Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers.
Authors | |
Keywords | |
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. |
Year of Publication | 2012
|
Journal | Nucleic Acids Res
|
Volume | 40
|
Issue | 15
|
Pages | e117
|
Date Published | 2012 Aug
|
ISSN | 1362-4962
|
URL | |
DOI | 10.1093/nar/gks624
|
PubMed ID | 22740649
|
PubMed Central ID | PMC3424587
|
Links | |
Grant list | T32 GM007753 / GM / NIGMS NIH HHS / United States
1P50 HG005550 / HG / NHGRI NIH HHS / United States
|