title: 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.
authors: Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. abriggs@genetics.med.harvard.edu
authors: Adrian W
authors: AW
authors: Briggs
authors: Xavier
authors: X
authors: Rios
authors: Raj
authors: R
authors: Chari
authors: Luhan
authors: L
authors: Yang
authors: Feng
authors: F
authors: Zhang
authors: Prashant
authors: P
authors: Mali
authors: George M
authors: GM
authors: Church
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism; D017930:Genes, Reporter; D005813:Genes, Synthetic; D059765:Homologous Recombination; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid
chemical_list: D004268:DNA-Binding Proteins; D004247:DNA; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific