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: 13.85). 08/2010; 20(8):1133-42. DOI: 10.1101/gr.106773.110
Source: PubMed

ABSTRACT 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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    • "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 · 5.64 Impact Factor
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    • "Additionally, no detectable transcriptional perturbations of the PPP1R12C and its flanking genes were observed after integration of transgenes in this locus, indicating that AAVS1 may represent a safe landing path for therapeutic transgene insertion in the human genome (Lombardo et al, 2011). These observations, together with the development of artificial zinc finger nucleases (ZFNs) that efficiently and selectively target the AAVS1 locus, have facilitated gene editing strategies aiming at inserting therapeutic transgenes in this locus, not only in immortalized cell lines but also in several primary human cell types, including induced pluripotent stem cells (hiPSCs; Hockemeyer et al, 2009; DeKelver et al, 2010; Lombardo et al, 2011; Zou et al, 2011b; Chang & Bouhassira, 2012). Because a defective FA pathway not only predisposes FA patients to cancer (Rosenberg et al, 2008) but also to the early development of bone marrow failure due to the progressive extinction of the HSCs (Larghero et al, 2002; Jacome et al, 2006), our final aim in these studies was the generation of gene-edited, disease-free FA-HSCs, obtained from non-hematopoietic tissues of the patient. "
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    ABSTRACT: Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies.
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