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

• Published in: Nucleic acids research Vol. 40; no. 15; p. e117
• Main Authors: Briggs, Adrian W, Rios, Xavier, Chari, Raj, Yang, Luhan, Zhang, Feng, Mali, Prashant, Church, George M
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01-08-2012
• Subjects: Base Sequence; Cell Line; Deoxyribonucleases, Type II Site-Specific - genetics; Deoxyribonucleases, Type II Site-Specific - metabolism; DNA - biosynthesis; DNA - chemistry; DNA-Binding Proteins - genetics; Genes, Reporter; Genes, Synthetic; Genomes; Homologous Recombination; Humans; Methods Online; Molecular Sequence Data; Monomers; Nuclease; Oligonucleotides; Protein Structure, Tertiary - genetics; Repeated DNA sequences; Repetitive Sequences, Nucleic Acid
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gks624

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.