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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    • "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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    ABSTRACT: CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.
    Frontiers in Genetics 10/2015; 6:300. DOI:10.3389/fgene.2015.00300
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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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    ABSTRACT: Methanosaeta harundinacea and Methanosarcina barkeri, known as classic acetoclastic methanogens, are capable of directly accepting electrons from Geobacter metallireducens for the reduction of carbon dioxide to methane, having been revealed as direct interspecies electron transfer (DIET) in the laboratory co-cultures. However, whether their co-occurrences are ubiquitous in the iron (III)-reducing environments and the other species of acetoclastic methanogens such as Methanosarcina mazei are capable of DIET are still unknown. Instead of initiating the co-cultures with pure cultures, two-step cultivation was employed to selectively enrich iron (III)-reducing microorganisms in a coastal gold mining river, Jiehe River, with rich iron content in the sediments. First, iron (III) reducers including Geobacteraceae were successfully enriched by 3-months successive culture on amorphous Fe(III) oxides as electron acceptor and acetate as electron donor. High-throughput Illumina sequencing, terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis based on 16S rRNA genes revealed that the enrichment cultures actively contained the bacteria belong to Geobacteraceae and Bacilli, exclusively dominated by the archaea belong to Methanosarcinaceae. Second, the enrichment cultures including methanogens and Geobacteraceae were transferred with ethanol as alternative electron donor. Remarkably, aggregates were successively formed in the enrichments after three transfers. The results revealed by RNA-based analysis demonstrate that the co-occurrence of Methanosarcina mazei and Geobacteraceae in an iron (III)-reducing enrichment culture. Furthermore, the aggregates, as close physical contact, formed in the enrichment culture, indicate that DIET could be a possible option for interspecies electron transfer in the aggregates.
    Frontiers in Microbiology 09/2015; 6. DOI:10.3389/fmicb.2015.00941 · 3.99 Impact Factor
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    • "Established methods in mutability analyses and mutagenesis assays have been successfully employed in the evaluation of how mutable a gene or genome is regarding the phenotypegenotype relationships and how potential mutagens cause gene mutations [11] [12] [13]. For screening or testing the mutagenesis effect of potential mutagens, a number of in vitro and in vivo assays have been developed and widely used, such as deep sequencing and mismatch Cel 1 or T7 [14] [15] [16] [17] [18] [19] [20] [21]. In general, these aforementioned methods are useful in evaluation of mutation spectra of either a gene, a genome, or a mutagen. "
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    ABSTRACT: Not all proteins are tolerable to mutations. Whether a specific protein can be a mutable target is of importance in the biotechnology and pharmaceutical industry. This study reported a novel mutagenesis assay using tandem NNT and NNC oligonucleotides to test the mutability of a candidate gene. These two tandem oligonucleotides avoid the risk of forming nonsense mutations and render flexibility of truncating or expanding the insertion size. As a reporter gene, ZeoR (zeocin resistance gene) was confirmed to have a high tolerance for mutagenesis by this new assay.
    07/2015; 2015:950873. DOI:10.1155/2015/950873
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