Article

FLASH assembly of TALENs for high-throughput genome editing

Molecular Pathology Unit, Center for Computational and Integrative Biology, and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
Nature Biotechnology (Impact Factor: 39.08). 04/2012; 30(5):460-5. DOI: 10.1038/nbt.2170
Source: PubMed

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.

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    • "r 4 bp ( NGG and NAG ) on average ( Cong et al . , 2013 ) . In contrast , PAM is not a requirement for ZFNs and TALENs . The target density is one in every 100 bp for ZFNs and one per bp for TALENs , and TALENs appears to be advantageous to both ZFNs and CRISPR / Cas9 in terms of high target density ( Sander et al . , 2011 ; Gupta et al . , 2012 ; Reyon et al . , 2012 ) ."
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