Article

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

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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    • "The presence of extensive identical repeat sequences confers a huge technical challenge to clone repeated TALE arrays for different DNA target sites. To this end, several modified methods have been developed to enable rapid TALE assembly, including the 'Golden Gate' platform[20,21], high-throughput solid-phase based sequential ligation systems[22,23], and ligation-independent cloning techniques[24]. For target site recognition, it's extremely important that the sequence of the TALE binding sites should start with a thymine (T). "
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    • "In the past, both approaches have been successfully performed using engineered zinc finger nucleases and transcription activatorlike effector-based nucleases (TALENs) to study individual gene function, and also meganucleases have been used exclusively for genome editing (Joung and Sander, 2013). However, it has been pointed out that due to the challenges in construct engineering for these systems, which rely on protein–DNA interactions for targeting, it is difficult to exploit their potential in large-scale screening approaches in which many different genes must be individually targeted (Reyon et al., 2012; Heintze et al., 2013). "
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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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