Document Detail


Engineering designer transcription activator-like effector nucleases (TALENs) by REAL or REAL-Fast assembly.
MedLine Citation:
PMID:  23026907     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Engineered transcription activator-like effector nucleases (TALENs) are broadly useful tools for performing targeted genome editing in a wide variety of organisms and cell types including plants, zebrafish, C. elegans, rat, human somatic cells, and human pluripotent stem cells. Here we describe detailed protocols for the serial, hierarchical assembly of TALENs that require neither PCR nor specialized multi-fragment ligations and that can be implemented by any laboratory. These restriction enzyme and ligation (REAL)-based protocols can be practiced using plasmid libraries and user-friendly, Web-based software that both identifies target sites in sequences of interest and generates printable graphical guides that facilitate assembly of TALENs. With the described platform of reagents, protocols, and software, researchers can easily engineer multiple TALENs within 2 weeks using standard cloning techniques.
Authors:
Deepak Reyon; Cyd Khayter; Maureen R Regan; J Keith Joung; Jeffry D Sander
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.    
Journal Detail:
Title:  Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]     Volume:  Chapter 12     ISSN:  1934-3647     ISO Abbreviation:  Curr Protoc Mol Biol     Publication Date:  2012 Oct 
Date Detail:
Created Date:  2012-10-02     Completed Date:  2013-02-11     Revised Date:  2013-10-17    
Medline Journal Info:
Nlm Unique ID:  8908160     Medline TA:  Curr Protoc Mol Biol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  Unit 12.15     Citation Subset:  IM    
Copyright Information:
2012 by John Wiley & Sons, Inc.
Affiliation:
Molecular Pathology Unit, Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
DNA Ligases / metabolism
DNA Restriction Enzymes / metabolism
Deoxyribonucleases / genetics*,  metabolism*
Humans
Molecular Biology / methods*
Plants
Recombination, Genetic
Transcriptional Activation*
Grant Support
ID/Acronym/Agency:
DP1 OD006862/OD/NIH HHS; DP1 OD006862/OD/NIH HHS; R01 GM088040/GM/NIGMS NIH HHS; R01 GM088040/GM/NIGMS NIH HHS; T32 CA009216/CA/NCI NIH HHS; T32 CA009216/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
EC 3.1.-/Deoxyribonucleases; EC 3.1.21.-/DNA Restriction Enzymes; EC 6.5.1.-/DNA Ligases
Comments/Corrections

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