Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers

Abstract

DNA built from modular repeats presents a challenge for gene synthesis. We present a solid surface-based sequential ligation approach, which we refer to as iterative capped assembly (ICA), that adds DNA repeat monomers individually to a growing chain while using hairpin 'capping' oligonucleotides to block incompletely extended chains, greatly increasing the frequency of full-length final products. Applying ICA to a model problem, construction of custom transcription activator-like effector nucleases (TALENs) for genome engineering, we demonstrate efficient synthesis of TALE DNA-binding domains up to 21 monomers long and their ligation into a nuclease-carrying backbone vector all within 3 h. We used ICA to synthesize 20 TALENs of varying DNA target site length and tested their ability to stimulate gene editing by a donor oligonucleotide in human cells. All the TALENS show activity, with the ones >15 monomers long tending to work best. Since ICA builds full-length constructs from individual monomers rather than large exhaustive libraries of pre-fabricated oligomers, it will be trivial to incorporate future modified TALE monomers with improved or expanded function or to synthesize other types of repeat-modular DNA where the diversity of possible monomers makes exhaustive oligomer libraries impractical.

Figure 1.

ICA of repeat-module DNA. ICA assembles repeat monomers into chains in a cycling A-B-C ligation-wash pattern with capping oligonucleotides to block incompletely extended chains. In each A step, monomer A is ligated to monomer C (except the first ABC cycle (shown) where A is ligated to a biotinylated initiator oligonucleotide immobilized on streptavidin beads). A hairpin capping oligonucleotide (black) is included to cap unligated monomer B (not relevant in the first cycle). In B steps, monomer B is ligated to monomer A and a capping oligo is included to cap unligated monomer C from the previous step (or unligated initiator in the first cycle). In C steps, monomer C is ligated to monomer B and a capping oligo is included to cap unligated monomer A from the previous step. This ABC cycling pattern continues until a final ligation with a terminator oligonucleotide (T, Final). A single exception occurs in the sixth step where a modified C monomer, Cseq, is used that allows later annealing of a sequencing primer. After the final ligation, products are eluted from the beads and briefly amplified by PCR before Golden-Gate assembly into a backbone vector, e.g. assembly of TALEs into a FokI nuclease-carrying plasmid, using BsaI. (a) Post-elution PCR products for TALES of length 11, 15, 18 and 19 monomers, plus the same 19-mer synthesized without capping oligos (nb). (b) AfeI digestions of TALE-nuclease (TALEN) plasmids from six randomly picked colonies per construct. Expected sizes of full-length TALE insert bands are labeled with red arrows, and the full-length clone frequencies are shown.

Figure 2.

Testing activity of TALEN variants on a human cell reporter system. (a) To generate a TALEN activity reporter system we integrated a barcoded (H₈, H = A/C/T) defective EGFP gene carrying a non-functional ACG start codon into the genome of 293 T cells. Gene editing by a 75-nt donor oligonucleotide stimulated by a TALEN-mediated double-strand break near the target site should correct the start codon and rescue EGFP expression. We designed 21 length variants of TALENs designed to flank the defective site. TALEN RVD sequences are shown. White monomer circles represent ‘0th’ positions of the DNA target sequence bound by the TALE N-terminus. (b) Post-elution PCR products of the ligated right TALE domains (for R11 see TAL-11, Figure 1b) and AfeI digestions of corresponding assembled and sequence-verified TALEN plasmids. (c) Epifluorescent microscopy and flow cytometry results after transfection of the reporter cells with the L19/R19 TALEN pair and the donor oligonucleotide. (d) Gene editing events (green sequences, functional start codon underlined) were confirmed by sequencing of the target site in EGFP+ cells, with widespread NHEJ (black sequences) also observed (red = unmodified sequence). Reporter barcodes (blue) identify from which of two genomic reporter copies each sequence is derived. (e) EGFP rescue activity of all successfully synthesized TALEN length variants. Each TALEN was transfected together with the donor and either L19 or R19, whichever was relevant. Bar colors indicate the DNA base located at the ‘0th’ position for each TALEN tested. Error bars show standard error (n = 3 for all).

Figure 3.

Accelerating TALEN production by ICA. (a) PCR amplification of repetitive DNA such as TALEs frequently generates artifacts, for example in colony PCR of an assembled TALEN (lane (i)). We found that using PCR primers in the vector far away (∼1400 bp) from the repetitive TALE region dramatically reduces artifacts resulting in clean PCR products (ii) that can be directly sequenced saving a day’s wait for plasmid preparation, or digested with AfeI (iii) to reveal the TALE insert length (red arrow). (b) We tested whether the PCR step after ICA ligation on beads can be omitted by performing Golden-Gate assembly of two 19-monomer long (excluding the 0th position) TALEs into the nuclease vector directly on the beads. (c) After transformation hundreds of colonies were generated, with the L19 and R19 constructs respectively generating 8/10 and 10/10 AfeI-digested colony PCRs with full-length inserts (red arrows). (d) A realistic three-day work schedule using the methods presented here from TALEN design to transfection of target cells.