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: 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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    ABSTRACT: Targeted genome editing mediated by clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) technology has emerged as one of the most powerful tools to study gene functions, and with potential to treat genetic disorders. Hearing loss is one of the most common sensory disorders, affecting approximately 1 in 500 newborns with no treatment. Mutations of inner ear genes contribute to the largest portion of genetic deafness. The simplicity and robustness of CRISPR/Cas9-directed genome editing in human cells and model organisms such as zebrafish, mice and primates make it a promising technology in hearing research. With CRISPR/Cas9 technology, functions of inner ear genes can be studied efficiently by the disruption of normal gene alleles through non-homologous-end-joining (NHEJ) mechanism. For genetic hearing loss, CRISPR/Cas9 has potential to repair gene mutations by homology-directed-repair (HDR) or to disrupt dominant mutations by NHEJ, which could restore hearing. Our recent work has shown CRISPR/Cas9-mediated genome editing can be efficiently performed in the mammalian inner ear in vivo. Thus, application of CRISPR/Cas9 in hearing research will open up new avenues for understanding the pathology of genetic hearing loss and provide new routes in the development of treatment to restore hearing. In this review, we describe major methodologies currently used for genome editing. We will highlight applications of these technologies in studies of genetic disorders and discuss issues pertaining to applications of CRISPR/Cas9 in auditory systems implicated in genetic hearing loss. Copyright © 2015. Published by Elsevier B.V.
    Hearing research 05/2015; 25. DOI:10.1016/j.heares.2015.04.016 · 2.85 Impact Factor
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    • "From the two protoplast transfections with the vector p35S- ALS-TALEN we obtained sequences with ALS-TALEN-induced mutations in clusters that aligned with each of the 5 reference sequences; however the highest mutation frequency was observed in cluster B (18.1%, Table 1). Our results indicated that ALS-TALEN was able to induce mutations in both genes and in different alleles, although some were targeted more often than others, which might be due to the fact that the TALEN architecture has been reported to be sensitive to the chromatin structure and methylation state of the target region (Reyon et al., 2012; Valton et al., 2012). 3.4. "
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    ABSTRACT: Potato is the third largest food crop in the world, however the high degree of heterozygosity, the tetrasomic inheritance and severe inbreeding depression are major difficulties for conventional potato breeding. The rapid development of modern breeding methods offers new possibilities to enhance breeding efficiency and precise improvement of desirable traits. New site-directed mutagenesis techniques that can directly edit the target genes without any integration of recombinant DNA are especially favorable. Here we present a successful pipeline for site-directed mutagenesis in tetraploid potato through transient TALEN expression in protoplasts. The transfection efficiency of protoplasts was 38-39% and the site-directed mutation frequency was 7-8% with a few base deletions as the predominant type of mutation. Among the protoplast-derived calli, 11-13% showed mutations and a similar frequency (10%) was observed in the regenerated shoots. Our results indicate that the site-directed mutagenesis technology could be used as a new breeding method in potato as well as for functional analysis of important genes to promote sustainable potato production. Copyright © 2015. Published by Elsevier B.V.
    Journal of Biotechnology 04/2015; 204. DOI:10.1016/j.jbiotec.2015.03.021 · 2.88 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.
    01/2015; 2015:950873. DOI:10.1155/2015/950873
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