DeKelver, RC, Choi, VM, Moehle, EA, Paschon, DE, Hockemeyer, D, Meijsing, SH et al.. Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome. Genome Res 20: 1133-1142

Sangamo BioSciences, Inc., Point Richmond Tech Center, Richmond, California 94804, USA.
Genome Research (Impact Factor: 14.63). 08/2010; 20(8):1133-42. DOI: 10.1101/gr.106773.110
Source: PubMed


Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a "safe harbor" locus known as AAVS1. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient transfection of both transformed and primary human cells, including fibroblasts and hES cells. When added to this locus, transgenes such as expression cassettes for shRNAs, small-molecule-responsive cDNA expression cassettes, and reporter constructs, exhibit consistent expression and sustained function over 50 cell generations. By avoiding random integration and drug selection, this method allows bona fide isogenic settings for high-throughput functional genomics, proteomics, and regulatory DNA analysis in essentially any transformed human cell type and in primary cells.

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    • "To overcome this problem, we generated GFAP::eGFP iPSC reporter lines (one healthy control and two ALS patient lines, SOD1 A4V and C9orf72) using zinc-finger nucleasemediated insertion of an eGFP transgene in the AAVS1 locus of the PPP1R12C gene located on chromosome 19 (Sadelain et al., 2012). This site is considered a safe harbor locus and has been shown to lead to high levels of transgene expression (DeKelver et al., 2010; Hockemeyer et al., 2009; Papapetrou et al., 2011; Smith et al., 2008). GFAP-driven expression of eGFP allows for enrichment of the differentiated eGFP-positive iPSC astrocytes using FACS. "
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    ABSTRACT: Astrocytes are instrumental to major brain functions, including metabolic support, extracellular ion regulation, the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental, psychiatric and neurodegenerative disorders. The generation of adult human fibroblast-derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human-derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes), we generated homogenous populations of iPS astrocytes using zinc-finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno-associated virus integration site 1 (AAVS1) locus in disease and control-derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP, S100β, NFIA and ALDH1L1. In addition, mature pure GFP-iPS astrocytes exhibited a well-described functional astrocytic activity in vitro characterized by neuron-dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP-iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion, the generation of GFAP::GFP-iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte-driven disease pathogenesis and therapy. GLIA 2015. © 2015 Wiley Periodicals, Inc.
    Glia 08/2015; DOI:10.1002/glia.22903
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    • "Then two donor template plasmids are coelectroporated with the AAVS1-TALEN constructs. The Puro-Cas9 donor plasmid contains a doxycycline-inducible Cas9 expression cassette selectable with puromycin, and the Neo-M2rtTA donor carries a constitutive reverse tetracycline transactivator (M2rtTA) expression cassette selectable with G418 (Geneticin) (DeKelver et al., 2010) (Fig. 11.2C). HDR of the DSBs allows simultaneous introduction of both Puro-Cas9 and Neo- M2rtTA cassettes into both AAVS1 alleles in trans. "
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    ABSTRACT: Human pluripotent stem cells (hPSCs) have the potential to generate all adult cell types, including rare or inaccessible human cell populations, thus providing a unique platform for disease studies. To realize this promise, it is essential to develop methods for efficient genetic manipulations in hPSCs. Established using TALEN (transcription activator-like effector nuclease) and CRISPRs (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) systems, the iCRISPR platform supports a variety of genome-engineering approaches with high efficiencies. Here, we first describe the establishment of the iCRISPR platform through TALEN-mediated targeting of inducible Cas9 expression cassettes into the AAVS1 locus. Next, we provide a series of technical procedures for using iCRISPR to achieve one-step knockout of one or multiple gene(s), “scarless” introduction of precise nucleotide alterations, as well as inducible knockout during hPSC differentiation. We present an optimized workflow, as well as guidelines for the selection of CRISPR targeting sequences and the design of single- stranded DNA (ssDNA) homology-directed DNA repair templates for the introduction of specific nucleotide alterations. We have successfully used these protocols in four dif- ferent hPSC lines, including human embryonic stem cells and induced pluripotent stem cells. Once the iCRISPR platform is established, clonal lines with desired genetic mod- ifications can be established in as little as 1 month. The methods described here enable a wide range of genome-engineering applications in hPSCs, thus providing a valuable resource for the creation of diverse hPSC-based disease models with superior speed and ease.
    The Use of CRISPR/cas9, ZFNs, TALENs in Generating Site Specific Genome Alterations, 1st edition edited by Doudna & Sontheimer, 11/2014: chapter The iCRISPR Platform for Rapid Genome Editing in Human Pluripotent Stem Cells; Methods in Enzymology., ISBN: 9780128011850
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    • "Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) (Takahashi et al., 2007), collectively referred to as human pluripotent stem cells (hPSCs), are currently used in disease modeling to address questions specific to humans and to complement insights gained from other model organisms (Soldner and Jaenisch, 2012; Soldner et al., 2011). Genetic engineering using site-specific nucleases was recently established in hPSCs (Dekelver et al., 2010; Hockemeyer et al., 2009, 2011; Yusa et al., 2011; Zou et al., 2009), allowing a level of genetic control that was previously limited to model systems. We can now target gene knockouts, generate tissue-specific cell lineage reporters, overexpress genes from a defined locus, and introduce or repair single-point mutations in hPSCs. "
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    ABSTRACT: Genetically engineered human pluripotent stem cells (hPSCs) have been proposed as a source for transplantation therapies and are rapidly becoming valuable tools for human disease modeling. However, many applications are limited due to the lack of robust differentiation paradigms that allow for the isolation of defined functional tissues. Here, using an endogenous LGR5-GFP reporter, we derived adult stem cells from hPSCs that gave rise to functional human intestinal tissue comprising all major cell types of the intestine. Histological and functional analyses revealed that such human organoid cultures could be derived with high purity and with a composition and morphology similar to those of cultures obtained from human biopsies. Importantly, hPSC-derived organoids responded to the canonical signaling pathways that control self-renewal and differentiation in the adult human intestinal stem cell compartment. This adult stem cell system provides a platform for studying human intestinal disease in vitro using genetically engineered hPSCs.
    Stem Cell Reports 06/2014; 2(6):838-52. DOI:10.1016/j.stemcr.2014.05.001
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