dc.contributor.author | Briggs, Adrian W. | |
dc.contributor.author | Rios, Xavier | |
dc.contributor.author | Chari, Raj | |
dc.contributor.author | Yang, Luhan | |
dc.contributor.author | Zhang, Feng | |
dc.contributor.author | Mali, Prashant | |
dc.contributor.author | Church, George M. | |
dc.date.accessioned | 2016-05-25T18:04:58Z | |
dc.date.available | 2016-05-25T18:04:58Z | |
dc.date.issued | 2012-06 | |
dc.date.submitted | 2012-06 | |
dc.identifier.issn | 0305-1048 | |
dc.identifier.issn | 1362-4962 | |
dc.identifier.uri | http://hdl.handle.net/1721.1/102681 | |
dc.description.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. | en_US |
dc.description.sponsorship | National Human Genome Research Institute (U.S.) ((NHGRI) 1P50 HG005550) | en_US |
dc.description.sponsorship | European Molecular Biology Organization (ALTF 91-2010) | en_US |
dc.description.sponsorship | Canadian Institutes of Health Research (Banting Postdoctoral fellowship) | en_US |
dc.language.iso | en_US | |
dc.publisher | Oxford University Press | en_US |
dc.relation.isversionof | http://dx.doi.org/10.1093/nar/gks624 | en_US |
dc.rights | Creative Commons Attribution Non-Commercial License | en_US |
dc.rights.uri | http://creativecommons.org/licenses/by-nc/3.0/ | en_US |
dc.source | Oxford University Press | en_US |
dc.title | Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers | en_US |
dc.type | Article | en_US |
dc.identifier.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. | en_US |
dc.contributor.department | Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences | en_US |
dc.contributor.mitauthor | Zhang, Feng | en_US |
dc.relation.journal | Nucleic Acids Research | en_US |
dc.eprint.version | Final published version | en_US |
dc.type.uri | http://purl.org/eprint/type/JournalArticle | en_US |
eprint.status | http://purl.org/eprint/status/PeerReviewed | en_US |
dspace.orderedauthors | Briggs, A. W.; Rios, X.; Chari, R.; Yang, L.; Zhang, F.; Mali, P.; Church, G. M. | en_US |
dspace.embargo.terms | N | en_US |
dc.identifier.orcid | https://orcid.org/0000-0003-2782-2509 | |
mit.license | PUBLISHER_CC | en_US |