An Integrated Chip for the High‐Throughput Synthesis of Transcription Activator‐like Effectors

Recent developments using transcription activator-like effectors (TALEs) provide an alternative approach to the design and synthesis of sequence-specific nucleases. TALEs, originally discovered in the plant pathogen Xanthomonas sp. , comprised of a variable number of tandem monomers (often 16 or more), each having 33–35 amino acids that specifically recognize one target nucleotide base. The sequence of the monomers is highly conserved, but they differ primarily at amino acids 12 and 13. This region, known as the repeat variable diresidue (RVD), is thought to determine the nucleotide-binding specificity of each TALE monomer (NI=A, HD=C, NG=T, NN=G or A). The well-delineated specificity between the RVD and its target base makes TALEs a powerful DNA-targeting tool with various applications. For example, fusion of the FokI nuclease domains to the C-termini of two paired synthetic TAL effectors (giving TALE nucleases, TALENs) allows efficient DNA sequence-specific endonuclease activity in a variety of cells and organisms, including human stem cells, zebrafish, and rats (Scheme 1). Once the paired TALENs bind to the target sites, the FokI catalytic domains dimerize between the paired binding sites and cleave the DNA, introducing double-strand breaks (DSBs). DSBs are generally repaired by the nonhomologous end-joining (NHEJ) pathway, which results in small deletions or insertions and functional gene knock-out. Alternatively, TALE-Fok1-mediated DSBs can also stimulate homologous recombination, enabling site-specific insertion of an exogenous DNA sequence. TALE transcription factors (TALE-TFs) are constructed by fusion of the TALE DNA-binding domain with the VP64 transcription activation domain (Scheme 1); these factors bind a specific site in the promoter region of a gene upstream of the transcription start site and recruit the transcription complex to initiate gene transcription. Despite these applications, the capability to fully utilize TALEN or TALE-TF remains limited because of the difficulty in constructing customized TALEs with specific arrangements of tandem repeat monomers. A few strategies for constructing TAL effector repeats have been reported, such as “hierarchical-ligation assembly”, “unit assembly”, “modular assembly”, “golden-gate clone”, and others. However, these methods require labor-intensive operations, including gel purification of products and selection of positive bacterial clones after each step of ligation. By taking advantage of solid-phase gene synthesis, we report herein a novel strategy of magnetic bead-based TALE assembly, which allowed the synthesis of over one hundred TALEs, comprised of 16 or 20 repeat units, in three days. We used a chip containing two components: a microwell array and magnetic microbeads coated with streptavidin (Figure 1A). Utilizing this chip allowed the simplification of large-scale TALE production to three steps: monomer ligation, enzymatic digestion, and purification (Figure 1B). Briefly, a biotinylated DNA double-strand adaptor with an SpeI restriction site on the one end was immobilized on streptavidin-coated magnetic beads. The first repeat unit was digested with SpeI and subsequently ligated to the adaptor using T4 DNA ligase on the magnetic beads. After rinsing with buffer three times, the ligated products (on the beads) Scheme 1. Two applications for TALEs. (Top) TALEs can be used to generate customized transcription factors (TALE-TFs) and modulate the transcription of endogenous genes. The TALE DNA-binding domain is fused to the synthetic VP64 transcriptional activator, which recruits RNA polymerase and other factors required to initiate transcription. (Bottom) TALE nucleases (TALENs) can be used to generate site-specific DNA double-strand breaks and facilitate genome editing through nonhomologous end joining or homologous recombination. Each TALE DNA-binding domain is fused to the catalytic domain of the FokI endonuclease; upon FokI dimerization, the endonuclease cuts the DNA between the left and right TALEN binding sites.