Conceived and designed the experiments: EW SM. Performed the experiments: EW RG SW CE. Wrote the paper: EW SM.
All authors are employed by Icon Gentics GmbH. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
Generation of customized DNA binding domains targeting unique sequences in complex genomes is crucial for many biotechnological applications. The recently described DNA binding domain of the transcription activator-like effectors (TALEs) from
The development of synthetic nucleases that cleave unique genomic sequences in living cells provides powerful tools for genome engineering, allowing targeted gene knockout and gene replacement
We present here an approach to assemble genes encoding TALE repeat domains based on the scaffold of AvrBs3, the first described and well characterized TALE family member
The dTALE assembly strategy described here uses the Golden Gate cloning method, which is based on the ability of type IIS enzymes to cleave outside of their recognition site. When type IIS recognition sites are placed to the far 5' and 3' end of any DNA fragment in inverse orientation, they are removed in the cleavage process, allowing two DNA fragments flanked by compatible sequence overhangs, termed fusion sites, to be ligated seamlessly (
(A) Golden Gate cloning principle applied for assembly of dTALEs. Plasmids encoding selected repeat modules (an example with only two modules, R1 and R2, is shown here due to space limitation) are mixed in one tube together with BsaI, T4 DNA ligase and the destination vector (containing a
We chose the native TALE AvrBs3 as a scaffold for customized assembly of dTALE constructs. The central DNA binding domain of AvrBs3 is formed by 17.5 tandemly arranged 34 amino acid repeats, with the last half repeat showing similarity to only the first 20 amino acids of a full repeat. In addition to the 17.5 repeats, AvrBs3 contains an N-terminally adjacent repeat 0 that is thought to be specific for a thymidine (as
Although 9 DNA fragments can be efficiently assembled in a single Golden Gate cloning reaction, cloning efficiency is significantly reduced for assembly of 17 repeat modules in a single cloning reaction (0 to 3 colonies out of 12). Therefore, we split the assembly in two successive steps. In the first cloning step, blocks of 5 or 6 repeats are assembled in three preassembly vectors, one for repeat module positions 1–6, one for positions 7–12 and one for positions 13–17 (pL1-TA1 to 3). The preassembly vectors confer ampicillin resistance (ApR) and encode a
To test functionality of the assembled dTALEs, we used transgenic
(A) Structure of the reporter construct present in transgenic
For construction of the 4 dTALE constructs, 12 parallel BsaI-based Golden Gate cloning reactions were set up with selected modules and the respective preassembly vectors pL1-TA1 to 3. For each reaction, plasmid DNA from two colonies was purified and sequenced, and all plasmids were found to contain the correct sequence. Preassembled repeat blocks were assembled to the final constructs dTALE-1 to 4 using a second BpiI-based Golden Gate cloning reaction (
We have shown here that constructs for dTALE proteins containing a 19 base DNA binding domain (consisting of 17 engineered full repeats, repeat 0 and the half repeat 17.5) can be easily assembled by two successive one-pot Golden Gate cloning reactions. We have prepared a set of 68 repeat modules that allows construction of DNA binding domains for any 17 base user-defined target sequence. The native half repeat 17.5 of AvrBs3, which contains a RVD specific for thymidine, was included in the C-terminal fragment of the final assembly vector. It would however be possible to also make half repeat modules with different RVD types to improve the binding of dTALE proteins for target sequences that do not have a T at this position. Such repeats could be assembled together with repeats 13 to 17 in a new preassembly vector replacing pL1-TA3. A new compatible final assembly vector lacking the half repeat should also be made.
In case 17 repeats are not sufficient to provide specific binding, dTALE proteins with additional repeats could easily be constructed. In order to expand the TALE modular cloning system to more than 17 repeats, new unique fusion sites have to be defined for each additional repeat, and one or more new preassembly vectors specific for the added fusion sites have to be constructed. A further option to increase dTALE specificity is the replacement of the NN RVD, which has an equal preference to A and G, by the highly G-specific NK RVD
The Golden Gate cloning method provides a perfect fit for dTALE protein engineering because it allows directional and seamless assembly of multiple DNA fragments. In addition, this cloning method is sequence-independent and allows assembly of repeats with identical or highly homologous sequences, since only the 4 base pair fusion sites at the end of the repeats have to be unique. Selection of fusion sites with unique sequence at the ends of successive repeats can be easily accomplished by either changing the codon usage of the ends of the repeats, or by shifting the fusion sites a few nucleotides at the ends of the various repeats. Since a complementary shift can be selected at the beginning of each following repeat (as shown in the result section/supporting information), seamless assembly of direct repeats can then be easily achieved.
Other alternative methods for seamless assembly of multiple DNA fragments include SLIC
In conclusion, the cloning system described here provides a simple and economical way of assembling constructs encoding dTALE proteins for genome engineering and other biotechnological applications.
Restriction enzymes used in this study were purchased from New England Biolabs (Ipswitch, MA) and Fermentas (Burlington, Canada). T4 DNA ligase was purchased from Promega (Fitchburg, WI). Plasmid DNA preparations were made by using the NucleoSpin Plasmid Quick Pure kit (Macherey-Nagel, Düren, Germany) following the manufacturer protocol. Plasmid DNA concentration was measured using a Nano Drop® Spectrophotometer ND-2000 (Peqlab, Erlangen, Germany). DNA sequences for the AvrBs3 N- and C-termini were codon-optimized using the
The repeat modules were made by annealing two partially overlapping primers and filling the single-stranded extensions using KOD polymerase (Merck, Darmstadt, Germany). The double-stranded products were digested with XhoI and cloned in the SalI site of a pUC19-derived vector conferring spectinomycin resistance and lacking BpiI and BsaI sites. For construction of the preassembly vectors pL1-TA1 to 3, a
One-step one-pot restriction/ligations were set up using approximately 30 fmol (∼100 ng for a 5 kb plasmid) of each plasmid in a mix containing Promega ligation buffer, 10 U of the selected restriction enzyme (BsaI or BpiI) and 10 U T4 DNA ligase, in a final reaction volume of 20 µl. The reactions were incubated for 2 hours at 37°C, 5 minutes at 50°C and 5 minutes at 80°C. The mix was then added to 100 µl chemical competent DH10b cells, incubated for 30 min on ice and transformed by heat shock. Two clones were analyzed by restriction analysis and, optionally, sequencing.
Sequence of the codon-optimized
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