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View additional product information for GeneArt™ Genomic Cleavage Detection Kit - FAQs (A24372)
66 product FAQs found
TALs or TALENs are transcription activator-like effector nuclease proteins that are naturally occurring transcriptional activators secreted by Xanthomonas spp. into their plant hosts. GeneArt TALs are derived from Xathomonas TAL effectors, the DNA-binding domain of which consists of a variable number of amino acid repeats. Each repeat contains 33–35 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). TAL effector repeats can be assembled in modular fashion, varying the RVDs to create a TAL protein that recognizes a specific target DNA sequence.
CRISPR-STOP is a method of inserting STOP codon sequences to generate knockouts.
Please refer to the following article: CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations.
Find additional tips, troubleshooting help, and resources within our Genome Editing Support Center.
We do offer a multiple cloning site sequence in the place of the effector domain sequence for our TAL MCS entry vector. This option allows you to insert any protein-coding sequence, and allows your resulting TAL protein to deliver the effector in a sequence-specific manner anywhere in the genome. We also provide gene synthesis services to generate any effector domain for which you don't have a template.
While our Invitrogen GeneArt Precision TALs required a T at the 5´end and 13-18 bp spacing between the forward and reverse TAL effectors for proper pairing of Fok1 nucleases, the Invitrogen GeneArt PerfectMatch TALs allow for targeting of any sequence across the genome and eliminates the 5´ T constraints. Additionally, the spacing between the two effectors is optimal at 15-16 bp.
The 19 bp binding domains perform better for the nucleases. The binding sites do not need to be the same size; however, best performance for the nucleases is with the 19 bp binding domains.
We have observed that as little as 3 bp mismatch affects the binding of the TALs. We recommend that there be an exact match between the design and the binding site. If you would like some help with the design, please email geneartsupport@lifetech.com.
Here are common problems, possible causes, and recommendations:
- Smear obtained: Lysate is too concentrated; Dilute lysate 2- to 4-fold and repeat the PCR reaction.
- Disparity in band intensity between amplicons: Lysate concentrations vary between samples; Purify the PCR products with the Invitrogen PureLink PCR Purification Kit. For best results in comparing samples, purify PCR and use the same quantity of DNA in each cleavage assay. 50 ng to 100 ng of DNA is enough for each reaction.
- No PCR product: Poor PCR primer design or GC-rich region; Redesign primers that are 18-22 bp, have 45-60% GC content, and a 52-58°C Tm range. For GC-rich regions, add 1-10 µL of 360 GC Enhancer in a 50 µL reaction and repeat the PCR amplification.
Samples are typically run on an agarose gel, such as an Invitrogen E-Gel EX gel, followed by analysis with image software or by microfluidic electrophoresis.
Yes, our GeneArt Genomic Cleavage Detection Kit can be used to determine the efficiency of nuclease cleavage at a given locus.
We recommend that you resuspend the vector in 50 µL of distilled water or 10 mM Tris-HCl (pH 8.0) and incubate for 1 hour at room temperature. Resuspend the vector DNA by gently pipetting up and down 5-10 times. Store the resuspended DNA at -20°C.
Please email our team, at outlicensing@lifetech.com.
No, we would recommend either lipid-mediated transfection or electroporation. Please review relevant references or consult with the supplier of your cell line for the optimal method of transfection.
Double-stranded DNA breaks can be created at your specified genomic locus by using a pair of Invitrogen GeneArt Precision TAL proteins that have been fused to the Fok1 endonuclease. Using a pair of Precision TAL proteins for the targeting reduces off-target effects. The breaks induced by the Fok1 nuclease domain are subsequently repaired through either of two endogenous cellular mechanisms: nonhomologous end joining (NHEJ), or homology-directed repair (HDR). NHEJ is prone to errors and often introduces a frameshift mutation when it occurs within the coding sequence of a protein-coding gene, effectively silencing the gene. Homologous DNA “donor sequences” can be used with HDR to introduce a defined new DNA sequence. Consequently, a Precision TAL protein fused to a Fok1 endonuclease can be used to induce gene silencing or to accurately insert an engineered DNA fragment into an exact location in the genome.
Truncated TAL Fok1: Recommended for mammalian cells
Native TAL Fok1: Recommended for plants
Native Activators: Higher performance in non-mammalian systems
Truncated MCS: Removing endogenous activator activity
All TALs are expressed as Entry clones (Gateway vector-compatible, flanked by attL1 and attL2).
Fok1 TALs:
- TALs tethered with the Fok1 nuclease may be used for targeting specific genes for silencing.
- Fok1 is a Type IIs restriction endonuclease from Flavobacterium okeanokoites, consisting of an N-terminal DNA-binding domain and a C-terminal nonspecific DNA cleavage domain.
- Fok1 acts as a nuclease pair and binds to the DNA duplex at target sites designated by the binding domains resulting in subsequent cleavage.
VP16 or VP64 TALs:
- Either VP16 or VP64 may be used to increase endogenous or recombinant gene expression levels.
- VP16 is a trans-acting protein originating from the herpes simplex virus, to form a complex with host transcription factors to induce immediate early gene transcription.
- VP64 is a tetrameric form of the VP16 minimal activation domain.
MCS TALs:
- The multiple cloning site (MCS) TAL allows the user to clone any desired effector domain for targeting to any locus within the genome.
Please follow standard plasmid DNA recommendations for transfection. We recommend our Invitrogen Lipofectamine 3000 and Invitrogen Lipofectamine 2000 reagents.
- Design Invitrogen GeneArt TALs for target DNA, and clone into Gateway entry or destination vectors
- In vitro validation of TALs (optional)
- Transfect cells with TAL expression vectors
- TAL-mediated target cleavage
- Cleavage analysis using Invitrogen GeneArt Genomic Cleavage Detection Kit
Yes, KO and KI strains involve editing the native genetic code by either mutating or deleting an encoded message or inserting a new piece of information at a desired site. Although this does manipulate the native genetic information, this technology, when used in a responsible manner, has very useful applications, including engineering yeasts for insulin production or engineering cells for more economically and clinically valuable products.
mRNA and DNA are best delivered via lipid-based transfection for standard test cell lines (i.e., 293, HeLa, etc.). mRNA delivery also reduces the risk of transgene integration. We offer products including our Invitrogen Lipofectamine MessengerMAX Reagent for delivery of mRNA, and Invitrogen Lipofectamine 3000 Reagent for delivery of DNA. For stem cells, electroporation is the best option.
Manufacturing takes place typically within 2 weeks after your order has been received.
By careful designing they can be engineered to be very specific. Recent publications show that 1-3 bp mismatches in target DNA sequences can be tolerated to a large extent.
Yes. If viral-based delivery is your preferred option, we recommend adenoviral systems over lentiviral systems for TAL delivery.
The TAL vector construct is 3.3 kb.
A recent paper (http://www.nature.com/nbt/journal/v31/n9/abs/nbt.2675.html) by Prashant Mali in Nature Biotechnology shows that TALENs are tolerant of 1-2 mismatches, but less tolerant to a large majority of 3 bp mismatches.
We offer the N-TAL Fok1 Entry Gateway vector or the Fok1 CMV promoter–driven vector for expression.
Invitrogen GeneArt PerfectMatch TALs are derived from Invitrogen GeneArt Precision TALs. PerfectMatch TALs have a new design that removes the 5′ base constraint, and therefore, can be designed to target any desired sequence in the genome. They contain 3 amino acids mutated at the N terminus of the TAL effector, which converts the 5′ binding motif to a universal binding motif able to bind to any base: A, G, C, or T. These PerfectMatch TALs can be designed with Fok1 nuclease in a Invitrogen Gateway entry vector or with CMV-driven expression for ready-to-express format for mammalian systems. PerfectMatch TALS perform as well as or better than our original Precision TALs. Currently, PerfectMatch TALs are only available with nuclease function (Fok1 Nuclease Pair), whereas Precision TALs are offered with nuclease function (Fok1 Nuclease Pair), activator function (VP16 or VP64), or custom function (MCS vector).
We compared cleavage efficiencies of PerfectMatch and Precision TALs designed for the HPRT locus using the Invitrogen GeneArt Genomic Cleavage Detection Kit, and found PerfectMatch TALs exhibit cleavage efficiencies equal to or better than the performance of Precision TALs on the same targeted region.
PerfectMatch TALs increase the flexibility of designing TAL effector targets and make it possible to keep the spacing distance between targets of TAL effector pairs at 15–16 bp to get maximal TAL effector efficiency.
Here are our suggestions:
Invitrogen GeneArtPrecision TALs allow the construction of TAL effector functional proteins directed to either 18- or 24-base DNA target sites.
Each target site must be preceded by a 5′ T because the N terminus of the TAL effector protein contains a conserved T-binding motif. The 5′ T does not count as one of the 18 or 24 bases to be selected for targeting your specific site.
Nuclease pairs need to be designed with a spacing of 13-18 bp between the target sites on opposite strands of the DNA.
For Invitrogen GeneArt PerfectMatch TALs, there are no restrictions for the 5′ base. We developed these second-generation TALs by mutating the N-terminal domain to reduce its specificity for 5′ T. Therefore, any 5′ base (T, G, C, or A) can be used with performance comparable to that of the original Precision TALs. We recommend that you design nuclease pairs with a 15-16 bp spacing between the two TAL effectors.
The contribution of individual binding motifs within the DNA-binding domain to TAL effector binding efficiency is thought to differ, since strong and weak binding motifs exist. The A- and T-binding motifs are thought to fall within the “weak binder” category, while the C- and G-binding motifs are thought to be “strong binders”. Stretches of more than 5 weak binders should be avoided at the extreme 5′ end of the binding domain (not counting the 5′ T), or if they are not flanked by Cs.
We recommended that you select a TAL effector with a DNA-binding domain composed of mixed binding motifs for best results. In the context of the living cell, DNA accessibility also determines TAL effector efficiency. It is possible that chromatin, DNA methylation, and/or proteins bound to the DNA may interfere with TAL binding.
Although promoter structure varies, and specific rules regarding design are currently lacking, it is recommended that TAL transcription factors used for transcriptional activation of natural promoters be positioned upstream of the TATA box, or in some cases downstream of the transcriptional start site. Selecting a target site directly over the TATA box or other known transcription factor binding site is not recommended. Be sure that the natural ATG is present, and that no premature ATG which may interfere with the natural translational start is transcribed.
Please view the available effector domains (https://www.thermofisher.com/us/en/home/life-science/cloning/gene-synthesis/geneart-precision-tals.html#table1).
We will ship you a clone with a verified, optimized sequence approximately 2 weeks after confirming your order.
Invitrogen GeneArt Precision TALs, in addition to gene deletion, down-regulation and integration, can also be used for gene activation. Additionally, the system is based on a protein-DNA system, in contrast to CRISPR, which is based on a RNA-DNA system. TALs can be used to target any gene in any cell, including mammalian, bacterial, yeast, plants, insect, stem cells and zebrafish. Lastly, off-target effects are low when using the TAL system. Please refer to the following paper (http://www.sciencedirect.com/science/article/pii/S016816561500200X) where the authors compared TALs and CRISPR technology.
- One-to-one relationship between two critical amino acids in each repeat and each DNA base in the target sequence
- Simple code for creating engineered TALs
- More predictable than zinc fingers
- Modular assembly of domains allows engineering of sequence-specific DNA-binding proteins
- Can be coded to deliver functionality to a specific locus for: nucleases, activators, repressors, chromatin modifiers
There are several ways to increase efficiency, for instance, adding antibiotic selection and/or FAC sorting to enrich for the transfected cells will both help.
PAM is a necessary requirement for CRISPR gene editing. However, in its absence, we recommend engineering a TAL effector to edit your desired gene efficiently. We offer GeneArt PerfectMatch TAL effectors. These are TAL effector nucleases that remove the 5´ base constraint and can be designed to target any desired sequence within the genome. Please go here for further details: https://www.thermofisher.com/us/en/home/life-science/genome-editing/geneart-tals.html
Genomic DNA at the locus being investigated must be PCR amplified prior to detection. Follow these recommended guidelines to ensure optimal amplification and subsequent detection:
- For best results, use primers with Tm >55°C.
- Design primers that are 18-22 bp in length and have 45-60% GC content.
- For efficient amplification, design primers to yield amplicon lengths between 400 and 500 bp.
- Design primers so that the potential cleavage site is not in the center of the amplicon and that the detection reaction will yield two distinct product bands.
The assay uses genomic DNA extracted from cells transfected with constructs expressing engineered nucleases. Following cleavage, indels are created by the cellular repair mechanisms. Loci where the gene-specific double-stranded breaks occur are amplified by PCR. The PCR product is denatured and reannealed so that mismatches are generated as strands with an indel reannealed to strands with no indel or a different indel. The mismatches are subsequently detected and cleaved by Detection Enzyme, and then the resultant bands are analyzed by gel electrophoresis and band densitometry.
The GeneArt Genomic Cleavage Detection Kit provides a simple, reliable, and rapid method for the detection of locus-specific double-strand break formation. This technique relies on mismatch detection endonucleases to detect genomic insertions or deletions (indels) incorporated during cellular NHEJ repair mechanisms.
GeneArtGenomic Cleavage Selection Kit
- Fast, live detection
- Visual indication (fluorescence)
- Proves editing tool works
- Allows for clone enrichment
GeneArt Genomic Cleavage Detection Kit
- Requires cells to be lysed
- Quantifiable results
- Negative result does not indicate whether editing tool works or not
- No enrichment capabilities
Yes, the Neon system does work for multiple gRNAs transfected at the same time.
We recommend starting at a ratio of 0.5 µg of Cas9 mRNA and 50 ng of each IVT gRNA per well in a 24-well format. You should determine the optimal ratio for your particular cell line via a dose-response study.
Create multiple gRNAs targeting the targets of your choice, followed by co-transfection with GeneArt CRISPR Nuclease mRNA or GeneArt Platinum Cas9 Nuclease. To make the gRNAs for Cas9 mRNA, use GeneArt CRISPR Strings DNA, U6 or IVT gRNAs (generated using either GeneArt CRISPR Strings DNA, T7 or the GeneArt Precision gRNA Synthesis Kit). For the Cas9 protein, use IVT gRNAs (generated using either GeneArt CRISPR Strings DNA, T7 or the GeneArt Precision gRNA Synthesis Kit).
Yes, if you use the current Invitrogen GeneArt CRISPR nuclease vectors the respective Limited-Use Label Licenses (LULLs) will apply.
No, the PAM sequence is unique to the bacterial species that was used to create the Cas9. In the Invitrogen GeneArt kits, we derived Cas9 from Streptococcus pyogenes.
PAM stands for the protospacer adjacent motif. It is necessary for Cas9 to bind to the DNA successfully. The PAM sequence for the Streptococcus pyogenes Cas9 in the Invitrogen GeneArt CRISPR kits is NGG.
With the CRISPR-Cas9 editing complex (DNA vector, mRNA or Protein), co-transfect a DNA repair template that contains high homology to the sequence of interest along with the desired sequence you would like to introduce into the DNA. By doing so HDR can occur, and your specific edits (mutation, insertion, etc.) can be incorporated into the genome.
Cleavage efficiency can be detected using the Invitrogen GeneArt Genomic Cleavage Detection Assay. This assay relies on mismatch detection endonucleases to detect insertions and deletions (indels) generated during cellular NHEJ repair.
An indel refers to the genomic insertion or deletion of bases, which are incorporated during either cellular NHEJ or HD repair mechanisms.
Since cleavage efficiency at a particular locus depends on the accessibility of the locus, chromatin state, and sequence, it is advisable to test multiple different loci/regions within a gene of interest. With CRISPR-Cas9-mediated genome editing, for each target of interest the user needs only to change the 19-20 bp target-specific oligo. After the cell lines have been screened and the sequence/locus with the highest cleavage efficiency has been identified, the biologically relevant mutations can be precisely created with high-specificity Invitrogen GeneArt TALs (https://www.thermofisher.com/us/en/home/life-science/genome-editing/geneart-tals.html).
Cleavage is precise, and, after binding of the Cas9 and gRNA complex to the target genomic sequence, the nuclease activity occurs 3 bases upstream of the PAM (NGG) site.
This would depend upon the half-life of the particular transcript in your cell. We typically start seeing reduction in mRNA levels as early as 24 hrs post transfection, with further reduction after 48-72 hrs. Hence, we recommend performing the genomic cleavage detection assay 48-72 hours post transfection.
Cas9 is transiently expressed and will therefore disappear over time with successive cell divisions.
Yes, it is possible but our system is not for prokaryotes, and has only been optimized for mammalian systems. Please also consult our CRISPR custom services for further inquiries (custom.services@lifetech.com).
We have only tested these in mammalian systems (human and mouse cells).
Yes, we do offer this service (https://www.thermofisher.com/us/en/home/life-science/genome-editing/genome-engineering-services/cell-line-engineering-services.html).p>
The gRNA oligo design strategy in the Invitrogen GeneArt CRISPR Nuclease User Guide (https://tools.thermofisher.com/content/sfs/manuals/GeneArt_CRISPR_nuclease_mRNA_man.pdf)describes how you can design the guide RNA to target the locus in which the neomycin cassette should be inserted. The cassette (neomycin) can be inserted via HDR, in which case the neomycin cassette should contain locus specific homology arms.
The first few exons would be best (closer to the promoter, resulting in premature transcript termination). Since the gRNA efficiency depends on the accessibility of the locus as well as the chromatin structure at that location, it is advisable to design and test a few target sites. Non-CRISPR-related mutations may be identified using gDNA isolated from non-CRISPR-treated cells as a control and performing a Invitrogen GeneArt Genomic Cleavage Detection Assay (https://www.thermofisher.com/order/catalog/product/A24372). Standard western blot analysis is a good measure for the verification of protein levels.
Yes, this should be possible using CRISPR technology combined with HDR.
Carefully designed crRNA target oligos and avoiding homology with other regions in the genome are critical for minimizing off-target effects.
HDR efficiency is very low, on average less than 2%.
Create a double-stranded DNA break using the GeneArt CRISPR Nuclease Vector (https://www.thermofisher.com/us/en/home/life-science/genome-editing/geneart-crispr/crispr-nuclease-vector.html), while simultaneously transfecting your plasmid-based donor repair template. Your donor repair template plasmid will contain the sequence you wish to introduce that is flanked by at least 500 bp (or more) of sequence, which results in efficient homologous recombination of your sequence.
All of them may work, but for better efficiency, a longer homology arm is better (at least 500 bp (or more) on either side of the exogenous DNA). The homology length is dependent on the size of the fragment and will need to be tested. ssDNA may be error-prone or choose NHEJ. We offer the Invitrogen GeneArt Strings dsDNA fragments (1-3 kb) to assist with this type of application.
Both HDR (homology directed repair) and NHEJ (non-homologous end joining) are cellular mechanisms through which double-stranded DNA lesions are repaired. When a repair template is not present, NHEJ occurs to ligate double-stranded breaks, leaving behind insertion/deletion (indel) mutations. HDR is an alternative repair pathway in which a repair template is used to copy the sequence to the double-stranded break. You can, therefore, introduce specific nucleotide changes or DNA fragments into your target gene by using HDR with a repair template.
Clonal isolation and a combined cleavage analysis and sequence verification of the edited clone is advisable.
As a simple two-component system that includes the Cas9 endonuclease and a noncoding guide RNA (gRNA), the engineered Type II CRISPR/Cas system can be leveraged 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). Both the gRNA and the PAM (NGG) motif guide the Cas9 nuclease to a specific genomic sequence to form a complex, followed by local strand separation (R-loop), at which the Cas9 nuclease creates a double-stranded DNA break (DSB) 3 nucleotides upstream from the PAM site. As a result, you may bring new functionality to the gene of interest via mutations, create knockouts, or introduce nonnative or synthetic genomic sequences to investigate novel applications.
CRISPR also allows for non-editing application flexibility such as gene regulation or RNAi-related studies. The Cas9 nuclease may be tethered to different functional domains (activators or repressors) or the gRNA may be designed to directly cleave miRNA.
TAL and CRISPR directly edit the genome by a combined cleavage and repair mechanism to impart permanent genomic change (deletion or frameshift mutation), and the resulting gene knockouts are very efficient. RNAi technology, on the other hand, is an indirect method in either down-regulating or shutting down a gene completely through direct interaction with RNA (coding or noncoding). Even in the case for stably expressed miRNA or shRNA systems, it may be difficult to effect complete penetrance (i.e., shRNA:mRNA ratio) since knock-down levels are dependent on the activity of the promoter (related to integration location).
With their highly flexible but specific targeting, CRISPR-Cas systems can be manipulated and redirected to become powerful tools for genome editing. CRISPR-Cas technology permits targeted gene cleavage and gene editing in a variety of eukaryotic cells, and because the endonuclease cleavage specificity in CRISPR-Cas 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.
CRISPR stands for clustered regularly interspaced short palindromic repeat; CRISPR-Cas (CRISPR-associated) systems are used for genome editing in various host organisms.