Autonomous Zinc-Finger Nuclease Pairs for Targeted Chromosomal Deletion The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Şöllü, Cem, Kaweh Pars, Tatjana I. Cornu, Stacey Thibodeau- Beganny, Morgan L. Maeder, J. Keith Joung, Regine Heilbronn, and Toni Cathomen. 2010. Autonomous zinc-finger nuclease pairs for targeted chromosomal deletion. Nucleic Acids Research 38(22): 8269-8276. Published Version doi:10.1093/nar/gkq720 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:4773990 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Published online 16 August 2010 Nucleic Acids Research, 2010, Vol. 38, No. 22 8269–8276 doi:10.1093/nar/gkq720 Autonomous zinc-finger nuclease pairs for targeted chromosomal deletion Cem S¸o¨ llu¨ 1,2, Kaweh Pars1,2, Tatjana I. Cornu2, Stacey Thibodeau-Beganny3, Morgan L. Maeder3,4, J. Keith Joung3,4,5, Regine Heilbronn2 and Toni Cathomen1,2,* 1Department of Experimental Hematology, Hannover Medical School, Hannover, 2Institute of Virology, Campus Benjamin Franklin, Charite´ Medical School, Berlin, Germany, 3Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, 4Biological and Biomedical Sciences Program and 5Department of Pathology, Harvard Medical School, Boston, MA, USA Received May 25, 2010; Revised July 27, 2010; Accepted July 28, 2010 ABSTRACT INTRODUCTION Zinc-finger nucleases (ZFNs) have been successfully Methods to introduce precise and stable modifications in used for rational genome engineering in a variety complex genomes hold great potential not only for the of cell types and organisms. ZFNs consist of a study of gene function but also for biotechnological and non-specific FokI endonuclease domain and a therapeutical applications. A promising approach for rational genome engineering is based on the zinc-finger specific zinc-finger DNA-binding domain. Because nuclease (ZFN) technology (1–3). ZFNs are artificial the catalytic domain must dimerize to become proteins that consist of a non-specific endonuclease active, two ZFN subunits are typically assembled at domain, derived from the FokI restriction enzyme, and the cleavage site. The generation of obligate a specific DNA-binding domain, which consists of heterodimeric ZFNs was shown to significantly tandem repeats of engineered zinc-finger domains, each reduce ZFN-associated cytotoxicity in single-site mediating binding to 3 nt of DNA (4,5). Because the genome editing strategies. To further expand the ap- catalytic FokI domain has to dimerize to become active, plication range of ZFNs, we employed a combination two ZFN subunits are assembled at a cleavage site (6). of in silico protein modeling, in vitro cleavage assays, Typically, a ZFN subunit contains three or four and in vivo recombination assays to identify autono- zinc-finger motifs that together recognize 9 or 12 bp, re- mous ZFN pairs that lack cross-reactivity between spectively. The combined target site is hence defined by a stretch of 18 or 24 bp, a sequence of sufficient length to be each other. In the context of ZFNs designed to statistically unique in a complex genome. recognize two adjacent sites in the human HOXB13 Several reports have highlighted the power of ZFNs for locus, we demonstrate that two autonomous studying biological systems in mammalian cells (7,8) or ZFN pairs can be directed simultaneously to two whole organisms, like the fruit fly (9), nematodes (10), different sites to induce a chromosomal deletion zebrafish (11–13) or rats (14,15). Moreover, the high fre- in 10% of alleles. Notably, the autonomous quency of gene editing at endogenous loci in plant cells ZFN pair induced a targeted chromosomal deletion (16–19), in mammalian cells classically used for drug with the same efficacy as previously published screening or protein production (20–22), or in primary obligate heterodimeric ZFNs but with significantly human cells, including pluripotent stem cells (23–26), less toxicity. These results demonstrate that autono- have demonstrated the wide application range of the mous ZFNs will prove useful in targeted genome ZFN technology in biology, biotechnology or human gene therapy. engineering approaches wherever an applica- Genome engineering by ZFNs is based on the enzyme’s tion requires the expression of two distinct ability to activate the cellular DNA repair machinery by ZFN pairs. creating a DNA double-strand break in the target locus. *To whom correspondence should be addressed. Tel: +49 511 532 5170; Fax: +49 511 532 5121; Email: [email protected] Present address Tatjana I. Cornu, Epiontis GmbH, Berlin, Germany. ß The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 8270 Nucleic Acids Research, 2010, Vol. 38, No. 22 In the presence of an appropriately designed donor DNA, letters indicated binding sites for EB2-N and BA1-N, ZFNs are used to stimulate homologous recombination respectively). The ZFN expression cassettes for the (HR) between the target locus and the donor DNA. HOXB13-specific ZFNs were generated by releasing the In the absence of a donor DNA, cleavage by ZFNs is zinc-finger domains from the OPEN selection plasmids harnessed to disrupt a gene locus through the non- (16) with XbaI/BamHI and then ligating them into homologous end-joining (NHEJ) pathway (2,3). Very the ‘2-in-1’ plasmid in either the first position with recently, the ability to create a targeted deletion in a NheI/BglII or the second position with XbaI/BamHI. chromosome by simultaneous expression of two ZFN The ‘2-in-1’ ZFN cassette is under control of a CMV pairs has been reported (21,27). promoter, and each ZFN contains a triple FLAG tag. Despite these successes, ZFN-induced toxicity by The ZFN subunits are separated by a sequence encoding cleavage at off-target sites has remained an important the T2A autoproteinase (Figure 4b for more details). issue (27–29). ZFN variants that combine high activity Mutations in the FokI domain were introduced using with reduced toxicity have been generated by improving site-directed mutagenesis. The relevant sequences are the DNA-binding specificities of the zinc-finger domains indicated in Figures 1a, 3a and Supplementary Figure S2. (16,30), by customizing the interdomain linkers (31,32), and/or by regulating the DNA-cleavage activity through In vitro cleavage assay re-design of the FokI protein–protein interface (33,34). In ZFNs were expressed in vitro using the TNT SP6 Quick the latter case, the ZFN dimer interface was altered such Coupled Transcription/Translation System according to that only heterodimeric ZFNs could form upon binding to the manufacturer’s instructions (Promega). To verify DNA. Here, we extend this approach and report the iden- equal expression levels, ZFNs were initially synthesized tification and characterization of ZFN variants that 35 in the presence of L-[ S]-methionine, separated by SDS– permit the simultaneous expression of two pairs of PAGE, and detected by autoradiography. The target obligate heterodimers, whose monomeric ZFNs do not DNA fragment was generated by PCR with Pfu polymer- cross-react with each other. This technological advance- ase (Stratagene) using plasmids pCMV.LacZsXXqGFP as ment is relevant for rational genome engineering templates and primers 50-tgcaccgctggataacgacat and approaches that are based on the concomitant expression 50-gaacttcagggtcagcttgc (Eurofins MWG Operon) to of two ZFN pairs, such as targeted chromosomal deletions amplify a 980-bp fragment. A target site for the restriction or editing the genome at two sites in parallel. We demon- enzyme AgeI was located next to the ZFN target site and strate that the combined expression of two autonomous used as a positive control. For in vitro cleavage, 1 mlof ZFN pairs was as effective at inducing a chromosomal each TNT lysate containing one ZFN subunit was mixed deletion as previously described obligate heterodimeric with 200 ng of the DNA template, 1 mg of BSA and ZFNs but that preventing cross-reaction between the in- NEBuffer 4 (50 mM potassium acetate, 20 mM Tris- dividual ZFN subunits reduced toxicity significantly. acetate pH 7.9, 10 mM magnesium acetate, 1 mM dithiothreitol; New England Biolabs) in a total volume MATERIALS AND METHODS of 10 ml. Where indicated, 100 mM NaCl was added to the reaction. After incubation at 37C for 90 min the In silico analysis reaction was analyzed on a 1.2% agarose gel. Protein modeling of the FokI cleavage domain was done with DeepView v4.0 (http://www.expasy.org/spdbv/) Quantitative cell-based recombination and toxicity assays (34,35). Energy calculations of the FokI dimers were per- Human HEK293T cells and the HEK293-based target cell formed using FoldX version 3.0beta (http://foldx.crg line 293EBBA were grown in DMEM (Gibco/Invitrogen) .es/references.jsp). The energy function includes terms supplemented with 10% fetal calf serum (Gibco/ that have been found to be important for protein stability, Invitrogen). The polyclonal target cell line 293EBBA including Van-der-Waals forces, intra- and inter- was generated by lentiviral transduction as previously molecular hydrogen-bond formation, electrostatic contri- described (40). To ensure that cells carry a single copy butions of charged groups, solvation energy and salt con- target, transduction was performed with an MOI <0.1. centration. Details are described elsewhere (34,36,37). The quantitative chromosomal recombination and toxicity assays were performed as previously described Plasmids (40).