A TALEN Genome-Editing System for Generating Human Stem Cell-based Disease Models

Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA.
Cell stem cell (Impact Factor: 22.27). 12/2012; 12(2). DOI: 10.1016/j.stem.2012.11.011
Source: PubMed


Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We report here the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells, the latter for which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease-dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy, motor-neuron death, and hepatitis C infection. We found little evidence of TALEN off-target effects, but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines, we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease.

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    • "A surrogate reporter system (Kim et al., 2011; Ramakrishna et al., 2014) can be used to enrich for cells containing indels induced by ZFNs or gRNAs through sorting based on fluorescence intensity. Other methods, including enrichment of TALEN-induced mutated cells by co-expressing TALENs and fluorescent proteins and subsequent sorting of TALEN expressing cells based on fluorescence intensity was reported (Ding et al., 2013). However, there are no reports regarding methods for enrichment of cells with chromosome deletions induced by CRISPR/Cas9. "
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    ABSTRACT: Production of nuclear donor cells with a high percentage of desired modifications is a critical step in the successful generation of genetically modified pigs through somatic cell nuclear transfer (SCNT). The CRISPR/Cas9 system has been used for efficient modification of the nuclear DNA in eukaryotic cells, including porcine cells. However, in vitro modified cells are often phenotypically indistinguishable from unmodified cells, hampering their enrichment. Here we investigate a dual fluorescence selection system for the efficient enrichment of porcine embryonic fibroblasts (PEFs) with CRISPR/Cas9-induced chromosomal deletions. Enrichment of cells with 170bp deletions reached a frequency of 74%, whilst enrichment of cells with a larger 5kb deletions achieved a frequency of 46%. This demonstrates the utility of a dual fluorescence reporter as an attractive tool for improving the efficiency of generating genome edited pigs. Copyright © 2015. Published by Elsevier B.V.
    Journal of Biotechnology 07/2015; DOI:10.1016/j.jbiotec.2015.07.011 · 2.87 Impact Factor
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    • "In particular, the transcription activator-like effector nuclease (TALEN) (Hockemeyer et al., 2011) and the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR associated 9 (Cas9) endonuclease systems (Cong et al., 2013; Mali et al., 2013) provide greater flexibility than meganucleases or zinc-finger nucleases (ZFNs) with regard to selecting the target regions of interest (Li et al., 2014). Several studies have demonstrated the effectiveness of TALENs (Hockemeyer et al., 2011; Ding et al., 2013a) and CRISPR (Ding et al., 2013b; Mali et al., 2013) in human induced pluripotent stem cells (iPSCs) for reporter knockin, gene knockout, and gene correction. In fact, corrections of disease mutation by nucleases in iPSCs have been reported for several diseases, including a1-antitrypsin deficiency (Choi et al., 2013), epidermolysis bullosa (Osborn et al., 2013), b-thalassemia (Ma et al., 2013), AIDS (Ye et al., 2014), and Niemann-Pick Type C (Maetzel et al., 2014). "
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    ABSTRACT: Duchenne muscular dystrophy (DMD) is a severe muscle-degenerative disease caused by a mutation in the dystrophin gene. Genetic correction of patient-derived induced pluripotent stem cells (iPSCs) by TALENs or CRISPR-Cas9 holds promise for DMD gene therapy; however, the safety of such nuclease treatment must be determined. Using a unique k-mer database, we systematically identified a unique target region that reduces off-target sites. To restore the dystrophin protein, we performed three correction methods (exon skipping, frameshifting, and exon knockin) in DMD-patient-derived iPSCs, and found that exon knockin was the most effective approach. We further investigated the genomic integrity by karyotyping, copy number variation array, and exome sequencing to identify clones with a minimal mutation load. Finally, we differentiated the corrected iPSCs toward skeletal muscle cells and successfully detected the expression of full-length dystrophin protein. These results provide an important framework for developing iPSC-based gene therapy for genetic disorders using programmable nucleases. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Stem Cell Reports 11/2014; 4(1). DOI:10.1016/j.stemcr.2014.10.013 · 5.37 Impact Factor
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    • "Recent advances show that TALE proteins consist of a modular DNA-binding domain (DBD) that can be rapidly synthesized de novo5. The ability to complement the DBD with a functional domain permits a wide range of applications that include gene activation, repression, and nucleotide deletion and insertion, in a variety of model organisms and cell types678910. "
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    ABSTRACT: The ability to perturb individual genes in genome-wide experiments has been instrumental in unraveling cellular and disease properties. Here we introduce, describe the assembly, and demonstrate the use of comprehensive and versatile transcription activator-like effector (TALE) libraries. As a proof of principle, we built an 11-mer library that covers all possible combinations of the nucleotides that determine the TALE-DNA binding specificity. We demonstrate the versatility of the methodology by constructing a constraint library, customized to bind to a known p53 motif. To verify the functionality in assays, we applied the 11-mer library in yeast-one-hybrid screens to discover TALEs that activate human SCN9A and miR-34b respectively. Additionally, we performed a genome-wide screen using the complete 11-mer library to confirm known genes that confer cycloheximide resistance in yeast. Considering the highly modular nature of TALEs and the versatility and ease of constructing these libraries we envision broad implications for high-throughput genomic assays.
    Scientific Reports 11/2014; 4:4857. DOI:10.1038/srep04857 · 5.58 Impact Factor
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