Table 1.
Primer sequences for monomer amplification.
Table 2.
Primer sequences for sequencing, subcloning and PCR verification.
Figure 1.
Timeline for the construction of TALE constructs.
Steps of the construction of TALENs and TALE-TFs are presented. TALE constructs can be generated in 3 days following the described steps. Samples of each step can be stored at −20°C for further use.
Table 3.
Primer sequences for TALE repeats amplification.
Figure 2.
Schematic representation of tetramer PCR mutation.
Cohesive ends of tetramers in the library can be changed after amplification with Tetramer-F1/Tetramer-R1, Tetramer-F2/Tetramer-R2 or Tetramer-F3/Tetramer-R3 for position specification. The original bases on tetramer plasmids are in blue and mismatches on primers are in red.
Figure 3.
Schematic representation of the cut/ligation reaction for tetramer plasmid construction.
4 selected monomer plasmids at each position (NI-1, HD-2, NG-3 and NN-4 for example in this Figure) are put into a reaction pool containing T4 DNA ligase, BsaI and ligase buffer. Once the temperature rises to 37°C, the right side of NI-1, both sides of HD-2, NG-3 and the left side of NN-4 are digested by BsaI. The unique cohesive ends facilitate the positioning of each monomer in the ligation product. T4 DNA ligase joins the fragments together at 16°C. The number of constructed tetramer plasmids increases as the cycles continue.
Figure 4.
Optional assembly of TALE repeats depending on PCR mutation.
Amplified with different primer pairs, repeat monomers, dimers, trimers or tetramers (1, 2, 3, 4 and N in this Figure) can be flexibly assembled in an optional order during a cut/ligation reaction. Some selected cohesive ends are shown and other possible cohesive ends can be designed by matching the last nucleotide coding Gly and three nucleotides coding Leu.
Figure 5.
Construction of customized TALENs or TALE-TFs depending on the tetramer library.
Three selected tetramer plasmids (Tetramer 1, Tetramer 2 and Tetramer 3) are amplified with separate primer pairs (Tetramer-F1/Tetramer-R1, Tetramer-F2/Tetramer-R2 and Tetramer-F3/Tetramer-R3) to be given unique cohesive ends specifying their positions in the assembly. The tetramers were then assembled and cloned into an expression backbone during a cut/ligation reaction.
Figure 6.
TALEN and TALE-TF expression backbones structure.
The TALEN backbone contains a TALE DNA binding domain fused to the wild type FokI nuclease. The TALEN is expressed under the control of the CMV promoter. The TALE-TF backbone contains a TALE DNA binding domain fused to the VP64 transcription activator. Both the TALE-TF and EGFP are expressed under the control of the CMV promoter and they are divided by 2A. A TALEN or a TALE-TF backbone contain a 0.5 repeat that binds the final nucleotide of the targeted DNA sequence (according to the TALE binding site); thus both the TALEN backbones and TALE-TF backbones come as 4 different plasmids with 4 different 0.5 repeats.
Table 4.
TALEN constructs.
Table 5.
TALE-TF constructs.