FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol

Molecular Pathology Unit, Center for Computational and Integrative Biology, and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
Nature Biotechnology (Impact Factor: 41.51). 04/2012; 30(5):460-5. DOI: 10.1038/nbt.2170
Source: PubMed


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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    • "th 5 bp barcodes . Triplicate reaction mixtures per sample were pooled , purified using the QIAquick PCR Purification kit ( QIAGEN ) , and normalized in equimolar amounts before pyrosequencing by means of a MiSeq sequencer ( Illumina ) . Raw reads of the bacterial 16S rRNA gene were processed using Trimmomatic ( Bolger et al . , 2014 ) and FLASH ( Reyon et al . , 2012 ) to merge the paired - end reads . The low quality sequences were filtered and chimeric sequences were removed by using USEARCH ( Edgar et al . , 2011 ) . Sequences were clustered into operational taxonomic units ( OTUs ) using CD - HIT ( Li and Godzik , 2006 ) with a cut - off of 97% sequence identity , and the most abundant sequence "
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    Frontiers in Microbiology 09/2015; 6. DOI:10.3389/fmicb.2015.00941 · 3.99 Impact Factor
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