GeneArt PerfectMatch TALs Precise and extraordinarily fl exible genome editing ®
PRODUCT GUIDE GeneArt PerfectMatch TALs ® GeneArt PerfectMatch TALs ® Precise and extraordinarily flexible genome editing GeneArt PerfectMatch TALs provide custom DNAbinding 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 flexibility of TAL effector target sites in the genome and prevents some specific 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 flexibility in target design. In addition, Thermo Fisher Scientific has recently secured rights to certain intellectual property around TALs, clarifying a path for you to move confidently forward and innovate. ® ® ® ® Product details Transcription activator-like (TAL) effectors are a widely used technology for precise and efficient gene editing in living cells. Native TAL effectors are DNAbinding 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. ® Before now, target sites for customized TAL effectors required a 5´ T in the target sequences for maximal binding activity [3–5] (Figure 1). The 5´ T constraint limited the flexibility of TAL effector target sites in the genome and prevented some specific sites in the genome from being targeted. Structure studies suggested the N-terminal domain (NTD) of the TAL effectors, not the central repeat domains, was responsible for the interaction with the 5´ T of the target [6]. We developed our second-generation TAL effectors, GeneArt PerfectMatch TALs, by mutating the N-terminal domain to relax its specificity for 5´ T. GeneArt PerfectMatch TALs can target DNA sequences with any 5´ base (T, G, C, or A) (Figure 1), with performance equal to or better than that of GeneArt Precision TALs. ® ® GeneArt PerfectMatch TALs are derived from GeneArt Precision TALs and contain a truncated TAL effector fused to a FokI nuclease domain. 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). With the mutation, GeneArt PerfectMatch TALs can be designed to precisely bind to any sequence across the genome in a sequence-specific manner. ® ® ® GeneArt PerfectMatch TALs are provided as lyophilized DNA in two kinds of vectors: a Gateway adapted entry vector and a CMV expression vector (Figures 2 and 3). ® ® attL2 okI mF t- er Ka na ® cin my ® N-TAL FokI entry vector 4,349 bp eat RV D TAL N -t e r m V 5 att L DNA-binding domain Functional domain FokI 13–18 bp TNNNNNNNNNNNNNNNNNN 1 Figure 2. Gateway adapted entry vector. Gateway adapted entry vectors allow easy transfer through an LR recombination reaction to destination vectors designed to facilitate high-level expression of the TAL effectors in your cells of choice. ® A pUC origin 18 or 24 rep GeneArt TALs engineered with the FokI nuclease can be used for targeting specific genes for editing (silencing, incorporation of exogenous DNA, or mutation). FokI is a type IIS restriction endonuclease from Flavobacterium okeanokoites, consisting of an N-terminal DNA-binding domain and a nonspecific DNA-cleavage domain at the C-terminus. A FokI nuclease pair binds to duplex DNA at the target sites designated by the DNA-binding domains, with each member of the pair cleaving one strand of the DNA target. TA LC ® ® NNNNNNNNNNNNNNNNNNT pU FokI 15–16 bp NNNNNNNNNNNNNNNNNNN 18 or 24 rep N-TAL FokI CMV expression vector NNNNNNNNNNNNNNNNNNN FokI cillin® Functional domain BGHpA 5,239 bp A m pi DNA-binding domain I Fok m r -te in rig Co B TA LC FokI eat RV D Figure 1. 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 13–18 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; 15–16 bp spacing between the two TAL effector targets is optimal for GeneArt PerfectMatch TALs. ® ® ® ® M V ® TAL N PC -te r m V 5 Figure 3. CMV expression vector. This vector contains a CMV promoter, which drives high-level expression of the TAL effector in mammalian systems. GeneArt PerfectMatch TALs provided in this vector can be directly used in mammalian systems without extra subcloning. Optional: A GeneArt PerfectMatch TAL cassette can be transferred into your favorite expression vector with restriction enzymes NotI and HindIII. ® ® Identify your target gene of interest Choose vector Place order Transfect Analyze Figure 4. Workflow for GeneArt PerfectMatch TALs. The GeneArt PerfectMatch TALs workflow is similar to the GeneArt Precision TALs workflow. ® ® ® Workflow The workflow for GeneArt PerfectMatch TALs is similar to that of the GeneArt Precision TALs (Figure 4). ® ® Step 1: Identify your target gene of interest. Design the targeting sequences for GeneArt PerfectMatch TALs yourself, or contact GeneArt support ([email protected]) for free design consultation. ® ® GeneArt PerfectMatch TAL binding site rules: ® • GeneArt PerfectMatch TAL offerings allow the construction of TAL functional proteins directed to either 18- or 24-base DNA target sites. ® • Each target site must be preceded by a 5´ “N” when you place the order, because the N-terminus of the GeneArt PerfectMatch TAL protein binds to any DNA base. The letter N represents any base—A, G, C, or T. The 5´ N does not count as one of the 18 or 24 bases to be selected for targeting your specific site. (This differs from GeneArt Precision TALs.) ® ® • Nuclease pairs need to be designed with a spacing of 13–18 bp between the target sites on opposite strands of the DNA. However, we recommend a spacing of 15–16 bp between the target sites to achieve maximal nuclease activity. The target sites can be either 18 or 24 bp in length. Figure 1B should be used as a reference for the orientation of the binding domains. (This differs from GeneArt Precision TALs.) ® • The contribution of individual binding motifs within the DNA-binding domain to TAL effector binding efficiency is thought to differ, because strong and weak binding motifs exist. The A- and T-binding motifs are thought to fall within the “weak binder” category, whereas 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´ “N”), or if they are not flanked by Cs. TAL effectors with DNA-binding domains composed of mixed binding motifs generally give the best results. • In the context of the living cell, DNA accessibility also determines TAL effector efficiency. Chromatin, DNA methylation, and/or proteins bound to the DNA may interfere with TAL effector binding. Step 2: Choose the vector to be received (Gateway adapted entry vector or CMV expression vector). ® Step 3: Place your order by downloading and completing the order form and emailing it to [email protected]. You will receive a clone with a verified, optimized sequence approximately 2 weeks after confirming your order. Step 4: Create an expression clone (optional). To create an expression clone, perform the LR recombination reaction to transfer the gene of interest into your destination vector of choice. If you chose the CMV expression vector in step 2, please directly go to step 5. If you chose the Gateway adapted entry vector in step 2, you need to complete this step. ® Step 5: Transfect. Step 6: Analyze the sample for gene modification 72 hours post-transfection using the GeneArt Genomic Cleavage Detection Kit (Cat. No. A24372). ® Figure 5 Analysis: data and applications Double-stranded DNA breaks can be created at your specified genomic locus by using a pair of GeneArt PerfectMatch TAL proteins that have been fused to the FokI endonuclease (see Figure 1B). Using a pair of TAL proteins for the targeting reduces off-target effects. The breaks induced by the FokI 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, GeneArt TAL proteins fused to FokI endonucleases can be used to induce gene silencing or to accurately insert an engineered DNA fragment into an exact location in the genome. The effectiveness of all genome-editing tools such as CRISPRs, TAL effectors, and zinc fingers depends on many factors, including the footprint of target sites (DNA sequences), spacing distance, DNA accessibility, cell type and condition, and transfection efficiency. Therefore, it is necessary to determine the cleavage efficiency prior to continuing with labor-intensive and expensive experiments. The GeneArt Genomic Cleavage Detection Kit (Cat. No. A24372) provides a simple, reliable, and rapid method to determine the nuclease cleavage efficiency at a given locus. HPRT ® ® ® To determine the effectiveness of GeneArt PerfectMatch TALs, we have compared the genome cleavage efficiencies of GeneArt PerfectMatch TALs to those of GeneArt Precision TALs at specific loci in a broad range of cell types. Target sequences for GeneArt PerfectMatch TALs and GeneArt Precision TALs were designed to be close to each other (within a few bases). In Figure 5, all TAL effector targets at the HPRT locus are shown as an example. Note that all TAL effectors tested in this comparison contain 18-mer TAL effector repeats, which bind to 18-base DNA target sites. GF AF CF TF GAACTCTAGC CAGAGTCTT ACTCTAGC CAGAGTCTTG C CTCTAGC CAGAGTCTTG CA TCTAGC CAGAGTCTTG CAT GAACTCTAGC CAGAGTCTTG CATTTCTCAG TCCTAAACAG GGTAATGGAC TGGGGCTGAA CTTGAGATCG GTCTCAGAAC GTAAAGAGTC AGGATTTGTC CCATTACCTG ACCCCGACTT CCATTACCTG ACCCCGACT C CCATTACCTG ACCCCGAC TC CCATTACCTG ACCCCGA GTC CCATTACCTG ACCCCG TR CR AR GR Figure 5. Target sites for GeneArt PerfectMatch TALs and GeneArt Precision TALs at the HPRT locus are shown here as an example of the similarities between the two targets. The DNA sequences in the pink boxes represent targeting sequences of TAL effectors. The DNA base in each light-pink box represents the 5´ base of the TAL effector target sequence. ® ® TF: TAL effector target site with a 5´ T on forward strand; TR: TAL effector target site with a 5´ T on reverse strand. CF: TAL effector target site with a 5´ C on forward strand; CR: TAL effector target site with a 5´ C on reverse strand. GF: TAL effector target site with a 5´ G on forward strand; GR: TAL effector target site with a 5´ G on reverse strand. AF: TAL effector target site with a 5´ A on forward strand; AR: TAL effector target site with a 5´ A on reverse strand. We compared cleavage efficiencies of GeneArt PerfectMatch and Precision TALs designed for the HPRT locus using the 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 function as well as or better than current TAL effectors in 293FT and HeLa cells when the targeting sequences of forward and reverse TAL effectors are preceded by different (nonidentical) bases (Figure 6). ® GeneArt PerfectMatch TALs function as well as or better than current TAL effectors in U2OS cells (Figure 7). GeneArt PerfectMatch TAL pairs with 15–16 bp spacing were compared with current TAL effectors in 7 loci in U2OS cells. ® ® A A AGCD Enzyme: GCD Enzyme: HPRT RELA GeneArt® Precision TAL GeneArt® PerfectMatch Negative TF/TR TAL CF/AR control HPRT GeneArt + -® Precision + TAL - GeneArt + -® PerfectMatch + - Negative + TF/TR TAL CF/AR control + - + - + - + - + - GeneArt® Precision TAL GeneArt® PerfectMatch Negative TF/TR TAL AF/TR control RELA GeneArt + -® Precision + TAL - GeneArt + -® PerfectMatch + - Negative + TF/TR TAL AF/TR control + - + - + - + - + - ACTB GeneArt® Precision TAL GeneArt® PerfectMatch Negative TF/TR TAL GF/CR control ACTB GeneArt + -® Precision + TAL - GeneArt + -® PerfectMatch + - Negative + TF/TR TAL GF/CR control + - + - + - + - + - Cleavage 18.1 efficiency (%) 19.2 21.6 25.0 4.9 5.0 16.1 14.3 32.8 37.5 47.6 50.1 Cleavage 18.1 efficiency (%) 19.2 21.6 25.0 4.9 5.0 16.1 14.3 32.8 37.5 47.6 50.1 Figure 6bGeneArt Precision TAL TF/TR GCD Enzyme: Figure 6bGeneArt + - Precision + TAL ® ® TF/TR GCD Enzyme: CCR5 GeneArt + -® PerfectMatch + - Negative + TAL CF/TR control + - + - + - + - + - B Cleavage 9.0 efficiency B (%) GeneArt Cleavage efficiency (%) IL2 GeneArt® PerfectMatch Negative TALIL2 CF/TR control 9.0 ® B 12.6 16.4 16.8 12.6 HPRT 16.4 16.8 Precision TAL GeneArt® PerfectMatch Negative TAL CF/AR control HPRT TF/TR GCD Enzyme: GeneArt + -® Precision + TAL - GeneArt + -® PerfectMatch + - Negative + TF/TR TAL CF/AR control GCD Enzyme: + - + - + - + - + - GeneArt® Precision TAL GeneArt® PerfectMatch Negative TF/TR TAL GF/TR control CCR5 GeneArt + -® Precision + TAL - GeneArt + -® PerfectMatch + - Negative + TF/TR TAL GF/TR control + - + - + - + - + 7.4 10.3 7.4 6.0 7.4 10.3 RELA 7.4 6.0 GeneArt® Precision TAL GeneArt® PerfectMatch Negative TF/TR TAL AF/TR control RELA GeneArt + -® Precision + TAL - GeneArt + -® PerfectMatch + - Negative + TF/TR TAL AF/TR control + - + - + - + - + - IP3R2 GeneArt® Precision TAL GeneArt® PerfectMatch Negative TF/TR TAL AF/GR control IP3R2 GeneArt + -® Precision + TAL - GeneArt + -® PerfectMatch + - Negative + TF/TR TAL AF/GR control + - + - + - + - + 6.6 6.6 13.0 12.9 6.6 6.6 ACTB 13.0 12.9 GeneArt® Precision TAL GeneArt® PerfectMatch Negative TF/TR TAL GF/CR control ACTB GeneArt + -® Precision + TAL - GeneArt + -® PerfectMatch + - Negative + TF/TR TAL GF/CR control + - + - + - + - + - Cleavage 23.6 efficiency (%) 25.3 35.6 35.3 4.5 1.5 12.1 11.1 15.1 14.3 22.0 31.2 Cleavage 23.6 efficiency (%) 25.3 35.6 35.3 4.5 1.5 12.1 11.1 15.1 14.3 22.0 31.2 IL2 CCR5 GeneArt® Precision TAL GeneArt® PerfectMatch Negative TF/TR TALIL2 CF/TR control GCD Enzyme: GeneArt + -® Precision + TAL - GeneArt + -® PerfectMatch + - Negative + TF/TR TAL CF/TR control GCD Enzyme: + - + - + - + - + - Cleavage 22.2 efficiency (%) 21.5 Cleavage 22.2 efficiency (%) 21.5 GeneArt® Precision TAL GeneArt® PerfectMatch Negative TF/TR TAL GF/TR control CCR5 GeneArt + -® Precision + TAL - GeneArt + -® PerfectMatch + - Negative + TF/TR TAL GF/TR control + - + - + - + - + - IP3R2 GeneArt® Precision TAL GeneArt® PerfectMatch Negative TF/TR TAL AF/GR control IP3R2 GeneArt + -® Precision + TAL - GeneArt + -® PerfectMatch + - Negative + TF/TR TAL AF/GR control + - + - + - + - + - 25.9 21.6 9.7 9.8 12.4 11.6 9.4 11.2 15.3 20.7 25.9 21.6 9.7 9.8 12.4 11.6 9.4 11.2 15.3 20.7 Figure 6. GeneArt PerfectMatch TALs function as well as or better than current TAL effectors in 293FT cells (A) and HeLa cells (B) when targeting sequences of forward and reverse TAL effectors are preceded by different (non-identical) bases. The red arrowheads point to the cleavage products of the genomic cleavage detection (GCD) enzyme if multiple bands were observed in a GCD assay. ® Figure 7 Figure Figure 7 7 Note: GeneArt PerfectMatch TALs exhibited ≥50% efficiency compared to current TAL effectors on the same targeted region. ® GeneArt® Precision TAL GeneArt TF/TR ® Precision TAL TF/TR ® Precision TAL GCD Enzyme: GeneArt + - + GCD Enzyme: TF/TR + - + GCD Enzyme: + - + - Cleavage 35.2 Cleavage efficiency (%) 35.2 Cleavage efficiency (%) 35.2 efficiency (%) 32.6 32.6 32.6 HPRT HPRT GeneArt PerfectMatch Negative HPRT GeneArt TAL GF/GRPerfectMatch Negative control GeneArt TAL GF/GRPerfectMatch Negative + GF/GR - + - control + TAL + - + - control + + - + - + ® ® ® 40.0 40.0 40.0 38.0 38.0 38.0 9.1 9.1 9.1 IL2 GeneArt® Precision TAL GeneArt TF/TR ® Precision TAL TF/TR ® Precision TAL GCD Enzyme: GeneArt + - + GCD Enzyme: TF/TR + - + GCD Enzyme: + - + - Cleavage 23.8 Cleavage efficiency (%) 23.8 Cleavage efficiency (%) 23.8 efficiency (%) 22.0 22.0 22.0 IL2® PerfectMatch GeneArt ® GeneArt PerfectMatch TALIL2 CF/CR ® GeneArt PerfectMatch TAL CF/CR + TAL CF/CR + - Negative Negative control Negative control + control + - + - + + - + - + - 19.7 19.7 19.7 24.4 24.4 24.4 AAVS-1 GeneArt® Precision TAL AAVS-1 GeneArt® PerfectMatch ® GeneArt GeneArt PerfectMatch TF/TR ® Precision TAL AAVS-1 TAL AF/AR ® ® GeneArt Precision TAL GeneArt PerfectMatch TAL AF/AR GCD Enzyme: TF/TR + + + + TF/TR TAL AF/AR GCD Enzyme: + - + - + - + GCD Enzyme: + - + - + - + - IP3R2 IP3R2 GeneArt PerfectMatch Negative IP3R2 GeneArt TAL CF/ARPerfectMatch Negative control GeneArt TAL CF/ARPerfectMatch Negative + CF/AR- + - control + TAL + - + - + - + - control + + - + - + - + - + - GeneArt® Precision TAL GeneArt TF/TR ® Precision TAL GeneArt TF/TR ® Precision TAL + - + TF/TR 10.2 10.2 10.2 ® ® ® 31.5 31.5 31.5 25.2 25.2 25.2 CCR5 GeneArt® Precision TAL GeneArt TF/TR ® Precision TAL GeneArt TF/TR ® Precision TAL + - + TF/TR ® CCR5 GeneArt PerfectMatch ® CCR5 GeneArt PerfectMatch TAL AF/AR ® GeneArt PerfectMatch TAL AF/AR + AF/AR- + TAL 16.6 16.6 16.6 11.9 11.9 11.9 + - + - + - + - + + - + - + - + - + - 17.6 17.6 17.6 21.8 21.8 21.8 20.1 20.1 20.1 8.5 8.5 8.5 GeneArt® Precision TAL GeneArt TF/TR ® Precision TAL GeneArt TF/TR ® Precision TAL + - + TF/TR ® ACTB GeneArt PerfectMatch ® ACTB GeneArt PerfectMatch TAL GF/GR ® GeneArt PerfectMatch TAL GF/GR + GF/GR - + TAL 23.3 23.3 23.3 25.2 25.2 25.2 13.8 13.8 13.8 27.0 27.0 27.0 ® ® ® 21.6 21.6 21.6 22.8 22.8 22.8 ACTB Negative Negative control Negative control + control + - + - + - + - + + - + - + - + - + 18.5 18.5 18.5 23.4 23.4 23.4 Figure 7. GeneArt PerfectMatch TALs function as well as or better than current TAL effectors in U2OS cells. GeneArt PerfectMatch TAL pairs with 15–16 bp spacing were compared with current TAL effectors at 7 loci in U2OS cells. The red arrowheads point to the cleavage products of the genomic cleavage detection (GCD) enzyme if multiple bands were observed in a GCD assay. ® Negative Negative control Negative control + control + + - ® Note: “Equal or better function” of GeneArt PerfectMatch TALs is when cleavage efficiency by GeneArt PerfectMatch TALs is ≥50% efficiency compared to current TAL effector performance on the same targeted region. ® Cleavage 11.0 Cleavage efficiency (%) 11.0 Cleavage efficiency (%) 11.0 efficiency (%) 13.0 13.0 13.0 IP3R1 IP3R1 GeneArt PerfectMatch Negative IP3R1 GeneArt TAL TF/ARPerfectMatch Negative control GeneArt TAL TF/ARPerfectMatch Negative + TF/AR- + - control + TAL + - + - + - + - control + + - + - + - + - + - GeneArt® Precision TAL GeneArt TF/TR ® Precision TAL GeneArt TF/TR ® Precision TAL + - + TF/TR Negative Negative control Negative control + control ® Conclusion GeneArt PerfectMatch TALs perform comparably to or better than GeneArt Precision TALs. GeneArt PerfectMatch TALs enable precise TAL effector–based targeting of any DNA sequence—there is no longer a 5´ T target sequence constraint, allowing you more freedom to innovate. 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. ® ® ® ® Ordering information The GeneArt PerfectMatch TALs order form is available at lifetechnologies.com/TALs ® Place your order by downloading and completing the order form and emailing it to [email protected] Related product ordering information Product Quantity Cat. No. GeneArt Genomic Cleavage Detection Kit 20 reactions A24372 Lipofectamine 2000 Transfection Reagent 1.5 mL 11668019 Lipofectamine 3000 Transfection Reagent 1.5 mL L3000015 ® ® ® References 1. Lamb BM, Mercer AC, Barbas CF 3rd (2013) Directed evolution of the TALE N-terminal domain for recognition of all 5´ bases. Nucleic Acids Res 41:9779–9785. 2. Bogdanove AJ, Schornack S, Lahaye T (2010) TAL effectors: finding plant genes for disease and defense. Curr Opin Plant Biol 13:394–401. 3. Bogdanove AJ, Voytas DF (2011) TAL effectors: customizable proteins for DNA targeting. Science 333:1843–1846. 4. Boch J, Scholze H, Schornack S, et al. (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326:1509–1512. 5. Cermak T, Doyle EL, Christian M, et al. (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39:e82. 6. Mak AN, Bradley P, Cernadas RA, et al. (2012) The crystal structure of TAL effector PthXo1 bound to its DNA target. Science 335:716–719. Find out more at lifetechnologies.com/tal For Research Use Only. Not for use in diagnostic procedures. © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. CO29200 0914
© Copyright 2024