Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers
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Zhang_Iterative capped assembly.pdf
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Author(s)
Briggs, Adrian W.
Rios, Xavier
Chari, Raj
Yang, Luhan
Zhang, Feng
Mali, Prashant
Church, George M.
Date Issued
June 2012
Journal
Nucleic Acids Research
Publisher
Oxford University Press
Citation
Briggs, A. W., X. Rios, R. Chari, L. Yang, F. Zhang, P. Mali, and G. M. Church. “Iterative Capped Assembly: Rapid and Scalable Synthesis of Repeat-Module DNA Such as TAL Effectors from Individual Monomers.” Nucleic Acids Research 40, no. 15 (June 26, 2012): e117–e117.
Version
Final published version
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.
MIT Department
Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences
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Creative Commons Attribution Non-Commercial License
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DOI of Published Version
https://doi.org/10.1093/nar/gks624
