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TALs

TALs or TALENs are transcription activator-like effector nuclease proteins that are naturally occurring transcriptional activators secreted by Xanthomonas spp. into their plant hosts. Invitrogen™ 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. 

1) One-to-one relationship between two critical amino acids in each repeat and each DNA base in the target sequence

2) Simple code for creating engineered TALs

3) More predictable than zinc fingers

4) Modular assembly of domains allows engineering of sequence-specific DNA-binding proteins

5) Can be coded to deliver functionality to a specific locus for: nucleases, activators, repressors, chromatin modifiers

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 where the authors compared TALEs and CRISPR technology.

We will ship you a clone with a verified, optimized sequence approximately 2 weeks after confirming your order.

Design

Here are our suggestions:

GeneArt Precision 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. The following image should be used as a reference for the orientation of the binding domains. (Figure A)

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 GeneArt Precision TALs. We recommend that you design nuclease pairs with a 15–16 bp spacing between the two TAL effectors. (Figure B)

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.

 

GeneArt PerfectMatch TALs are derived from GeneArt Precision TALs. GeneArt 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, GeneArt 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), repressor function (KRAB), or custom function (MCS vector).

We compared cleavage efficiencies of GeneArt PerfectMatch and Precision TALs designed for the HPRT locus using the Invitrogen™ GeneArt™ Genomic Cleavage Detection Kit, and found GeneArt PerfectMatch TALs exhibit cleavage efficiencies equal to or better than the performance of GeneArt Precision TALs on the same targeted region.

GeneArt 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.

We offer the N-TAL Fok1 Entry Gateway vector or the Fok1 CMV promoter–driven vector for expression.

A recent paper 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.

The TAL vector construct is 3.3 kb.

Yes. If viral-based delivery is your preferred option, we recommend adenoviral systems over lentiviral systems for TAL delivery.

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.

Manufacturing takes place typically within 2 weeks after your order has been received.

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.

Yes, KO and KI 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.

  1. Design GeneArt TALs for target DNA, and clone into Gateway™ entry or destination vectors
  2. In vitro validation of TALs (optional)
  3. Transfect cells with TAL expression vectors
  4. TAL-mediated target cleavage
  5. Cleavage analysis using GeneArt Genomic Cleavage Detection Kit

Please follow standard plasmid DNA recommendations for transfection. We recommend our Lipofectamine 3000 and Lipofectamine 2000 reagents. Please see the graph below showing transfection of GeneArt Precision TALs or CRISPR with Lipofectamine 3000 or 2000 reagent.

All TALs are expressed as Entry clones (Gateway vector–compatible, flanked by attL1 and attL2).

1. 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.

2. 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. 

3. KRAB TALs

The KRAB repressor can be used to down-regulate/repress endogenous or recombinant gene expression. 

4. 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.

TAL format

Considerations

Truncated TAL Fok1

Recommended for mammalian cells

Native TAL Fok1

Recommended for plants

Native Activators

Higher performance in nonmammalian systems

Truncated MCS

Removing endogenous activator activity

Double-stranded DNA breaks can be created at your specified genomic locus by using a pair of 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 GeneArt 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.

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.

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.

Yes, KO and KI 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.

Invitrogen™ GeneArt™ Genomic Cleavage Selection Kit

The GeneArt Genomic Cleavage Selection Kit can be used to:

  • Screen for functionality of your engineered nucleases as early as 24 hours post-transfection using fluorescence microscopy
  • Enrich for modified cells using fluorescence-activated cell sorting (FACS) or Invitrogen™ Dynabeads™ CD4 magnetic beads

The vector in the kit has an OFP gene that is interrupted by the insertion of 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. Stop codons after the N-terminal OFP sequence ensure no expression of the reporter prior to nuclease activity. The CD4 gene is out of frame for expression when the OFP gene is interrupted by the cloning site. Double-stranded breaks cause the complementary strands of the end sequences of the OFP gene to recombine, and OFP expression is restored. The CD4 gene is now in frame for expression and can be screened for via FACs analysis or Dynabeads magnetic bead selection.

Yes, the GeneArt Genomic Cleavage Selection Kit can be used with either TALs or CRISPR constructs.

Please see the comparison table below:

GeneArt Genomic Cleavage Selection Kit

GeneArt Genomic Cleavage Detection Kit

Fast, live detection

Requires cells to be lysed

Visual indication (fluorescence)

Quantifiable results

Proves editing tool works

Negative result does not indicate whether editing tool works or not

Allows for clone enrichment

No enrichment capabilities

It is possible to verify cleavage as early as 24 hours post-transfection by checking for OFP expression of the transfected cells under the microscope. The percentage of OFP-positive cells indicates the cleavage activity of TAL or CRISPR-Cas9.

The GeneArt genomic cleavage selection vector also contains the membrane protein CD4 coding gene that is fused with OFP through the T2A self-cleavage peptide, allowing nuclease-modified cells to be enriched through cell sorting or CD4 antibody–conjugated Dynabeads. This cleavage selection vector allows simple, rapid evaluation of the functionality of the programmable nuclease, and direct enrichment of the genome-modified cells.

OFP and CD4 expression are considered an estimation rather than absolute quantification of genomic cleavage. We recommend using our GeneArt Genomic Cleavage Detection Kit to verify cleavage on endogenous genomic locus.

OFP has peak excitation of 548 nm and emission of 560 nm. A 488 nm laser is recommended for efficient excitation. Standard 530/30, 574/26 and 603/48 emission filters are recommended for detection.

Invitrogen™ GeneArt™ Genomic Cleavage Detection Kit

Yes, our GeneArt Genomic Cleavage Detection Kit can be used to determine the efficiency of nuclease cleavage at a given locus. 

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. These mismatches are recognized and cleaved by the Detection Enzyme. This cleavage is both easily detectable and quantifiable using gel analysis.

Please use the formula below: 

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.