A desirable way to synthesize long repetitive DNA, which is refractory to current high-throughput DNA synthesis methods (6
), would be to sequentially couple DNA repeat modules by ligation on a solid surface, in a process analogous to chemical oligonucleotide synthesis from single nucleotides (5
). However, in contrast to oligonucleotide synthesis, which uses chemical reactions with coupling efficiencies >99.5% (5
), enzymatic ligation of relatively large DNA monomers may not be similarly efficient. This is critical since a drop in coupling efficiency just to 90% would result in only ~12% full-length chains after 20 consecutive ligations. In addition, although nucleotides can be chemically protected and deprotected to prevent self-coupling during single attachment steps (4
), analogous processes are not trivial for oligonucleotide ligation, likely requiring further enzymatic steps with sub-optimal efficiencies. Our ICA method overcomes these problems by alternating between three versions (A–C) of each monomer in the ligation steps, which as well as preventing monomer self-ligation permits inclusion in each step of a ‘capping’ oligonucleotide that can ligate to incompletely extended chains from the previous step. The capping oligonucleotides are hairpin-based short single strands, which makes them stoichiometrically perfect, rapidly diffusible and cheap to use in high molar concentrations, leading to high capping efficiency.
Here we have tested ICA on custom TALE genes. Due to their highly repetitive nature, custom TALEs have mostly been synthesized by multi-step hierarchical ligation and cloning. Most recent methods (15
) use variations of Golden-Gate cloning (22
), in which a Type IIS restriction enzyme allows the creation of position-specific overhangs on monomers (prepared from PCR or plasmids) for multi-piece, defined-sequence ligation in a single reaction. While effective, these methods required extensive initial formatting of the monomers or pre-fabricated dimers into many different position-specific forms whether through PCR (18
) or construction of large (>70) plasmid libraries (15
). Due to limits in the number of pieces Golden-Gate ligation can assemble in one reaction, the studies cited above required multiple rounds of assembly and hence between 2 and 5 days to produce new TALEs with target sites >14
bp. Recently, Reyon et al
. presented FLASH (19
), the first automated method of TALE synthesis which like ICA builds TALEs by sequential ligation on magnetic beads. FLASH is a powerful method that can produce dozens of TALENs per day in parallel using an automated liquid handing station. However, FLASH builds TALEs from oligomers (mostly 4-mers) rather than monomers, picked from an exhaustive pre-fabricated library of 376 plasmids. This reduces the number of ligation reactions needed to make full-length TALEs, which is likely necessary given that capping oligonucleotides are not used in FLASH (although in theory they could be introduced to this method to improve efficiency). However, reliance on the oligomer library will make it cumbersome to replace individual monomers with new ones of improved design and combinatorially virtually impossible to expand the routine repertoire of monomers much beyond 4. We envisage multiple reasons to add new TALE monomers to the standard repertoire, such as incorporation of monomers tagged with fluorescent or activatable moieties or ones that target epigenetically modified bases, or even the creation of hybrid DNA-binding domains that combine TALEs with other motifs such as zinc fingers. ICA is, to our knowledge, unique in providing an automatable platform that can produce full-length TALEs from individual monomers, with only three or four PCRs needed to introduce any new module to the repertoire. In addition, since each ligation step picks from only four standard monomers rather than hundreds of oligomers and takes place rapidly (2
min) at room temperature, ICA is likely to be applicable to current to microarray printing technology to allow production of libraries of thousands of TALE constructs. We note that for effective production of large TALE libraries, the error rate of individual constructs () must be reduced. However, since most errors we observed likely stem from primer or polymerase errors introduced during monomer PCR, the majority of these might be removed by preparing monomers in future from digested plasmids (15
) rather than PCR.
In testing the TALENs we produced by ICA, we used an oligonucleotide donor to genomically modify a reporter gene after a TALEN-stimulated chromosomal break, representing to our knowledge the first report of TALEN-oligonucleotide gene editing. Sequence analysis showed that the L19/R19 TALEN pair very efficiently cut the target site, with up to 80% of alleles showing evidence of NHEJ 4 days after TALEN transfection. However, the frequency of donor oligonucleotide incorporation was considerably lower, with only 1–2% of cells becoming EGFP
positive. Since oligonucleotides carry advantages over longer donor DNA in terms of cost and design simplicity, future work into increasing the ratio of gene editing to NHEJ after TALEN cutting would be extremely useful. By testing our TALEN length variants in human cells, we found that all TALENs tested showed activity, although TALENs shorter than ~15 monomers long tended to be less effective than longer ones, supporting the need for a synthesis method that can produce TALENs with sufficiently long DNA target sites. In our assay, we observed similar target repair activity among several TALENs with different bases at the 0th position, indicating that a thymine at this position is not strictly required for TALEN activity. This result, which contrasts with previous bioinformatic, crystal structure and some empirical data (10
) but agrees with others (13
), may have several explanations. TALENs as long as the ones we used may be tolerant of the decrease in binding affinity at any single site including the 0th position. We also note that using EGFP
rescue of our reporter system as a measurement of TALEN activity may become less accurate once TALEN cutting efficiency approaches 100%, since donor DNA incorporation will become the limiting factor in the rescue rather than target site cutting. In addition, since at least one of each TALEN pair tested did overlap a 0th position thymine, FOKI recruitment effects might be acting to increase activity of the other TALEN. Nevertheless, our results generally support and extend recent findings (19
) that TALENs can be confidently adapted for new targets with minimal design constraints, supporting the usefulness of high-throughput synthesis of TALENs and other TALE-fusion genes. In summary, ICA is a fast, efficient and scalable method of producing repetitive modular DNA of defined sequence. Although here we have applied the method to TALE-gene synthesis, we believe the approach should be useful for production of other modular constructs in synthetic biology (29
) where there is increasing demand for rapid DNA assembly-from-parts.