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Gene Knockout and Knockin by Zinc-Finger Nucleases: Current Status and Perspectives

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Gene Knockout and Knockin by Zinc-Finger Nucleases: Current Status and Perspectives Gene knockout and knockin by zinc-finger nucleases: current status and perspectives J. Hauschild-Quintern, B. Petersen, G. J. Cost & H. Niemann Cellular and Molecular Life Sciences ISSN 1420-682X Cell. Mol. Life Sci. DOI 10.1007/s00018-012-1204-1 1 23 Your article is protected by copyright and all rights are held exclusively by Springer Basel. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your work, please use the accepted author’s version for posting to your own website or your institution’s repository. You may further deposit the accepted author’s version on a funder’s repository at a funder’s request, provided it is not made publicly available until 12 months after publication. 1 23 Author's personal copy Cell. Mol. Life Sci. DOI 10.1007/s00018-012-1204-1 Cellular and Molecular Life Sciences REVIEW Gene knockout and knockin by zinc-finger nucleases: current status and perspectives J. Hauschild-Quintern • B. Petersen • G. J. Cost • H. Niemann Received: 29 June 2012 / Revised: 19 October 2012 / Accepted: 22 October 2012 Ó Springer Basel 2012 Abstract Zinc-finger nucleases (ZFNs) are engineered CHO Chinese hamster ovary site-specific DNA cleavage enzymes that may be designed Dat Dopamine transporter to recognize long target sites and thus cut DNA with high DHFR Dihydrofolate reductase specificity. ZFNs mediate permanent and targeted genetic DSB Double-strand break alteration via induction of a double-strand break at a spe- ES cells Embryonic stem cells cific genomic site. Compared to conventional homology- FACS Fluorescence-activated cell sorting based gene targeting, ZFNs can increase the targeting rate FUT8 a-1,6-fucosyltransferase by up to 100,000-fold; gene disruption via mutagenic DNA GFP Green fluorescence protein repair is similarly efficient. The utility of ZFNs has been GGTA1 a1,3-galactosyltransferase (Gal) shown in many organisms, including insects, amphibians, GPI-AP Glycosyl-phosphatidyl-inositol anchored proteins plants, nematodes, and several mammals, including GS Glutamine synthetase humans. This broad range of tractable species renders HDR Homology-direct repair ZFNs a useful tool for improving the understanding of hFIX Human factor IX complex physiological systems, to produce transgenic hif1 a Hypoxia-inducible factor 1a animals, cell lines, and plants, and to treat human disease. HR Homologous recombination HSPCs Hematopoietic stem progenitor cells Keywords Zinc-finger nucleases Á IDLV Integrase-defective lentivirus Homology-directed repair Á Transgenic animals Á IgM Immunoglobulin M Gene knockout Á Targeting efficiency IL2Rc Interleukin-2 receptor common c-chain iPS cells Induced pluripotent stem cells Abbreviations Jag1 Jagged 1 BKG Beta-lactoglobulin KO Knockout CCR5 Human chemokine receptors 5 gene KI Knockin Mdr1a Multidrug-resistant 1a NHEJ Non-homologous end joining J. Hauschild-Quintern Á B. Petersen Á H. Niemann (&) Notch3 Notch homolog 3 Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, OPEN Oligomerized pool engineering Mariensee, Hoeltystrasse 10, 31535 Neustadt a. Rbge., Germany ORF Open reading frame e-mail: heiner.niemann@fli.bund.de OTS Off-target site H. Niemann Ppar-c Peroxisome proliferator-activated receptor-c Rebirth, Cluster of Excellence, Hannover Medical School, SCID Severe combined immune deficiency Hannover, Germany SCNT Somatic cell nuclear transfer tfr2 Telomerase, transferrin receptor 2 G. J. Cost Sangamo BioSciences, 501 Canal Blvd., WT Wild type Richmond, CA 94804, USA ZF Zinc-finger 123 Author's personal copy J. Hauschild-Quintern et al. ZFN Zinc-finger nucleases almost any triplet [3]. Several ZFs can be combined to ZFP Zinc-finger proteins form a larger DNA-recognition domain. The invention of zinc-finger nucleases (ZFNs) Introduction While the zinc-finger motif was discovered in the 1980s [2], ZFNs have a shorter history. Prior to the invention of Genetic modification often starts with the creation of a ZFNs, naturally occurring restriction enzymes were the double-strand break (DSB) in DNA. The efficiency of main class of site-specific nucleases. These endonucleases targeted genetic modification can be significantly enhanced cut many times in even the smallest genome due to their by creation of a site-specific DSB [1]. An effective tool for short recognition sites (8 bp or less). Meganucleases such the induction of specific DNA cleavage is the ZFN, as the as the naturally occurring I-Sce-I recognize very long target variable DNA binding domain of a ZFN can be designed to sequences (as much as 18 bp), but have proven difficult to bind to an investigator-specified DNA sequence. By engineer to recognize new DNA targets. A ZFN consists of selecting for different outcomes of DNA repair, either gene a site-specific zinc-finger DNA binding domain fused to the knockout (KO) or targeted transgene insertion (KI) can be nonspecific cleavage domain of the FokI endonuclease first obtained. In this review, we describe zinc-finger nucleases described in 1996 by the group of Dr. Chandrasegaran [5]. and their application to genetic modification in a variety of Since the FokI nuclease must dimerize to cut the DNA, two organisms and cell types. We begin with the discovery of ZFN molecules are usually required, doubling the number the zinc-finger domain and the invention of the ZFN, and of specifically recognized bp [6]. Moreover, for productive then proceed to describe how to modify a genome with dimerization and cleavage, the two ZFN molecules bind to ZFNs. Further, we discuss different methods to generate the targeted DNA on opposite strands in a tail-to-tail ori- ZFNs. Finally, we survey the growing literature describing entation separated by 5–7 bp, with double-stranded DNA the utility of ZFNs for targeted genome modification. cleavage occurring in this spacer region (Fig. 2). As described below, ZFNs are regularly used as tools to Structural aspects of zinc-fingers (ZFs) introduce double-strand breaks (DSBs) at specific sites of the genome to induce mutations via non homologous end The first zinc-finger (ZF) protein with a specific binding joining (NHEJ) or to insert exogenous DNA via homology- affinity to DNA was discovered as part of transcription directed repair (HDR). Initial studies on the ZFN function factor IIIa in Xenopus oocytes [2]. A typical zinc-finger in multicellular organisms were performed in Xenopus (Cys2 His2) consists of *30 amino acids that form two oocytes in 2001 [7]. A plasmid containing the ZFN target anti-parallel b sheets opposing an a-helix (Fig. 1)[3]. The sequence was co-injected with ZFN mRNA resulting in an domain is stabilized by two cysteine and two histidine extraordinary high homologous recombination (HR) effi- residues binding a zinc ion, thus forming a compact ciency of 46 %. These first preliminary experiments globular domain. The zinc-finger motif uses residues in the showed that ZFNs could work in a eukaryotic cell when the a-helix to bind to approximately 3 specific bp in the major target sequence was supplied on a plasmid. Successful groove of the DNA [4]. ZFs can be designed for binding to endogenous gene targeting mediated by ZFNs was A B Fig. 1 a The zinc-finger molecule consists of one a-helix and two b molecules fused to a nuclease form a ZFN. Two ZFNs have to bind sheets. The zinc ion (black ball) is bound by two cysteines of the b the targeted region in tail to tail direction to allow dimerization and sheets and two histidines of the a-helix leading to a stabilization of cleavage of the FokI nuclease domain the fold. Residues of the a-helix contact 3 bp of the DNA. b Four ZF 123 Author's personal copy Gene knockout and knockin by zinc-finger nucleases Fig. 2 After two ZFN molecules, each consisting of five zinc fingers, gene knockout by frame shift. If a donor DNA (grey) is added with bind specifically to their target sequences, the nuclease domains homologous arms (white letters on grey background) to the targeted dimerize and cut the DNA. Double-strand break repair by non region, the sequence information between the homology arms of the homologous end joining (NHEJ) can induce mutations leading to a donor DNA is copied into the genome and a gene knockin occurs accomplished in Drosophila melanogaster (the drosophilas of the targeted gene. The Cel-I assay (Fig. 4, Surveyor yellow (y) gene) and was reported in 2002 and led to a nuclease assay) is a suitable in vitro system to evaluate functional gene knockout by small deletions and/or inser- ZFN-mediated DNA modification by NHEJ [11]. The tions at the targeted site [8]. The following years saw a enzyme used in this assay recognizes and cuts base pair burst of research activity in this field. mismatches (loops) formed when wild-type DNA and mutated DNA are hybridized. From the extent of digestion Modification of a genome using ZFNs (ratio of cleaved to uncleaved product) one can calculate the NHEJ frequency in a given cell population. Since the To employ a ZFN for genetic engineering, the plasmid frequency of ZFN-mediated gene modification is generally DNA or mRNA encoding a specific ZFN is introduced into [1 % even in the absence of selection for the desired cells or embryos via microinjection or transfection (Fig. 3). events, isolation of knockout cells is readily achieved by After translation, the ZFN pair binds to its specific target interrogation of cell clones generated by limiting dilution facilitating the DNA-binding dependent dimerization of the [12–15]. For non-immortalized and other poorly clonable FokI catalytic domains, and resulting in DNA cleavage. cell lines, fluorescence activated cell sorting (FACS) or ZFN activity can be enhanced by incubating transfected magnetic bead selection have been successfully employed cells at 30 °C for a few days as proven in human cells for to enrich the targeted cells [16–18].
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