailed information about both formats on the following pages.
GeneArt CRISPR-Cas9 toolsRapid and efficient editing with multiplexing capabilities
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15Life Technologies | Genome editing
LABTalks: CRISPR-Cas9: simple and versatile genome editing tool
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16 Life Technologies | Genome editing
GeneArt CRISPR Nuclease Vector Kits are reporter vector systems for expression of the functional components needed for CRISPR-Cas9 genome editing in mammalian cells. This system offers a simple, ready-to-use, all-in-one expression vector system consisting of both a Cas9 nuclease expression cassette and a guide RNA (gRNA) cloning cassette for rapid and efficient cloning of DNA that encodes target-specific
CRISPR RNA (crRNA). These kits are available with two different reporters (Figure 4). GeneArt CRISPR nuclease vectors with orange fluorescent protein (OFP) allow fluorescence-activated cell sorting (FACS) of cell populations expressing Cas9 and gRNA, whereas GeneArt CRISPR nuclease vectors with CD4 enable bead-based enrichment of cells expressing Cas9 and gRNA. The linearized GeneArt CRISPR nuclease vectors provide a rapid and efficient way to clone double-stranded oligonucleotides encoding a crRNA representing a desired target into an expression cassette that allows sequence-specific targeting of the Cas9 nuclease.
Want us to design your target oligonucleotide and clone it for you?
Let us know at [email protected], and well design the sequence and provide you with 100 g of transfection-quality DNA.
Find out more or place an order at lifetechnologies.com/crisprvectors
GeneArt CRISPR Nuclease Vector KitsEfficient cloning, streamlined workflow, faster results
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17Life Technologies | Genome editing
Ordering information
Product Quantity Cat. No.
GeneArt CRISPR Nuclease Vector: OFP Reporter 10 reactions A21174
GeneArt CRISPR Nuclease Vector: OFP Reporter with Competent Cells (Combo)
10 reactions A21178
GeneArt CRISPR Nuclease Vector: CD4 Enrichment 10 reactions A21175
GeneArt CRISPR Nuclease Vector: CD4 Enrichment with Competent Cells (Combo)
10 reactions A21177
Figure 4 . GeneArt CRISPR nuclease vector maps. The vector is prelinearized with 5 bp overhangs for easy cloning of your double-stranded DNA oligo that encodes a target-specific crRNA. Maps are shown of the vectors with (A) an OFP reporter and (B) a CD4 reporter. The gRNA, Cas9, and reporter are expressed from the same vector. Cas9 is directed to the nucleus by nuclear localization signals (NLS1 and NLS2).
TK pA
U6 promoterCACCG
CAAAAPol IIIterm
tracrRNA
F1 origin
SD4
2A
Cas9 (with NLS1 and NLS2)
PCMV
Ampicillin
pUC origin
CRISPR Nuclease CD4 Reporter 9,822 bp
B
TK pA
U6 promoterCACCG
CAAAAPol IIIterm
tracrRNA
F1 origin
OFP
2A
Cas9 (with NLS1 and NLS2)
PCMV
Ampicillin
pUC origin
CRISPR Nuclease OFP Reporter 9,219 bp
A
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18 Life Technologies | Genome editing
In the GeneArt CRISPR Nuclease mRNA System, crRNA and tracrRNA are expressed together as a single gRNA that mimics the natural crRNA-tracrRNA hybrid in bacterial systems. The gRNA is encoded by a custom-synthesized DNA fragment (GeneArt CRISPR Strings DNA) containing either a U6 or T7 promoter. GeneArt CRISPR U6 Strings DNA is synthesized with
a U6 promoter and can be directly introduced into mammalian cells for expression of the gRNA. GeneArt CRISPR T7 Strings DNA is synthesized with a T7 promoter and can be used as a template for in vitro transcription of the gRNA. Either of these target-specific gRNAs is cotransfected into cells with GeneArt CRISPR Nuclease mRNA, which encodes the Cas9 endonuclease (Figure 5). The system is versatile and simple to use, and changing target specificity only requires a change in the design of the GeneArt CRISPR Strings DNA.
A custom service option is available when a ready-to-use, complete RNA format is desired. Submit your gene of interest to [email protected] and receive your target-specific IVT gRNA that is ready for use in your applications.
Find out more at lifetechnologies.com/CRISPRmRNA
GeneArt CRISPR Nuclease mRNA SystemHigh-efficiency CRISPR genome editing tools for multiplex editing
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19Life Technologies | Genome editing
Figure 4
A. 293FT cells, HPRT locus
Clea
vage
efficien
cy (%
)
60
50
40
30
20
10
01 2 3 4
500 bp400 bp300 bp
200 bp
Cleavage efficiency: 32% 36% 51% 0% 11% 25% 51% 0% 10% 9% 32% 0% 4% 17% 49% 0%
CRISPR-Cas9 format Lane
All-in-one plasmid 1
Cas9 mRNA + Strings DNA 2
Cas9 mRNA + IVT gRNA 3
Control (Cas9 mRNA only) 4
C. U2OS cells, RELA locus
Clea
vage
efficien
cy (%
)
60
50
40
30
20
10
01 2 3 4
300 bp200 bp
CRISPR-Cas9 format Lane
All-in-one plasmid 1
Cas9 mRNA + Strings DNA 2
Cas9 mRNA + IVT gRNA 3
Control (Cas9 mRNA only) 4
Clea
vage
efficien
cy (%
)
500 bp400 bp300 bp
200 bp
CRISPR-Cas9 format Lane
HPRT RELA
All-in-one plasmid 1 5
Cas9 mRNA + Strings DNA 2 6
Cas9 mRNA + IVT gRNA 3 7
Control (Cas9 mRNA only) 4 8
B. HCT116 cells, HPRT and RELA loci
60HPRT RELA
50
40
30
20
10
0 1 2 3 4 5 6 7 8
Assay for:
GeneArt CRISPR Nuclease mRNA SystemHigh-efficiency CRISPR genome editing tools for multiplex editing
Ordering information
Product Quantity Cat. No.
GeneArt CRISPR Nuclease mRNA 15 g A25640
GeneArt Strings U6 DNA >200 ng Contact [email protected]
GeneArt Strings T7 DNA >200 ng Contact [email protected]
Custom in vitro transcribed gRNA 250 nmol Contact [email protected]
Figure 5. The GeneArt CRISPR Nuclease mRNA System demonstrates efficient genome editing in a broad range of cell types. (A) 293FT, (B) HCT116, and (C) U2OS human cell lines were transfected in 24-well plates using the indicated CRISPR-Cas9 format, targeting the HPRT or RELA locus. The CRISPR-Cas9 formats and corresponding sample lane numbers are listed in the tables. The GeneArt CRISPR Nuclease Vector with OFP reporter was transfected using Lipofectamine 3000 Transfection Reagent, the Strings DNA format was transfected using Lipofectamine 2000 Transfection Reagent, and the IVT gRNA format was in vitro transcribed using delivered MEGAshortscript T7 Transcription Kit (Cat. No. AM1354) and MEGAclear Transcription Clean-Up Kit (Cat. No. AM1908), and then transfected using Lipofectamine MessengerMAX Reagent. At 72 hours post-transfection, cells were harvested and genome editing efficiency was quantified using the GeneArt Genomic Cleavage Detection Kit (Cat. No. A24372). The cleavage products are indicated with arrowheads.
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20 Life Technologies | Genome editing
Further your knowledge by downloading the latest technical product guide or viewing one of our on-demand webinars.
For detailed design strategy and target specificity guidelines, refer to our technical product guide.
TECHNICAL PRODUCT BULLETIN GeneArt CRISPR Nuclease Vector Kit
GeneArt CRISPR Nuclease Vector KitRapid and efficient genome editing from a single vector
IntroductionThe Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) system is the latest addition to the genome editing toolbox, offering a simple, rapid, and efficient solution. The CRISPR/Cas system is a prokaryotic adaptive immune system that uses an RNA-guided DNA nuclease to silence viral nucleic acids [1]. The type II CRISPR/Cas system from the bacterium Streptococcus pyogenes has been modified to enable editing of mammalian genomes [2, 3]. As a simple two-component system composed of Cas9 protein and a non-coding guide RNA (gRNA), the engineered type II CRISPR/Cas system can be utilized to cleave genomic DNA at a predefined target sequence of interest.
The gRNA has two molecular components, a target complementary CRISPR RNA (crRNA), and an auxiliary trans-activating crRNA (tracrRNA). The gRNA unit guides the Cas9 nuclease to a specific genomic locus and the Cas9 protein induces a double-strand break (DSB) at the specific genomic target sequence (Figure 1). Following CRISPR/Cas9-induced DNA cleavage, the DSB can be repaired by the cellular repair machinery using either non-homologous end joining (NHEJ) or a homology-directed repair mechanism. This product bulletin provides detailed information about our GeneArt CRISPR Nuclease Vector technology along with recommendations for using the kits.
Target-specific crRNA
Target genomic loci PAM
C G T A A A G C C A T A C G T A T A C T A C C
G C A T T T C G G T A T G C A T A T G A N G G
G G GGC C CA A
A
AU U U UG
AA UU U GA U
tracrRNAA A A A A A
AAAAA
A
A
AAAAA
A AA
G
G
GG
G
G
G G G
G
G
GG
GGG
G
G
G C C C CC
C
C
C
CC
CC
UU
U U U
UU
U
U
UUUU
U
U U
UUU
AA
A AA
U
UU
U
Cas9
Figure 1. A CRISPR/Cas9 targeted double-strand break. Cleavage occurs on both strands, 3 bp upstream of the NGG proto-spacer adjacent motif (PAM) sequence on the 3 end of the target sequence.
Find out more at lifetechnologies.com/crispr
PG1321-PJ5212-CO28372-CRISPR Technical Product Bulletin (Global).indd 1 11/15/13 1:23 PM
CRISPR-Cas9 Technical ResourcesLet our knowledge work for you
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21Life Technologies | Genome editing
Watch this webinar to get an overview of CRISPR-Cas9mediated genome editing and learn about the GeneArt CRISPR Nuclease System. The presenter, Namritha Ravinder, is a staff scientist with our Synthetic Biology Divisions Genetic Circuits and Cell Engineering team.
CRISPR-Cas9 Technical ResourcesLet our knowledge work for you
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22 Life Technologies | Genome editing
GeneArt CRISPR Nuclease mRNA SystemHigh-efficiency, multiplex-compatible genome editing tool for a broad range of cell types
PRODUCT BULLETIN GeneArt CRISPR Nuclease mRNA System
IntroductionClustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins have revolutionized the field of cell engineering. Derived from components of a simple bacterial immune system, the CRISPR-Cas9 system can be manipulated and redirected for highly flexible but specific genome editing in eukaryotes. The CRISPR-Cas9 system is composed of a short noncoding guide RNA (gRNA) that has two molecular components: a target-specific CRISPR RNA (crRNA) and an auxiliary trans-activating crRNA (tracrRNA). The gRNA unit guides the Cas9 protein to a specific genomic locus via base pairing between the crRNA sequence and the target sequence (Figure 1).
Upon binding to the target sequence, the Cas9 protein induces a specific double-strand break. Following CRISPR-Cas9induced DNA cleavage, the break can be repaired by the cellular repair machinery using either non-homologous end joining (NHEJ) or a homology-directed repair mechanism. With target specificity defined by a very short RNA-coding region, the CRISPR-Cas9 system greatly simplifies genome editing and has great promise in broad applications such as stem cell engineering, gene therapy, tissue and animal disease models, and engineering disease-resistant transgenic plants.
In the GeneArt CRISPR Nuclease mRNA System, crRNA and tracrRNA are expressed together as a single gRNA that mimics the natural crRNA-tracrRNA hybrid in bacterial systems. The gRNA is encoded by a custom-synthesized DNA fragment containing either a U6 or T7 promoter. GeneArt CRISPR U6 Strings DNA is synthesized with a U6 promoter and can be directly introduced into mammalian cells for expression of the gRNA. GeneArt CRISPR T7 Strings DNA is synthesized with a T7 promoter and is used as a template for in vitro transcription of the gRNA. This target-specific gRNA is cotransfected into cells with GeneArt CRISPR Nuclease mRNA, which encodes the Cas9 endonuclease. The
Target genomic locus
Figure 1. A CRISPR-Cas9 targeted double-strand break. Cleavage occurs on both strands, 3 bp upstream of the NGG proto-spacer adjacent motif (PAM) sequence on the 3 end of the target sequence.
Further your knowledge by downloading the latest technical product guide or viewing one of our on-demand webinars at your leisure.
Did you know?
You can find a lot of data and application examples in our GeneArt CRISPR Nuclease mRNA product bulletin.
CRISPR-Cas9 technical resourcesLet our knowledge work for you
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23Life Technologies | Genome editing
Watch this webinar on simple and versatile CRISPR-based genome editing and its promise in large-scale cell engineering applications.
CRISPR-Cas9 technical resourcesLet our knowledge work for you
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24 Life Technologies | Genome editing
Technology overviewTranscription activatorlike (TAL) effector proteins are produced by bacteria in the genus Xanthomonas, which are widely distributed plant pathogens. Natural TAL effectors bind to specific sequences of host DNA, altering the infected plants gene expression in ways that further the disease process. The natural TAL effector proteins have two distinct domains: an effector domain and an extraordinarily specific DNA-binding domain. The DNA-binding domain consists of a variable number of amino acid repeats (Figure 6). Each repeat contains 3335 amino acids and recognizes a single DNA base pair. The DNA recognition occurs via 2 hypervariable amino acid residues at positions 12 and 13 within each repeat, called repeat-variable di-residues (RVDs).
Watch this online tutorial, presented by Dr. Jon Chesnut, research fellow and R&D lead, to learn more about GeneArt Precision TAL technology.
TAL effector technologyPrecise and extraordinarily flexible genome editing
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25Life Technologies | Genome editing
Working with stem cells? Check out these new resources!Stem cell engineering resources
On-demand webinar: Watch this webinar as we examine genetic engineering of iPSCs derived from Parkinsons disease samples, which provide model systems to study disease mechanisms in cell types not previously available.
White paper: Parkinsons disease cell modelspart 3: Genome editing of Parkinsons disease donor-derived iPSC lines using GeneArt Precision TAL technology
Video: Moving forward: future directions for understanding Parkinsons disease
Figure 6. TAL effector DNA-binding domain. The structure of the DNA-binding domain can be manipulated to produce a protein domain that binds specifically to any DNA sequence in the genome. TAL effector repeats can be assembled modularly, varying the RVDs to create a TAL protein that recognizes a specific target DNA sequence. Linking the repeats is straightforward, and long TAL effectors can be designed to specifically target any locus in the genome.
Repeat-variable di-residues (RVD)
Genome editing of Parkinsons disease donor-derived iPSC lines using GeneArt Precision TALs technology
Parkinsons disease cell modelspart 3
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26 Life Technologies | Genome editing
TAL effectors are a widely used technology for precise and efficient gene editing in live cells. The deciphering of the TAL effector code led to the engineering of designer TAL effector proteins. We have recently secured rights to certain intellectual property (e.g., patented methods) around TALs, clarifying a path for you to move confidently forward and innovate. GeneArt TALs provide custom DNA binding proteins engineered and designed for accurate DNA targeting and precise genome editing. GeneArt TALs offer site-specific delivery of nucleases, activators, repressors, chromatin modifiers, genomic labels, and crosslinking molecules (Figure 7). This genome editing technology is known to function in a variety of host systems, including bacteria, yeast, plants, insects, fish, and mammals. Based on your research needs, you can select from our two different formats of TAL effector tools: GeneArt Precision TALs or GeneArt PerfectMatch TALs (Figure 8). Choose GeneArt Precision TALs when working with plants, or if you have no design constraints. Choose GeneArt PerfectMatch TALs when you need complete flexibility in target design, removing the 5 T constraint for targets. GeneArt PerfectMatch TALs are derived from GeneArt Precision TALs and contain a truncated TAL effector fused to a FokI nuclease domain. GeneArt PerfectMatch TALs contain 3 amino acids mutated at the N terminus of the TAL effector of a GeneArt Precision TAL. The mutation converts the 5 T binding motif at the TAL effector N terminus to a universal binding motif (it will bind to any base: A, G, C, or T).
GeneArt TAL effector toolsPrecise and flexible editing with freedom to innovate
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27Life Technologies | Genome editing
Figure 8. Designing target sites for customized TAL effectors for maximal binding. (A) GeneArt Precision TALs encode a DNA-binding protein specific to a customer-submitted sequence, fused to a FokI nuclease domain for genome editing. The sequence targeted by our first-generation TAL effectors must have a T at its 5 end, and spacing between forward and reverse TALs must be 1318 bp for proper pairing of the FokI nucleases and creation of a double-stranded break. (B) GeneArt PerfectMatch TALs eliminate the 5 T constraint of GeneArt Precision TALs. GeneArt PerfectMatch TALs allow targeting of any sequences across the genome; 1516 bp spacing between the two TAL effector targets is optimal for GeneArt PerfectMatch TALs.
DNA binding domainFunctionaldomain
FokI
FokI
TNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNT
1318 bp
DNA binding domainFunctionaldomain
FokI
FokI
NNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNN
1516 bp
A B
DNA binding domainFunctionaldomain
Fok1
Fok1
An MCS version of the GeneArt Precision TAL vector allows you to customize the resulting TAL fusion protein with an effector domain of your choice.
Precision TALs fused to a Fok1 nuclease allow sequence-specific, double-stranded DNA breaks to be introduced.
Precision TALs fused to a vp16/KRAB functional domain allow specific activation/repression of gene expression.
Gene targeting (Fok1 nuclease pair) Silencing
Incorporation of exogenous DNA
Activation of transcription (Activator vp16
or vp64)
Increasing the expression level of endogenous gene isoforms
Epigenetic repression of transcription
(KRAB)
Heritable knockdown of gene expression
Steric repression and custom design
(MCS vector)
Transient knockdown of gene expression
Target any locus in the genome with the effector domain of your choice (multiple cloning site vector)
Figure 7. Available effector domains
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28 Life Technologies | Genome editing
GeneArt TAL effector toolsPrecise and flexible editing with freedom to innovate
Figure 9. Cleavage efficiencies of GeneArt PerfectMatch TALs compared to GeneArt Precision TALs. To determine the functionality of GeneArt PerfectMatch TALs, we have compared the genome cleavage efficiencies of GeneArt PerfectMatch TALs to those of GeneArt Precision TALs at specific loci using the GeneArt Genomic Cleavage Detection Kit (Cat. No. A24372). GeneArt PerfectMatch TALs function is equal to or better than GeneArt Precision TALs in 293FT cells when targeting sequences of forward and reverse TAL effectors are preceded by different (nonidentical) bases. The red arrowheads point to the cleavage products by GCD enzyme if multiple bands were shown in a GCD assay. TF: TAL effector target site with 5 T on forward strand; TR: TAL effector target site with 5 T on reverse strand. CF: TAL effector target site with 5 C on forward strand; CR: TAL effector target site with 5 C on reverse strand. GF: TAL effector target site with 5 G on forward strand; GR: TAL effector target site with 5 G on reverse strand. AF: TAL effector target site with 5 A on forward strand; AR: TAL effector target site with 5 A on reverse strand.
HPRTGeneArt Precision TAL TF/TR
GeneArt PerfectMatch TAL CF/AR
Negative control
GCD Enzyme: + - + - + - + - + -
Cleavage efficiency (%)
18.1 19.2 21.6 25.0
IL2GeneArt Precision TAL TF/TR
GeneArt PerfectMatch TAL CF/TR
Negative control
GCD Enzyme: + - + - + - + - + -
Cleavage efficiency (%)
9.0 12.6 16.4 16.8
RELAGeneArt Precision TAL TF/TR
GeneArt PerfectMatch TAL AF/TR
Negative control
+ - + - + - + - + -
4.9 5.0 16.1 14.3
CCR5GeneArt Precision TAL TF/TR
GeneArt PerfectMatch TAL GF/TR
Negative control
+ - + - + - + - + -
7.4 10.3 7.4 6.0
ACTBGeneArt Precision TAL TF/TR
GeneArt PerfectMatch TAL GF/CR
Negative control
+ - + - + - + - + -
32.8 37.5 47.6 50.1
IP3R2GeneArt Precision TAL TF/TR
GeneArt PerfectMatch TAL AF/GR
Negative control
+ - + - + - + - + -
6.6 6.6 13.0 12.9
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29Life Technologies | Genome editing
Ordering GeneArt TALsThe fastest and easiest way to design, edit, optimize, and order GeneArt Precision TALs is through the GeneArt portal. For GeneArt PerfectMatch TALs, you can download and complete the TAL order form and email it to [email protected].
The ordering process is simple; just follow these three steps:
1. Select the functionality (effector domain) of your TAL from Table 1. For details, refer to the TAL functionality overview.
2. Select the product name from Table 1.
3. Then place your order through our online design tool within the GeneArt portal, or you can download (.xls) and complete the TAL order form and email it to [email protected]
If you have a question or need free design consultation, contact us and well be happy to assist you.
We will ship you a clone with a verified, optimized sequence approximately 2 weeks after confirming your order.
Find out more or place an order at lifetechnologies.com/tals
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30 Life Technologies | Genome editing
TAL effector technical resourcesLet our knowledge work for you
Watch this webinar, The power of GeneArt PerfectMatch TALs: editing tool targeting any DNA sequence.
Watch this webinar, TAL effectors tool box for precision genome editing, for an introduction to designer TAL effectors and their applications for targeted gene editing.
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31Life Technologies | Genome editing
Doing plant engineering? These resources could help you get started.Precise plant genome editing and better crop engineering
Generating transgenic plants is key to introducing new crop traits and carrying out gene studies, both in discovery and applied settings. The development of genetically modified plants often requires a complex design of DNA elements to achieve optimum effects on expression. Learn more.
GeneArt TALs are ideal for plant research scientists, offering research-use access with a clear licensing path for commercial use through the Two Blades Foundation.
Webinar: Watch this webinar in which Dr. Neal Stewart, professor and director at the Tennessee Plant Research Center, provides novel insights into the potential applications of synthetic TAL effectors for targeted gene activation of transgenes in plants
Webinar: Presented by Dr. Jon Chesnut, research fellow and lead scientist at Thermo Fisher Scientific, this webinar introduces the latest genome editing technology for plant science researchers
GeneArt PerfectMatch TALs provide custom DNA-binding proteins designed for accurate DNA targeting and precise genome editing. Unlike other technologies that limit the choice of targets, or provide ambiguous results, GeneArt PerfectMatch TALs help enable the targeting of any locus in the genome. Previous versions of GeneArt Precision TALs required a thymine nucleotide (T) at the 5 end of each target sequence. The 5 T constraint limits the fl exibility of TAL effector target sites in the genome and prevents some specifi c sites in the genome from being targeted[1]. With our new GeneArt PerfectMatch TALs, the 5 T requirement is removed, making it possible to design a TAL effector pair for anywhere in the genome, giving you broad fl exibility in target design. In addition, Thermo Fisher Scientifi c has recently secured rights to certain intellectual property around TALs, clarifying a path for you to move confi dently forward and innovate.
Product detailsTranscription activator-like (TAL) effectors are a widely used technology for precise and effi cient gene editing in living cells. Native TAL effectors are DNA-binding proteins produced by plant pathogens of the genus Xanthomonas. When these bacteria infect plants, the TAL effector proteins bind to selected regulatory DNA sequences and directly modulate host gene expression[2]. The DNA-binding domain of TAL effectors consists of a variable number of amino acid repeats, called repeat-variable di-residues (RVDs), which recognize a single DNA base pair. We provide GeneArt TALs with special functional domains (nuclease, activator, or repressor domains) that are designed to interact with unique DNA targets in large, complex genomes.
PRODUCT GUIDE GeneArt PerfectMatch TALs
GeneArt PerfectMatch TALs Precise and extraordinarily exible genome editing
Find more data and application examples in our GeneArt PerfectMatch product guide.
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32 Life Technologies | Genome editing
Site-specific gene integration toolsTargeted integration of your gene of interest into specific integration sites
Jump-In targeted integration kitsEfficiently produce high-expressing mammalian cell lines
Whether you are experienced at cell engineering or need engineered cells for your projects, the Jump-In cell engineering platform can accelerate your projects by enabling you to generate engineered cell lines in much less time than traditional methods. Jump-In technology helps accelerate stable cell line development via the targeted integration of genetic material into specific integration sites using PhiC31 integrase.
Unlike better-known recombinases such as Cre and Flp, PhiC31 integrase catalyzes recombination between two nonidentical sites and, because it lacks a corresponding excision enzyme, makes the integration unidirectional and virtually irreversible. In addition, combining PhiC31 with R4 integrase allows for isogenic expression from a defined genomic locus, providing the ideal solution for comparative analysis of gene families, isoforms, or orthologs. Depending on your needs and experience, we offer multiple options for accessing Jump-In technology, allowing you to choose the method that best uses your available resources and fits your desired timelines.
Find out more at lifetechnologies.com/jumpin
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33Life Technologies | Genome editing
Site-specific gene integration toolsTargeted integration of your gene of interest into specific integration sites
Ordering information
Product Quantity Cat. No.
Jump-In Fast Gateway System 20 reactions A10893
Jump-In Fast Gateway Core Kit 1 kit A10894
Jump-In TI Gateway System 20 reactions A10895
Jump-In TI Gateway Vector Kit 1 kit A10896
Jump-In TI Platform Kit 1 kit A10897
Jump-In CHO-K1 Kit 1 kit A14148
Jump In T-REx HEK 293 Kit 1 kit A15008
Jump-In Custom Services [email protected]
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34 Life Technologies | Genome editing
Flp-In systemEfficient integration into a specific genomic location
The Flp-In system allows stable integration and expression of your gene of interest, to deliver single-copy isogenic cell lines. Flp-In expression involves introduction of a Flp recombination target (FRT) site into the genome of the mammalian cell line of choice. An expression vector containing your gene of interest is then integrated into the genome via Flp recombinasemediated DNA recombination at the FRT site.
We offer a wide selection of Flp-In products, including expression vectors and systems, as well as parental cell lines with a stably integrated FRT site. Designed for rapid generation of stable cell lines, these products help ensure high-level expression of your protein of interest from a Flp-In expression vector.
Find out more at lifetechnologies.com/flpin
Gene of interest
+ pOG44(Flp recombinase)
ATG
SV40
pA
BGH pA
SV40
pA
FRT FRTPSV40 PCMV goi IacZ-ZeocinTMhygropUC ori Amp Amp pUC ori
Flp-InTM expression cell line (hygromycin-resistant, ZeocinTM-sensitive)
pcDNATM 5/FRTgoi = gene of interest cloned
into pcDNATM 5/FRT
Flp-InTMExpressionVector
BGH pA
SV40 pA
puc ori
Ampicillin
P CMV
Hygrom
ycin
FRT
Site-specific gene integration toolsTargeted integration of your gene of interest into specific integration sites
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35Life Technologies | Genome editing
Ordering information
Product Quantity Cat. No.
Flp-In Complete System 1 kit K601001
Flp-In Core System 1 kit K601002
Flp-In T-REx Core Kit 1 kit K650001
Flp-In T-REx 293 Cell Line 1 mL R78007
Flp-In -BHK Cell Line 1 mL R76007
Flp-In -CHO Cell Line 1 mL R75807
Flp-In -CV-1 Cell Line 1 mL R75207
Flp-In -293 Cell Line 1 mL R75007
Flp-In -Jurkat Cell Line 1 mL R76207
Flp-In -3T3 Cell Line 1 mL R76107
Site-specific gene integration toolsTargeted integration of your gene of interest into specific integration sites
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36 Life Technologies | Genome editing
Gene modulation RNAi toolsTransient knockdown of multiple transcripts
RNA interference (RNAi) is a specific, potent, and highly successful approach for loss-of-function studies in virtually all eukaryotic organisms. We have developed two types of small RNAs that function in RNAi: short interfering RNA (siRNA) and microRNA (miRNA). We offer products for RNAi analysis in vitro and in vivo, including libraries for high-throughput applications. Your choice of tool depends on your model system, the length of time you require knockdown, and other experimental parameters.
Ambion siRNA Superior siRNAs for in vitro applications
RNAi is the best way to effectively knock down gene expression to study protein function in a wide range of cell types. Traditional RNAi methods for gene knockdown in mammalian cells involve the use of synthetic RNA duplexes consisting of two unmodified 21-mer oligonucleotides annealed together to form small interfering RNAs (siRNAs). Silencer Select siRNA products incorporate the latest improvements in siRNA design, off-target effect prediction algorithms, and chemistry, to offer:
High potencyimproved siRNA prediction accuracy compared to Silencer siRNA (Table 1) Minimal off-target effectsLocked Nucleic Acid (LNA) chemical modifications
reduce off-target effects by up to 90% Guaranteed100% guaranteed to silence, for increased confidence in your reagents** Open access65,000 siRNA sequences and associated data on PubChem from
our Silencer Select siRNA library** siRNA guarantee(a) Silencer Select siRNA: Thermo Fisher Scientific guarantees that when you purchase two Silencer Select Pre-Designed siRNAs to the same target, then those two siRNAs will silence the target mRNA by 70% or more. To qualify for the guarantee, siRNAs must have been transfected at 5 nM and mRNA levels detected 48 hours post-transfection. Real-time RT-PCR is recommended but not required for this application. Customers must also show sufficient knockdown with a positive control siRNA to demonstrate transfection efficiency. If the guaranteed level of knockdown is not observed and an appropriate positive control is successful, a new Silencer Select siRNA sequence will be synthesized free of charge. This guarantee does not extend to any replacement product.(b) Stealth RNAi: Thermo Fisher Scientific guarantees that when you purchase three Stealth RNAi Pre-Designed siRNAs to the same target, then at least two of those three independent, non-overlapping siRNAs will silence the target mRNA by 70% or more. To qualify for the guarantee, siRNAs must have been transfected at 20 nM and mRNA levels detected 48 hours post-transfection. Real-time RT-PCR is recommended but not required for this application. Customers must also show sufficient knockdown with a positive control siRNA to demonstrate transfection efficiency. If the guaranteed level of knockdown is not observed and an appropriate positive control is successful, a new Silencer siRNA sequence will be synthesized free of charge. This guarantee does not extend to any replacement product. We also recommend the use of an appropriate negative control, such as one of the three Stealth RNAi Negative Controls to normalize message knockdown.(c) Silencer products: Thermo Fisher Scientific guarantees that when you purchase three Silencer Pre-Designed siRNAs to the same target, at least two of the siRNAs will reduce target mRNA levels in cultured cells by 70% or more when measured 48 hours after transfection at 100 nM or higher final siRNA concentration under the conditions described below. If at least two of the three siRNAs do not induce >70% target mRNA knockdown, Thermo Fisher Scientific will provide a one-time replacement of up to three Silencer Pre-Designed siRNAs per target at no additional charge. Requests for replacement product must be made within one hundred and eighty (180) days from the date of delivery of the Silencer Pre-Designed siRNAs. Optimum transfection efficiency must be confirmed using good laboratory practices and a proven-to-work siRNA to an endogenous message, such as Ambion Silencer GAPDH siRNA Control. To assess knockdown, target mRNA levels in treated samples must be compared to that of cells transfected with a nontargeting control siRNA, such as Silencer Negative Control #1. We recommend TaqMan Gene Expression Assays to quantify mRNA levels.
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37Life Technologies | Genome editing
siRNA controlsProper controls are essential to help ensure success in every RNAi experiment. The number and types of controls chosen depend on the ultimate research goal, and we offer positive and negative controls, as well as GeneArt optimized gene synthesis for siRNA-resistant genes that can be used in RNAi rescue experiments.
For more information, go to lifetechnologies.com/sirnacontrols and lifetechnologies.com/geneartgenesynthesis
Table 1. Ambion siRNA selection guide.
Cost-effective siRNAGood knockdown, low off-target effects
Highest knockdown, lowest off-target effects
Silencer siRNA Stealth RNAi siRNA Silencer Select siRNA
Potency 100 nM recommended concentration
20 nM recommended concentration
5 nM recommended concentration
Efficacy (>70% knockdown) 2 of 3 siRNA guaranteed 2 of 3 siRNA guaranteed 2 of 2 siRNA guaranteed
Target specificity Moderate High Highest
Coverage Coding RNA Coding RNA Coding and noncoding RNA
Target species Human, mouse, rat (use custom tool for other species)
** siRNA guarantee(a) Silencer Select siRNA: Thermo Fisher Scientific guarantees that when you purchase two Silencer Select Pre-Designed siRNAs to the same target, then those two siRNAs will silence the target mRNA by 70% or more. To qualify for the guarantee, siRNAs must have been transfected at 5 nM and mRNA levels detected 48 hours post-transfection. Real-time RT-PCR is recommended but not required for this application. Customers must also show sufficient knockdown with a positive control siRNA to demonstrate transfection efficiency. If the guaranteed level of knockdown is not observed and an appropriate positive control is successful, a new Silencer Select siRNA sequence will be synthesized free of charge. This guarantee does not extend to any replacement product.(b) Stealth RNAi: Thermo Fisher Scientific guarantees that when you purchase three Stealth RNAi Pre-Designed siRNAs to the same target, then at least two of those three independent, non-overlapping siRNAs will silence the target mRNA by 70% or more. To qualify for the guarantee, siRNAs must have been transfected at 20 nM and mRNA levels detected 48 hours post-transfection. Real-time RT-PCR is recommended but not required for this application. Customers must also show sufficient knockdown with a positive control siRNA to demonstrate transfection efficiency. If the guaranteed level of knockdown is not observed and an appropriate positive control is successful, a new Silencer siRNA sequence will be synthesized free of charge. This guarantee does not extend to any replacement product. We also recommend the use of an appropriate negative control, such as one of the three Stealth RNAi Negative Controls to normalize message knockdown.(c) Silencer products: Thermo Fisher Scientific guarantees that when you purchase three Silencer Pre-Designed siRNAs to the same target, at least two of the siRNAs will reduce target mRNA levels in cultured cells by 70% or more when measured 48 hours after transfection at 100 nM or higher final siRNA concentration under the conditions described below. If at least two of the three siRNAs do not induce >70% target mRNA knockdown, Thermo Fisher Scientific will provide a one-time replacement of up to three Silencer Pre-Designed siRNAs per target at no additional charge. Requests for replacement product must be made within one hundred and eighty (180) days from the date of delivery of the Silencer Pre-Designed siRNAs. Optimum transfection efficiency must be confirmed using good laboratory practices and a proven-to-work siRNA to an endogenous message, such as Ambion Silencer GAPDH siRNA Control. To assess knockdown, target mRNA levels in treated samples must be compared to that of cells transfected with a nontargeting control siRNA, such as Silencer Negative Control #1. We recommend TaqMan Gene Expression Assays to quantify mRNA levels.
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38 Life Technologies | Genome editing
Silencer Select siRNA librariesWe also offer predefined collections of Silencer Select siRNAs against popular human gene classeskinase, phosphatase, GPCR, ion channel, nuclear hormone receptor, and protease, as well as the genome and druggable genome. Custom libraries are also available for all human, mouse, and rat genes. For more information, please contact [email protected]. For more information, go to lifetechnologies.com/sirnalibraries.html
Meet the inventor: Susan Magdaleno discusses Silencer Select siRNA technology
Gene modulation RNAi toolsTransient knockdown of multiple transcripts
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39Life Technologies | Genome editing
Dr. Gavin Robertson describes how Silencer Select siRNA technology is advancing melanoma research.
To learn more or place an order, go to lifetechnologies.com/rnai
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40 Life Technologies | Genome editing
mirVana miRNA mimics and inhibitorsFor artificial regulation of target mRNA translation
mirVana miRNA mimics and inhibitors are chemically modified, synthetic nucleic acids designed to either mimic mature miRNAs, or to bind to and inhibit endogenous miRNAs. These products provide a means to functionally study the role of specific miRNAs within cellular systems, or to validate the role of miRNAs in regulating target genes. mirVana miRNA mimics and inhibitors have been validated with Lipofectamine RNAiMAX Transfection Reagent for use in cell-based systems, and with Invivofectamine 2.0 Reagent for in vivo delivery. In vivoready mirVana miRNA mimics and inhibitors have been purified by HPLC and dialysis, making them ready for immediate use. mirVana miRNA mimics and inhibitors are:
Versatilefunctionally study specific miRNAs using in vitro or in vivo systems
Potentvalidate miRNA regulation of gene expression with minimal off-target effects (Figure 10)
High throughputcompatiblegenerate libraries for effective screening of multiple miRNAs simultaneously
Currentcontent is regularly updated based on the miRBase miRNA database
Gene modulation RNAi toolsTransient knockdown of multiple transcripts
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41Life Technologies | Genome editing
Figure 10. mirVana miRNA inhibitors effectively suppress miRNA in vivo. miR122 or Negative Control #1 mirVana miRNA inhibitors were complexed with Invivofectamine 2.0 Reagent and delivered to BALB/c mouse liver via tail vein injection on 3 consecutive days at a dose of 7 mg per kilogram of body weight. Expression of four mRNA targets (AldoA, Hfe2, Slc35a4, and Lass6), natural targets of miR122, were measured in transfected livers of mice injected with miR122 miRNA inhibitor or Negative Control #1 (Neg 1) and livers of mice that were not transfected (NT) using TaqMan MicroRNA Assays. This indicates that mirVana miRNA inhibitors are efficiently delivered to the liver with Invivofectamine 2.0 Reagent, leading to upregulation of genes naturally suppressed by miR122.
0
50
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200250
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miR122
NT Neg1 miR122
NT Neg1 miR122
NT Neg1 miR122
NT Neg1
AldoA Hfe2 Slc35a4 Lass6
mRN
A up
regu
lation
%
miR122 = miR122 miRNA inhibitorNT = Not transfectedNeg1 = Negative Control #1
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42 Life Technologies | Genome editing
Dr. Susan Magdaleno: miRNAs are a fascinating field.
mirVana miRNA librariesComplete mirVana libraries containing mimics and inhibitors for every human, mouse, and rat miRNA are available. For information on all our predefined and custom miRNAs libraries, contact us at [email protected]
Gene modulation RNAi toolsTransient knockdown of multiple transcripts
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43Life Technologies | Genome editing
Ordering information
Product Quantity Cat. No.
mirVana Predesigned miRNA Mimic 5 nmol 4464066
mirVana Predesigned miRNA Mimic, in vivo use 250 nmol 4464070
mirVana Predesigned miRNA Inhibitor 5 nmol 4464084
mirVana Predesigned miRNA Inhibitor, in vivo use 250 nmol 4464088
mirVana miRNA Mimic, Negative Control #1 5 nmol 4464058
mirVana miRNA Inhibitor, Negative Control #1 5 nmol 4464076
mirVana miRNA Mimic, miR-1 Positive Control 5 nmol 4464062
mirVana miRNA Inhibitor, let-7c Positive Control 5 nmol 4464080
To learn more or place an order, go to lifetechnologies.com/mirna
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44 Life Technologies | Genome editing
Nucleic acid transfection techniques for mammalian cells vary widely and include lipid-based transfection and physical methods such as electroporation. Lipofectamine transfection reagents are the most cited and trusted in the world because of their superior transfection performance and broad cell spectrum. An overview of our most effective transfection products is shown in Table 2 to help you choose the solution thats right for you.
Superior Transfection ReagentsEnhanced gene editing outcomes with superior transfection reagents
Table 2. Transfection selection guide. More blocks represent higher transfection efficiency into a greater number of cell types
Transfection selection guide
Table 1. Transfection selection guidemore blocks represent higher transfection efficiency into a greater number of cell types.
Transfection product DNA mRNA RNAiCo-delivery*
Easy-to-transfect cells
Difficult-to- transfect cells
Primary cells Stem cells
Suspension cells
Superior transfection reagentsLipofectamine 3000 Transfection Reagent
Lipofectamine RNAiMAX Transfection ReagentLipofectamine MessengerMAX Transfection Reagent
ExpiFectamine 293 Transfection Kit Recommended for Expi293F
cells
Broad-spectrum transfection reagentLipofectamine 2000 Transfection Reagent
ElectroporationNeon Transfection System
In vivo deliveryInvivofectamine 2.0 reagent In vivo delivery to liver following tail vein injection
Transfection is the process by which nucleic acids are introduced into eukaryotic cells. Techniques vary widely and include lipid-based transfection and physical methods such as electroporation. Lipofectamine transfection reagents are the most trusted and cited in the scientific literature due to their superior transfection performance and broad cell spectrum. An overview of our most effective transfection products is shown in Table 1 to help you choose the solution thats right for you. 5,000
10,000
15,000
20,000
25,000
30,000
35,000
40,000
45,000
50,000
0
19961997199819992000200120022003200420052006200720082009
2010201120122013
2014
Cumulative number of publications citing the use of Lipofectamine family of reagents since 1996.
4
*Cotransfection of RNAi vector and siRNA.* Cotransfection of RNAi vector and siRNA
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45Life Technologies | Genome editing
Superior Transfection ReagentsEnhanced gene editing outcomes with superior transfection reagents
Watch this videoA persistent challenge in genome editing is obtaining high transfection efficiency that is reproducible and that yields cells with high viability, so that they can be used in a variety of downstream applications.
We have been working with our existing transfection reagent for quite some time now and there are situations where its efficiency drops fairly significantly, namely when using primary hepatocytes or difficult-to-transfect cell lines, like C2C12 muscle cells.
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46 Life Technologies | Genome editing
Superior Transfection Reagents
Improve gene editing outcomesLipofectamine 3000 reagent was developed to break through the boundaries of traditional delivery methods and facilitate new technologies, such as genome engineering, in more biologically-relevant systems. With this reagent, GeneArt CRISPR vectors targeting the AAVS1 locus in HepG2 and U2OS cells show improved transfection efficiency (Figure 11), mean fluorescence intensity, and genomic cleavage. High transfection and genome editing efficiency is also observed with GeneArt Precision TALs. These advancements in delivery help minimize painstaking downstream workflows, enable easier stem cell manipulation, and enhance site-specific insertion of transgenes into the genome.
Mea
n O
FP in
tens
ity
0
50,000
100,000
150,000
3 L Lipofectamine 2000 1.5 L Lipofectamine 3000
B
Mea
n O
FP in
tens
ity
0
50,000
100,000
150,000
200,000
3 L Lipofectamine 2000 1.5 L Lipofectamine 3000
A
Figure 11. Transfection efficiency and protein expression using GeneArt CRISPR Nuclease Vector. The vector contained an orange fluorescent protein (OFP) reporter gene and was transfected with Lipofectamine 2000 or Lipofectamine 3000 reagent into (A) U2OS and (B) HepG2 cell lines. Bar graphs show reporter gene expression; images show fluorescence of corresponding cells expressing OFP.
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47Life Technologies | Genome editing
0
5
10
15
20
25
30
35
40
LipofectamineMessengerMAX
reagent
Competitor A Competitor B Lipofectamine 3000 Cells only
Cleavage efficiency (%
)
Cas9 mRNA + IVT gRNA
All-in-one CRISPR plasmid
GeneArt CRISPR Nuclease mRNA
In vitro transcribe gRNAsUse MEGAshortsciptT7 Transcription Kitfor gRNA synthesis
IVT gRNAs
TRANSFECT with Lipofectamine
MessengerMAX reagent
Up to 10x higher cleavage efficiency with mRNA CRISPRsLipofectamine MessengerMAX reagent helps increase the likelihood of cleavage and recombination with GeneArt CRISPR Nuclease mRNA through highly efficient transfection, maximizing the efficiency of genetic modifications and simplifying the downstream processes (Figure 12).
Figure 12 . Cleavage efficiency of various GeneArt CRISPR formats targeting the HPRT locus in Gibco iPS cells in a 12-well format. Lipofectamine 3000 reagent was used to deliver CRISPR nuclease all-in-one plasmid DNA; Lipofectamine MessengerMAX and two leading mRNA delivery reagents were used to deliver an all-RNA CRISPR format (Cas9 mRNA + IVT gRNA). Cleavage efficiency was determined using the GeneArt Genomic Cleavage Detection Kit 72 hours posttransfection.
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48 Life Technologies | Genome editing
When using genome editing tools such as CRISPRs, TAL effectors, or zinc finger nucleases to obtain targeted mutations, it is necessary to determine the efficiency with which these nucleases cleave the target sequence, prior to continuing with labor-intensive and expensive experiments. We have developed a set of tools that will enable you to quickly determine which cells have been edited.
Detect and analyzeEssential tools for monitoring the efficiency of your genome editing experiments
Table 3. Comparison of the three genomic analysis methodologies.Methodology Advantages Limitations When to use
GeneArt Genomic Cleavage Detection Assay
Can detect small changes in genomic DNANHEJ and HDR editing
Limited by detection sensitive to the low rate of modification
Triaging colonies from editing via NHEJ repair
GeneArt Genomic Cleavage Selection Assay
Fast-live detection as early as 24 hours post-transfection
Visual indication and allows clone enrichment
Detecting cleavage on the reporter construct not actually genomic loci
Triaging colonies from editing via NHEJ repair
TaqMan SNP Genotyping Assay
Fast
Clearly distinguishes changes in allele status
Only detects changes in alleles that the assay is designed formay not detect indels from NHEJ repair
Triaging colonies from editing via homologous recombination
Ion PGM sequencing Can specifically detect all changes in a population
Quantitative results
Higher cost compared to other assays
Longer workflow
Best used as a secondary assayfor confirmation and quantitation of editing in populations identified from primary screens
When combined, these genetic analysis methodologies can streamline the genome editing process. Table 3 compares the advantages, limitations, and suggested applications of each method.
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49Life Technologies | Genome editing
Detect and analyzeEssential tools for monitoring the efficiency of your genome editing experiments
Harvest cells for analysis
Extract genomic DNA
Perform 1st round of PCR amplification
Perform 2nd nested PCR amplification
TaqMan SNPGenotyping
Analysis sub-workflow
Analysis Expansion
GeneArt
GenomicCleavageDetection
GeneArt
GenomicCleavageDetection
Ion PGM
sequencing
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50 Life Technologies | Genome editing
When using genome editing tools to obtain targeted mutations, it is necessary to determine how efficiently they cleave the target sequence, particularly prior to proceeding to the more laborious and expensive cell line cloning and sequencing processes. The GeneArt Genomic Cleavage Detection Kit provides a relatively quick, simple, and reliable assay that allows the assessment of the cleavage efficiency of genome
editing tools at a given locus (Figure 13). A sample of the edited cell population is used as a direct PCR template for amplification with primers specific to the targeted region. The PCR product is then denatured and reannealed to produce heteroduplex mismatches where double-strand breaks have occurred resulting in indel introduction. The mismatches are recognized and cleaved by the detection enzyme. Using gel analysis, this cleavage is both easily detectable and quantifiable.
Easywith direct PCR amplification, theres no need for genomic DNA isolation
Rapid5-hour total processing time
Quantitativegel band density is directly correlated to target indel introduction
GeneArt Genomic Cleavage Detection KitA quick, simple, and reliable method for detecting and quantifying locus-specific double-strand breaks
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51Life Technologies | Genome editing
GeneArt Genomic Cleavage Detection KitA quick, simple, and reliable method for detecting and quantifying locus-specific double-strand breaks
Figure 13. GeneArt genomic cleavage detection assay. To detect either an indel or a mutation within a specific sequence of DNA, the region is first amplified using primers specific for that region. A second nested PCR can be performed to increase sensitivity. After heating the sample and reannealing the PCR products, amplicons containing indels or other changes in sequence will result in the formation of heteroduplexes with amplicons containing unmodified sequences. When these heteroduplexes are treated with an endonuclease that only cleaves in the presence of a mismatch, two pieces of DNA of known size are generated, which can be detected by agarose gel electrophoresis.
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52 Life Technologies | Genome editing
GeneArt Genomic Cleavage Detection Kit
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Figure 14. Cleavage efficiency of GeneArt TALs and CRISPRs. The TALs and CRISPRs targeted the AAVS1 locus in (A) U2OS cells, derived from human bone osteosarcoma, and (B) HepG2 cells, derived from a human hepatocellular carcinoma, after transfection with either Lipofectamine 2000 or 3000 reagent. Cleavage was assayed using the GeneArt Genomic Cleavage Detection Kit. Both cell lines showed improved transfection efficiency and protein expression compared to transfection using Lipofectamine 2000 reagent. Transfection efficiency and protein expression were assessed using a CRISPR construct that contains the orange fluorescent protein (OFP) reporter gene. We observed increased TAL- and CRISPR-mediated cleavage for the AAVS1 target locus in both cell lines transfected with Lipofectamine 3000 reagent, demonstrating that increasing the transfection efficiency and, by implication, protein expression, will increase the cleavage rate of TALs and CRISPRs. U2OS cells transfected with Lipofectamine 3000 reagent showed 1.5-fold improved TAL cleavage efficiency and slightly improved CRISPR cleavage (A). HepG2 cells had 3-fold higher cleavage efficiency for TAL effectors and 8-fold higher for CRISPRs (B).
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53Life Technologies | Genome editing
Application example: Improve genome editing outcomes in biologically-relevant cell models. Read the full application note here.
Ordering information
Product Quantity Cat. No.
GeneArt Genomic Cleavage Detection Kit 20 reactions A24372
IntroductionWith increasing expansion into research areas of more biological relevance, existing molecular and cellular techniques need to be improved. Lipofectamine 3000, a new transfection reagent developed to improve delivery and enable use of new technologies, can be used in more relevant systems enabling faster and more reliable outcomes. The area of genome editing is rapidly growing and requires more advanced techniques to maximize its potential applications. Transcriptional activator-like effector nucleases (TALENs) and technology derived from clustered regularly interspaced short palindromic repeats (CRISPRs) allow precise cleavage of DNA at specifi c loci. However, the effectiveness of these tools is contingent upon the intrinsic properties of the locus of interest, effi cient delivery, and the painstaking downstream processes of generating stable cell lines and knockout models to study the phenotypic effects of such genetic modifi cations. During the development of Lipofectamine 3000 Reagent, we assessed transfection of HepG2 and U2OS cell lines with TALEN and CRISPR vectors designed using GeneArt Gene Synthesis services to target a specifi c locus. We observed improvements in transfection effi ciency, mean fl uorescence intensity, and genomic cleavage. These advancements in delivery help minimize painstaking downstream workfl ows, enable easier stem cell manipulation, and enhance site-specifi c insertion of transgenes into the cellular genome.
Materials and methodsPlasmid design and preparationGeneArt Precision TALs and GeneArt CRISPR Nuclease Vectors were designed using the Life Technologies GeneArt web design tool(lifetechnologies.com/us/en/home/life-science/cloning/gene-synthesis/geneart-precision-tals.html). The forward and reverse TALENs contain the FokI nuclease and target the AAVS1 safe harbor locus. The all-in-one CRISPR vector system contains a Cas9 nuclease expression cassette and a guide RNA cloning cassette that target the AAVS1 safe harbor locus, combined with a downstream orange fl uorescent protein (OFP) reporter. A negative control plasmid, pcDNA3.3, was also used throughout the assay. The plasmids were transformed into competent E. coli cells. Clones were analyzed and sequenced for specifi city and then purifi ed using a PureLink HiPure Plasmid Filter Maxiprep Kit to ensure low endotoxin activity and high-quality DNA.
Improve genome editing outcomes in biologically relevant cell models
APPLICATION NOTE Lipofectamine 3000
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54 Life Technologies | Genome editing
GeneArt Genomic Cleavage Selection Kit A quick method for screening the functionality of nuclease cleavage with enriching capabilities
The GeneArt Genomic Cleavage Selection Kit is a rapid and reliable tool for detecting functionality of engineered nucleases in transfected cells as well as enriching for modified cells. When using engineered nucleases to create double- stranded breaks in genomic DNA, it is necessary to know whether or not the designed nucleases are functional. Furthermore, to efficiently screen for modified cells, a way to enrich for the edited cells is also necessary, particularly if the engineered nuclease has low efficiency or the cell line used is difficult to transfect. The GeneArt Genomic Cleavage Selection Kit contains a vector with the orange fluorescent protein (OFP) gene for a quick visual check of the functionality of the engineered nuclease. In addition, the reporter genes OFP and CD4 can be used to enrich for edited cells. It can be used in conjunction with genome editing tools such as zinc finger nucleases (ZFNs), transcriptional activator-like (TAL) effector nucleases, and CRISPRs.
Screenfor functionality of engineered nucleases as early as 24 hours post-transfection using fluorescence standard microscopy
Enrichfor modified cells using fluorescence-activated cell sorting (FACS) or Dynabeads CD4 magnetic beads
Ordering information
Product Quantity Cat. No.
GeneArt Genomic Cleavage Selection Kit 10 reactions A27663
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55Life Technologies | Genome editing
GeneArt Genomic Cleavage Selection Kit A quick method for screening the functionality of nuclease cleavage with enriching capabilities
Engineered nuclease recognition sequences
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Double-strand break repair
Restore OFP and CD4 expression
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Figure 15. Simple, rapid evaluation of the functionality of the programmable nuclease and direct enrichment of the genome-modified cells. The GeneArt Genomic Cleavage Selection Vector contains an orange fluorescent protein (OFP) reporter gene for quickly detecting the functionality of the engineered nucleases in the transfected cells. The OFP and CD4 reporters can be used for enrichment of the nuclease-modified cells using fluorescence-activated cell sorting (FACS) or CD4 antibodyconjugated Dynabeads particles. The vector has been constructed such that the N-terminal and C-terminal ends of the OFP gene are separated by a cloning site for the target sequence of the programmable nuclease. The upstream sequence coding for the N-terminal portion of the OFP gene contains a region complementary to the 5 end of the C-terminal region of the OFP gene. Three stop codons are inserted following the upstream coding sequence of the N-terminal OFP sequence to ensure no expression of the reporter OFP prior to nuclease activity. The CD4 gene is out of frame for expression when the OFP gene is interrupted by the cloning site. When a double-strand break is introduced into the target sequence by the programmable nuclease, the complementary strands from each end sequence of OFP will recombine to restore OFP expression, and the CD4 gene is now in frame for expression. Thus, cleavage by TAL, CRISPR, or zinc finger nucleases can be checked as early as 24 hours posttransfection by simply viewing the transfected cells under a fluorescence microscope. The percentage of OFP-positive cells will reflect the cleavage activity of the TAL, CRISPR, or zinc finger nuclease. The OFP-positive cells can be enriched through FACS. Additionally, the membrane protein CD4 coding gene is fused to OFP through T2A self-cleavage peptide, so the nuclease-modified cells can be enriched using Dynabeads particles conjugated with anti-CD4 antibody.
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56 Life Technologies | Genome editing
TaqMan SNP Genotyping AssayA simple, reliable, and rapid method for the detection of locus-specific double-strand break formation
TaqMan SNP Genotyping Assays provide a fast and simple way to perform single nucleotide polymorphism (SNP) genotyping analysis using genomic DNA. The workflow is shown schematically in Figure 16A. Discrimination between alleles is accomplished using two different TaqMan fluorescent reporter probes (Figure 16B).
Genome editing analysis tools
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57Life Technologies | Genome editing
Figure 16. TaqMan SNP Genotyping Assay. (A) TaqMan SNP Genotyping Assays workflow. Genotyping is performed on the QuantStudio 12K Flex Real-Time PCR System using the TaqMan SNP Genotyping Assay and the 400-bp amplicon containing the region targeted for TAL editing. Results are analyzed with the TaqMan Genotyper Software. (B) TaqMan SNP Genotyping Assays technology. Discrimination between two SNP alleles (or allelic discrimination) is achieved by selective annealing of two TaqMan probes, with each probe specific for one allele and carrying a different fluorescent reporter. During PCR amplification, the probe hybridized on its target allele degrades and generates a specific fluorescence signal. The signal from each reporter is recorded during real-time PCR and plotted on an allelic discrimination plot: the relative fluorescence signal from allele 1 is plotted along the x-axis of the allelic discrimination plot and the relative fluorescence signal from allele 2 is plotted along the y-axis. Colonies homozygous for allele 1 will plot along the x-axis. Colonies homozygous for allele 2 will plot along the y-axis. Heterozygous colonies will plot on a diagonal.
A BDesign/Order TaqMan
Genotyping Assay
Load master mix, assays, and samples into 96-well plate
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Allelic discrimination achieved by selectiveannealing of TaqMan probes
PrOducT BullETIN TaqMan Genotyping Assays
Introduction Genomic sequencing of a large number of organisms has produced a tremendous amount of information enabling further advances in genomic analysis, including SNP genotyping. Applied Biosystems Custom TaqMan SNP Genotyping Assays is a design service that provides optimized, ready-to-run assays for any genome, any organism. These custom assays are designed, synthesized, formulated, quality control tested, and delivered in a convenient, single-tube format. Custom assays use TaqMan minor groove-binding (MGB) probe-based assays for superior allelic discrimination and assay design flexibility. Simply submit your target DNA sequence information from any organism, and we will develop your ready-to-use SNP genotyping assay.
Custom assays eliminate the time-consuming manual steps of probe and primer design, formulation, and testing to simplify your genomic project plan.
The easy workflow entails adding an assay plus TaqMan Universal PCR Master Mix or TaqMan Genotyping Master Mix with enzyme to your DNA sample and one-step PCR amplification. TaqMan Assays enable you to focus on your strain identification, disease association, marker selection, quanti-tative trait loci, linkage mapping, and other SNP genotyping studies.
Assay design ServiceThe Custom TaqMan SNP Genotyping Assays Service provides secure and confidential access to Applied Biosystems assay design expertise. The service uses the proven Applied Biosystems Assay Design Pipeline to create assays designed to your specific-ations. This versatile resource provides genotyping assays for any possible SNP, MNP (up to six base pairs in length), and InDel (up to six base pairs in length).
Applied Biosystems synthesizes and tests all assay components in our state-
Custom TaqMan SNP Genotyping Assays Simplify Your Genomic Projects
Any genome, any organism
Versatility to detect single nucleotide polymorphisms (SNPs), insertion/deletions (InDels), and multi-nucleotide polymorphisms (MNPs)
Proven TaqMan MGB probe-based assays
Made to your specifications
One tube, one seamless workflow
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58 Life Technologies | Genome editing
Ion PGM sequencingTo expedite detection and quantitation of TAL editing events, we developed an amplicon sequencing method on the Ion PGM System (Figure 17). This method allows you to generate thousands of sequencing reads from any one sample and to process up to 96 samples in one sequencing run. With this method, you are able to determine whether an isolated iPSC colony harbors any cells that contain a desired mutation