Table 2.
TALEN Assembly Methods
| Class/Name | Description | Minimum Length of Protocol† |
Size RVD assembly (bps) |
Addgene Name | Reference |
|---|---|---|---|---|---|
| Golden Gate (GG) | Original Golden Gate | 5 days | 12–31 | Golden Gate 2.0 | 27 |
| Modified Golden Gate | Original GG with FusB4 library | 5 days | 15 | TBA | 25 |
| Original GG with 6-RVD FusA | 5 days | 12–31 | TALEN construction and evaluation accessory pack | 43 | |
| Original GG with tetramer/trimer library | 3 days | 15 | N/A | 23 | |
| GG/PCR assembly of multimers | 3 days | 14–19 | N/A | 156 | |
| GG tetramer/dimer/monomer library | 3 days | 15–19 | N/A | 14 | |
| Hierarchical PCR/ligation | GG followed by PCR/ligation (human codon optimized) | 3 days | 14–25 | Zhang lab TALE Toolbox | 68 |
| Modular Assembly | GG-like/8-mer assembly | 5 days | 16 or 24 | N/A | 157 |
| Unit Assembly | Units assembled via icosaudamer restriction digest/ligation | 7 days | 10–17 | N/A | 44 |
| FLASH | Libraries of 2-, 3-, and 4-mer repeats/solid-phase magnetic beads | 3 days | Unlimited | N/A | 40 |
| REAL* | Restriction digest/ligation (non-GG) | 9 days | 13–17 | Joung Lab REAL assembly TALEN kit | 54 |
| REAL-Fast* | FLASH libraries/cloning hybrid | 7 days | 13–17 | N/A | 54 |
| Ligation-independent cloning | T4 DNA polymerase endonuclease activity to assemble multimers | 3 days | 16 or 19 | LIC TAL effector assembly kit | 42 |
| Iterative Cap Assembly | Sequential construction using monomers on solid- state beads | 3 days | Unlimited | N/A | 38 |
*
= For TALENs longer than 17 RVDs, two additional days are needed
†
= Differences in lengths a result of the number of overnight bacterial cultures