title: FLASH assembly of TALENs for high-throughput genome editing.
abstract: Engineered transcription activator–like effector nucleases (TALENs) have shown promise as facile and broadly applicable genome editing tools. However, no publicly available high-throughput method for constructing TALENs has been published, and large-scale assessments of the success rate and targeting range of the technology remain lacking. Here we describe the fast ligation-based automatable solid-phase high-throughput (FLASH) system, a rapid and cost-effective method for large-scale assembly of TALENs. We tested 48 FLASH-assembled TALEN pairs in a human cell–based EGFP reporter system and found that all 48 possessed efficient gene-modification activities. We also used FLASH to assemble TALENs for 96 endogenous human genes implicated in cancer and/or epigenetic regulation and found that 84 pairs were able to efficiently introduce targeted alterations. Our results establish the robustness of TALEN technology and demonstrate that FLASH facilitates high-throughput genome editing at a scale not currently possible with other genome modification technologies.
authors.affiliation: Molecular Pathology Unit, Center for Computational and Integrative Biology, and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
authors.forename: Deepak
authors.initials: D
authors.lastname: Reyon
authors.forename: Shengdar Q
authors.initials: SQ
authors.lastname: Tsai
authors.forename: Cyd
authors.initials: C
authors.lastname: Khayter
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Foden
authors.forename: Jeffry D
authors.initials: JD
authors.lastname: Sander
authors.forename: J Keith
authors.initials: JK
authors.lastname: Joung
mesh_terms: D001331:Automation; D001483:Base Sequence; D019921:Biotinylation; D019295:Computational Biology / Q000379:methods; D004262:DNA Restriction Enzymes / Q000235:genetics; D004720:Endonucleases / Q000235:genetics*; D044127:Epigenesis, Genetic; D018390:Gene Targeting / Q000379:methods; D005821:Genetic Techniques; D015894:Genome, Human; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009369:Neoplasms / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
chemical_list: D049452:Green Fluorescent Proteins; D004720:Endonucleases; D004262:DNA Restriction Enzymes