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TALEN and TAL Effector Products and Services

Site-specific genome editing at will   

Introduction

Transcription activator-like (TAL) effectors are DNA binding proteins produced by Xanthomonas bacteria when they infect plants. These proteins can activate the expression of plant genes by recognizing and binding host plant promoter sequences through a central repeat domain consisting of a variable number of ~34 amino acid repeats. The residues at the 12th and 13th positions of each repeat are hyper-variable. There appears to be a simple one-to-one code between these two critical amino acids in each repeat and each DNA base in the target sequence, e.g. NI = A, HD = C, NG = T, and NN = G or A.

TAL effectors have been utilized to create site-specific gene-editing tools by fusing target sequence-specific TAL effectors to nucleases (TALENs), transcription factors (TALE-TFs) and other functional domains. These fusion proteins can recognize and bind chromosome target sequences specifically to execute their gene editing functions, such as gene knockout, knockin (with donor plasmid), mutagenesis, activation, repression and more. Unlike zinc fingers’ nucleotide triplet recognition, TAL effector domains recognize single nucleotides, which allows researchers to be able to specifically target whatever sequence they want.

Advantages

  • Target any sequence in any cell
  • Highly sequence-specific genome editing
  • For gene knockout, knockin, mutagenesis, activation, repression and more
  • Flexible TAL effector design of binding and functional domains, such as TALEN and TALE-TF

 

Figure 1. Illustration of TALEN design

 

Figure 2. Illustration of TALE-TF design

Comparison between TALEN and CRISPR-Cas9 Systems

Property TALEN CRISPR-Cas9
Type of recognition Protein-DNA RNA-DNA
Methylation sensitive Sensitive Not sensitive
Chromotin structure sensitive Sensitive Sensitive
Off-target effect Less observed off-target effects More potential off-target effects than TALENs and ZFNs
Multiplexing Rarely used Capable

 

References

  1. Boch, J. et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science. 2009 326(5959):1509-12
  2. Moscou, M. et al. A simple cipher governs DNA recognition by TAL effectors. Science. 2009 326(5959):1501
  3. Christian, M. et al. Targeting DNA Double-Strand Breaks with TAL Effector Nucleases. DOI: 10.1534/genetics.110.120717
  4. Morbitzera, R. et al. Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE)-type transcription factors. www.pnas.org/cgi/doi/10.1073/pnas.1013133107
  5. Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Research, 2011, Vol. 39, No. 12   e82 doi:10.1093/nar/gkr218
  6. Li, T. et al. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acids Research, 2011, Vol. 39, No. 14   6315–6325 doi:10.1093/nar/gkr188
  7. Zhang, F. et al. Programmable Sequence-Specific Transcriptional Regulation of Mammalian Genome Using Designer TAL Effectors. Nat Biotechnol. 2011 February ; 29(2): 149–153. doi:10.1038/nbt.1775.

Applications

Genome Editing Applications

TAL Effectors and CRISPR-Cas9 are efficient systems for precise genome modifications in cell lines or model organisms. The table below shows some gene editing applications.

Genome modifications Description Genome editing tools Donor DNA
Gene tagging Add a fusion tag (e.g. luciferase, GFP) to track an endogenous promoter activity or an endogenous protein expression and location TALEN or CRISPR Required
Gene mutagenesis Introduce point mutations to an endogenous gene TALEN or CRISPR Required
Gene knockout Introduce deletions or insertions (e.g. a selection marker) to knockout an endogenous gene TALEN or CRISPR Highly recommended for selection of knockout cell lines
Gene activation Activate an endogenous gene expression TALE-TF or CRISPR-TF N/A
Gene repression Repress an endogenous gene expression TALE-R or CRISPR-R N/A
Safe harbor knockin Knockin an exogenous ORF or other genetic element to safe harbor sites of human or mouse genome TALEN or CRISPR Required

 

 

TALEN

TALEN services

   

A TAL effector nuclease (TALEN) contains a TALE DNA binding domain fused to the FokI nuclease. Two TALENs must bind on each side of the target site for FokI to dimerize and cut. TALENs induce a site-specific double-strand break (DSB), which is repaired by non-homologous end joining (NHEJ), which is error-prone and frequently causes small insertions or deletions ("indels") near the DSB site. These indels often cause frameshift mutations that can efficiently knock a gene out. Alternatively, an exogenous double-stranded donor DNA fragment can be introduced into the genome at the DSB by homologous recombination (HR). TALENs have been used to generate stably modified human embryonic stem cell and induced pluripotent stem cell (IPSCs) clones, and to generate knockout organisms such as mice, C. elegans, and zebrafish.

Figure 1. Illustration of TALEN design

 

TALEN-mediated_gene_knockout_A-m TALEN-mediated_gene_knockout_B-m
   
Figure 2. TALEN-mediated gene knockout. (left) TALEN-created DSB are repaired by NHEJ. (right) TALEN-created DSB are repaired by the insertion of selection markers (or other genetic element) from a donor plasmid through HR.

 

Figure 3. In vitro target DNA cleavage by EGFP-TALENs. (A) The TALEN target plasmid (6110 bp) contains an unique EcoRI site and an eGFP TALEN target site. The two sites are1066 bp apart. (B) 1 µg of the plasmid was incubated with the indicated enzymes for 30 min at 37°C.  0.5 volume of  the digestion reaction was analyzed by the agarose gel electrophoresis. * The  indicated fragment was checked by PCR, data not shown.

 

Figure 4. TALENs knockdown eGFP expression. (A) eGFP TALENs expression validation: ~80% confluence HEK293T cells  were transfected with 0.8 µg plasmid per well in a 6-well plate. The cells were harvested 48 hrs post-transfection. 1/20th of the cell lysate per well was analyzed for western blot using anti-Flag antibody in a SDS-PAGE (8% resolve gel) , with the untransfected cell lysate as the negative control. (B) TALENs knockdown eGFP expression: HEK293T cells in a 6-well plate were co-transfected with EX-EGFP-Lv105 and TALEN plasmids or control plasmid. EGFP expression was checked under microscope(Nikon Eclipse Ti, exposure time: 600ms) 48hrs post-transfection.

 

(A))

*The surrogated reporter plasmid was constructed by disrupting a CMV-driven Gaussia luciferase (GLuc) with a in-frame stop codon followed by eGFP TALEN target  sequences. 

**The donor plasmid contains a promoter-less wild type GLuc, which can replace the interrupted GLuc in the surrogate reporter plasmid and restore the GLuc expression through homologous recombination, which is enhanced by TALEN cleavage. 

(B)

Figure 5. Restore disrupted Gaussia luciferase function via TALEN-mediated homologous recombination. (A) Illustration of experimental design. (B) HEK293T cells in a 6-well plate were co-transfected with the eGFP-TALEN pair (1 µg), the surrogate reporter plasmid (0.5 µg) and the donor plasmid (0.5 µg). 48hours post-transfection, the  restored Gluc  activity was determined to evaluate the TALEN function. Internal control SEAP activity was used for normalization.

TALE-TF

TALE-TF services

 

A key application for TALEs is the targeted activation and repression of target genes in cells by fusing transactivation domains to TALE DNA binding domains . The TALE-TF construct is a powerful tool to selectively modulate gene expression in eukaryotic cells with exquisite specificity. The TALE-TF contains a TALE DNA binding domain fused to the VP64 transcription activator.

Figure 1. Illustration of TALE-TF design

TALE-TF control clone

Buy Catalog# Product Selection Marker Reporter Gene Price
CTR001-TF-01 Scrambled control for TALE-TF Hygro N/A inquire

 

 

Figure 2. NTF3 TALE-TF regulates endogenous NTF3 transcription. Endogenous NTF3 transcription activation by TALE-TF: HEK 293T cells transfected with the NTF3 TALE-TF (6 well plate, 1 µg plasmid per well) exhibited a 17-fold increase in the amount of NTF3 mRNA compared to cells transfected with an empty vector. Measurements were performed in triplicate.

 

Figure 3. Synthetic TALE activators act synergistically to express human genes. (a) Cartoon of a TALE. The indicated amino acids in each repeat recognize the base below. NLS, nuclear localization sequence; VP64/p65, activation domains (ADs). (b) Single TALEs induce target human genes with variable efficiencies. (c) Combinations of TALEs targeting either DNA strand allow for much higher gene induction rates. (Nature Methods. 2013 Vol. 10. No. 3: 207-208)

 

References

  1. Richter, A. et al. Designer TALEs team up for highly efficient gene induction. Nature Methods. 2013 Vol. 10. No. 3: 207-208
  2. Perez-Pinera, P. et al. Synergistic and tunable human gene activation by combinations of synthetic transcription factors. Nature Methods. 2013 Vol. 10. No. 3: 239-242
  3. Maeder, M.L. et al. Robust synergistic regulation of human gene expression using TALE activators. Nature Methods. 2013 Vol. 10. No. 3: 243-245

Services

 

TAL effector custom services

 

GeneCopoeia offers complete custom services for TALE-based targeted genomic modification, including TALEN or TALE-TF design and construction, donor design and construction, functional validations, stable cell line establishment, and transgenic mouse generation.

Advantages

  • Fast delivery of TAL effector plasmids or mRNA transcripts
  • Fully sequence-verified and transfection-ready
  • Functional validations available
  • Knockin donor plasmid also available
  • Stable cell line or transgenic mouse services available

 

Service details

Services Description Application
Validation Services T7 endonuclease I cleavage assay Chromosomal-level functional validation. It can detect the presence of the indels created by TALEN-mediated NHEJ repairs at the specific target site of the chromosome. TALEN
qPCR assay Chromosomal-level functional validation. It can measure the change in expression level of the target gene induced by site-specific TALE-TF transcription activator. TALE-TF
Donor clone services Donor clone design and construction
Customized plasmids designed to specifically transfer your gene of interest, selection marker or other genetic element into targeted site through homologous recombination (HR) induced by our genome editing tools.
We offer various donor vector choices with different selection markers and genetic elements built in for your experiment purpose.
TALEN
Stable cell line services Single colony Single clone of stable cell line with TALEN-mediated genome modifications. TALEN
Cell bank Creating cell bank of monoclonal stable cell line with TALEN-mediated genome modifications. TALEN
Transgenic mouse services Transgenic mouse development Transgenic mice with TALEN-mediated genome modifications TALEN
 

Donor vector types

Vector Promoter Reporter Gene Selection Marker LoxP site Vector Map
pDonor-D01 EFa1 copGFP Puromycin N/A get_info
pDonor-D02 CMV copGFP Neomycin N/A get_info
pDonor-D03 CMV N/A Neomycin N/A get_info
pDonor-D04 CMV N/A Puromycin N/A get_info
pDonor-D05 EFa1 N/A Neomycin N/A get_info
pDonor-D07 EFa1 copGFP Puromycin/TK LoxP get_info
pDonor-D08 CMV copGFP Neomycin/TK LoxP get_info
pDonor-D09 EFa1 N/A Puromycin/TK LoxP get_info
pDonor-D10 CMV N/A Neomycin/TK LoxP get_info
pDonor-D11 PGK copGFP Puromycin/TK LoxP get_info
pDonor-D12 EF1a copGFP Hygromycin/TK LoxP get_info
pDonor-D13 PGK copGFP Neomycin/TK LoxP get_info
pDonor-D14 PGK N/A Puromycin/TK LoxP get_info