DOCSLIB.ORG

Improved Cell-Penetrating Zinc-Finger Nuclease Proteins for Precision Genome Engineering

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Improved Cell-Penetrating Zinc-Finger Nuclease Proteins for Precision Genome Engineering Citation: Molecular Therapy—Nucleic Acids (2015) 4, e232; doi:10.1038/mtna.2015.6 © 2015 The American Society of Gene & Cell Therapy All rights reserved 2162-2531/15 www.nature.com/mtna Improved Cell-Penetrating Zinc-Finger Nuclease Proteins for Precision Genome Engineering Jia Liu1,2,3,4, Thomas Gaj1,2,3, Mark C Wallen1,2,3 and Carlos F Barbas III1,2,3,5 Safe, efficient, and broadly applicable methods for delivering site-specific nucleases into cells are needed in order for targeted genome editing to reach its full potential for basic research and medicine. We previously reported that zinc-finger nuclease (ZFN) proteins have the innate capacity to cross cell membranes and induce genome modification via their direct application to human cells. Here, we show that incorporation of tandem nuclear localization signal (NLS) repeats into the ZFN protein backbone enhances cell permeability nearly 13-fold and that single administration of multi-NLS ZFN proteins leads to genome modification rates of up to 26% in CD4+ T cells and 17% in CD34+ hematopoietic stem/progenitor cells. In addition, we show that multi-NLS ZFN proteins attenuate off-target effects and that codelivery of ZFN protein pairs facilitates dual gene modification frequencies of 20–30% in CD4+ T cells. These results illustrate the applicability of ZFN protein delivery for precision genome engineering. Molecular Therapy—Nucleic Acids (2015) 4, e232; doi:10.1038/mtna.2015.6; advance online publication 10 March 2015 Subject Category: Gene insertion, deletion & modification Gene vectors The emergence of site-specific DNA endonucleases and expression from DNA could lead to increased off-target genome editing has empowered researchers with the activity,26 a detrimental side effect that might have serious unprecedented ability to manipulate virtually any gene consequences for therapeutic genome editing. Indeed, in across a broad spectrum of cell types and organisms.1–3 order for nuclease-mediated genome engineering to reach The technologies most commonly used to achieve this— its full potential, new methods that improve their specificity homing endonucleases,4,5 zinc-finger nucleases (ZFNs),6–8 and safety are needed. TAL effector nucleases (TALENs),9–11 and CRISPR/Cas912– The direct delivery of nuclease proteins into mammalian 14—have the potential to revolutionize basic biological cells has been shown to be a viable alternative to DNA or research and transform medicine, as evidenced by recent mRNA-based delivery systems. Unlike methods that rely on clinical trials for HIV/AIDS based on ZFN-mediated modifi- expression from nucleic acids, protein delivery poses no risk cation of the HIV-1 coreceptor chemokine (C-C motif) recep- for chromosomal insertions and only exposes the cell to the tor 5 (CCR5).15 These systems are generally configured nuclease for a short period of time, thereby reducing off-tar- to induce targeted DNA double-strand breaks (DSBs) that get effects.27 To date, nuclease proteins have been admin- stimulate the cellular DNA repair machinery, typically lead- istered directly into cells via cell-penetrating peptides,28–30 ing to one of two outcomes: gene knockout16 via mutagenic ligand-mediated endocytosis,31 electroporation,32,33 or trans- nonhomologous end joining (NHEJ) or, in the presence of an fection34 and retro-35 or lentivirus particles.36–38 In particular, accompanying donor template, targeted gene integration17 we previously showed that ZFN proteins are inherently cell- or correction8,18 by homology-directed repair (HDR). While permeable and capable of inducing genome modifications recent technological advances have helped make genome via their direct application to human cells.27 ZFNs are chi- editing a routine endeavor for nonspecialized laboratories, meric endonucleases consisting of the cleavage domain of establishing safe and efficient delivery methods for sensi- the FokI restriction endonuclease and a custom-designed 39,40 tive applications remains challenging. Current methods for Cys2-His2 zinc-finger DNA-binding protein, the latter of achieving this are based on transfection or electroporation19 which possesses the innate ability to cross cell membranes of nuclease-encoded DNA or mRNA or the use of viral vec- likely due to its high overall positive charge. Due to its sim- tor delivery systems.20 These strategies, however, are lim- plicity and ability to improve the specificity of genome editing, ited by a number of factors: viral vectors are time-consuming ZFN protein delivery represents a promising approach for to produce, capable of chromosomal integration, hampered modifying cells for ex vivo genome engineering applications. by repetitive elements (e.g., TAL effector repeats)21–23 and, in However, for most cell-types, consecutive protein treatments the case of adenoassociated virus (AAV), size-constrained. are necessary to achieve high levels of genomic modifica- Nonviral delivery systems, on the other hand, are restricted tion, a drawback that limits the scope and scalability of this to certain cell types and have been reported to show toxicity methodology. from electroporation24 or transfection.25 In addition, regard- Here, we explore the use of nuclear localization signals less of the delivery strategy used, high levels of nuclease (NLS)—highly positively charged peptide domains that have 1The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA; 2Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA; 3Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA; 4Shanghai Institute for Advanced Immunochemi- cal Studies (SIAIS), ShanghaiTech University, Shanghai, China; 5Deceased. Correspondence: The first two authors contributed equally to this work. Jia Liu, Shanghai Institute for Advanced Immunochemical Studies (SIAIS), ShanghaiTech University, Shanghai, China. E-mail: [email protected] or Thomas Gaj, Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, California, USA. E-mail: [email protected] Keywords: genome editing; protein delivery; zinc-finger nuclease Received 26 January 2015; accepted 27 January 2015; advance online publication 10 March 2015. doi:10.1038/mtna.2015.6 Improved Cell-Penetrating ZFN Proteins for Genome Editing Liu et al 2 the innate ability to cross cell membranes—as a means to evident for either ZFN protein in cell culture (Supplemen- enhance ZFN protein cell permeability. We demonstrate that tary Figure S2). incorporation of tandem NLS repeats into the ZFN protein ZFNs typically contain a single N-terminal Simian vacuo- backbone enhances ZFN cell-penetrating activity and leads lating virus 40 (SV40) NLS sequence (PKKKRKV) that medi- to highly efficient genome modification in a diverse range of ates nuclear import but does not measurably contribute to cell types, including primary CD4+ T cells, CD34+ hematopoi- its intrinsic cell-penetrating activity.27 Because in some con- etic stem/progenitor cells (HSPCs) and induced pluripotent texts NLS sequences possess an innate ability to cross cell stem cells (iPSCs). In addition, we show that multi-NLS ZFN membranes45 and mediate protein transfection,46 we hypoth- proteins retain the ability to mitigate off-target effects and esized that tandem NLS repeats could enhance ZFN protein mediate high levels of dual gene modification in CD4+ T cells, cell-permeability. To test this, we fused one, two, three, or illustrating the potential of ZFN protein delivery for ex vivo four additional repeats of the SV40 NLS to the N-terminus genome engineering processes. of ZFN proteins that already contained one NLS and were designed to target the human CCR5 gene (Figure 1a).47 We generated ZFN proteins in high yield (>2 mg/l) and >80% Results purity from the soluble fraction of Escherichia coli lysates but observed varying levels of proteolysis of three-, four- and Improving ZFN protein delivery via tandem NLS repeats five-NLS ZFN proteins (Supplementary Figure S3). Com- As a means to enhance the innate cell-penetrating activity pared to native one-NLS ZFN protein, only four- and five- of ZFN proteins, we explored the possibility of genetically NLS proteins showed a decrease in cleavage activity in vitro fusing protein transduction domains (PTDs) to the N-ter- (Supplementary Figure S3). In particular, low-levels of non- minus of ZFNs. We27 and others29 previously reported that specific cleavage were evident for the five-NLS ZFN proteins incorporation of the cell-penetrating peptide sequence from (Supplementary Figure S3), likely due to nonspecific asso- the HIV-1 TAT protein41 or the poly-Arg peptide42 impairs ciation between the highly positively charged N-terminus of ZFN protein expression. We thus expanded the scope of the ZFN protein and the DNA backbone. this approach by separately incorporating two additional We evaluated the ability of these multi-NLS ZFN proteins PTDs, penetratin43 and transportan,44 into the ZFN protein to enter cells and stimulate mutagenesis using a previously backbone. While both fusion proteins could be expressed in described human embryonic kidney (HEK) 293 reporter yields sufficient for downstream analysis (Supplementary cell line (Figure 1b).27,48 This system features an integrated Figure S1), reduced activity was observed for both proteins EGFP gene whose expression has been disabled by the in vitro and no improvement in genomic modification was presence of a frame-shift mutation introduced by a ZFN acPKKKRKV 12 0.5 μmol/l One-NLS Zinc-finger Fokl 10 1.0 μmol/l 2.0 μmol/l Two-NLS Zinc-finger Fokl 8 4.0 μmol/l 6 Three-NLS Zinc-finger Fokl 4 Four-NLS Zinc-finger Fokl 2 Five-NLS Zinc-finger cells (%) EGFP-positive Fokl 0 One Two Three Four Five NLS repeats bde 10 1X 100 * * 8 * * * 2X 80 * CMV EGFP CCR5-R CCR5-R EGFP * * 3X 6 * * 60 Insertion (+2, 5, 8... bps) or NHEJ-mediated * deletion (-1, 4, 7...bps) mutagenesis 4 40 * CMV EGFP 2 20 EGFP-positive cells (%) EGFP-positive 0 cells (%) FITC-positive 0 o One Two Three Four Five One Tw Four Five Mock Three NLS repeats NLS repeats Figure 1 Tandem NLS repeats enhance ZFN protein activity.
Load more

Recommended publications
  • Improving Treatment of Genetic Diseases with Crispr-Cas9 Rna-Guided Genome Editing
    Sanchez 3:00 Team R06 Disclaimer: This paper partially fulfills a writing requirement for first-year (freshmen) engineering students at the University of Pittsburgh Swanson School of Engineering. This paper is a student paper, not a professional paper. This paper is not intended for publication or public circulation. This paper is based on publicly available information, and while this paper might contain the names of actual companies, products, and people, it cannot and does not contain all relevant information/data or analyses related to companies, products, and people named. All conclusions drawn by the authors are the opinions of the authors, first- year (freshmen) students completing this paper to fulfill a university writing requirement. If this paper or the information therein is used for any purpose other than the authors' partial fulfillment of a writing requirement for first-year (freshmen) engineering students at the University of Pittsburgh Swanson School of Engineering, the users are doing so at their own--not at the students', at the Swanson School's, or at the University of Pittsburgh's--risk. IMPROVING TREATMENT OF GENETIC DISEASES WITH CRISPR-CAS9 RNA-GUIDED GENOME EDITING Arijit Dutta [email protected] , Benjamin Ahlmark [email protected], Nate Majer [email protected] Abstract—Genetic illnesses are among the most difficult to treat as it is challenging to safely and effectively alter DNA. INTRODUCTION: THE WHAT, WHY, AND DNA is the basic code for all hereditary traits, so any HOW OF CRISPR-CAS9 alteration to DNA risks fundamentally altering the way someone’s genes are expressed. This change could lead to What Is CRISPR-Cas9? unintended consequences for both the individual whose DNA was altered and any offspring they may have in the future, CRISPR-Cas9 is an acronym that stands for “Clustered compounding the risk.
    [Show full text]
  • Strategies for Precision Modulation of Gene Expression by Epigenome Editing: an Overview Benjamin I
    Laufer and Singh Epigenetics & Chromatin (2015) 8:34 DOI 10.1186/s13072-015-0023-7 REVIEW Open Access Strategies for precision modulation of gene expression by epigenome editing: an overview Benjamin I. Laufer* and Shiva M. Singh Abstract Genome editing technology has evolved rather quickly and become accessible to most researchers. It has resulted in far reaching implications and a number of novel designer systems including epigenome editing. Epigenome editing utilizes a combination of nuclease-null genome editing systems and effector domains to modulate gene expression. In particular, Zinc Finger, Transcription-Activator-Like Effector, and CRISPR/Cas9 have emerged as modular systems that can be modified to allow for precision manipulation of epigenetic marks without altering underlying DNA sequence. This review contains a comprehensive catalog of effector domains that can be used with components of genome editing systems to achieve epigenome editing. Ultimately, the evidence-based design of epigenome editing offers a novel improvement to the limited attenuation strategies. There is much potential for editing and/or correcting gene expression in somatic cells toward a new era of functional genomics and personalized medicine. Keywords: Regulation of gene expression, Functional genomics, Stem cells, dCas9, CRISPR/Cas9, Zinc Finger, Transcription-Activator-Like Effector (TALE), Synthetic biology Background editing systems rely on two components, a DNA-bind- The modulation of gene expression can be achieved by ing element, and nuclease, to modify the targeted DNA a variety of biotechnologies such as RNA interference, sequence. Genome editing can be used to study protein non-precision drugs, and artificial transcription factors function by altering coding sequence or achieve tran- (ATFs).
    [Show full text]
  • CRISPR Foki Dead Cas9 System: Principles and Applications in Genome Engineering
    cells Review CRISPR FokI Dead Cas9 System: Principles and Applications in Genome Engineering Maryam Saifaldeen y, Dana E. Al-Ansari y , Dindial Ramotar * and Mustapha Aouida * College of Health and Life Sciences, Division of Biological and Biomedical Sciences, Hamad Bin Khalifa University, Education City, Qatar Foundation, Doha P.O.Box 34110, Qatar; [email protected] (M.S.); [email protected] (D.E.A.-A.) * Correspondence: [email protected] (D.R.); [email protected] (M.A.) These authors contributed equally to this work. y Received: 27 September 2020; Accepted: 19 November 2020; Published: 21 November 2020 Abstract: The identification of the robust clustered regularly interspersed short palindromic repeats (CRISPR) associated endonuclease (Cas9) system gene-editing tool has opened up a wide range of potential therapeutic applications that were restricted by more complex tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Nevertheless, the high frequency of CRISPR system off-target activity still limits its applications, and, thus, advanced strategies for highly specific CRISPR/Cas9-mediated genome editing are continuously under development including CRISPR–FokI dead Cas9 (fdCas9). fdCas9 system is derived from linking a FokI endonuclease catalytic domain to an inactive Cas9 protein and requires a pair of guide sgRNAs that bind to the sense and antisense strands of the DNA in a protospacer adjacent motif (PAM)-out orientation, with a defined spacer sequence range around the target site. The dimerization of FokI domains generates DNA double-strand breaks, which activates the DNA repair machinery and results in genomic edit. So far, all the engineered fdCas9 variants have shown promising gene-editing activities in human cells when compared to other platforms.
    [Show full text]
  • Zinc Finger Nucleases (Zfns) and Transcription Activator-Like Effector Nucleases (Talens) Deepak Reyon Iowa State University
    Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2011 Genome engineering using DNA-binding proteins: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) Deepak Reyon Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Bioinformatics Commons, Computational Biology Commons, and the Genomics Commons Recommended Citation Reyon, Deepak, "Genome engineering using DNA-binding proteins: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)" (2011). Graduate Theses and Dissertations. 14745. https://lib.dr.iastate.edu/etd/14745 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Genome engineering using DNA-binding proteins: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) by Deepak Reyon A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Bioinformatics and Computational Biology Program of Study Committee: Drena Dobbs, Co-Major Professor Edward Yu, Co-Major Professor Vasant Honavar Amy Andreotti Gaya Amarasinghe Iowa State University Ames,
    [Show full text]
  • An Update on the Tools for Creating Transgenic Animal Models of Human Diseases – Focus on Atherosclerosis
    Brazilian Journal of Medical and Biological Research (2019) 52(5): e8108, http://dx.doi.org/10.1590/1414-431X20198108 ISSN 1414-431X Review 1/7 An update on the tools for creating transgenic animal models of human diseases – focus on atherosclerosis A.S. Volobueva2, A.N. Orekhov 1,3,4, and A.V. Deykin 1 1Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia 2Laboratory of Gene Therapy, Biocad Biotechnology Company, Strelnya, Russia 3Laboratory of Angiopathology, Institute of General Pathology and Pathophysiology, Moscow, Russia 4Institute for Atherosclerosis Research, Skolkovo Innovative Center, Moscow, Russia Abstract Animal models of diseases are invaluable tools of modern medicine. More than forty years have passed since the first successful experiments and the spectrum of available models, as well as the list of methods for creating them, have expanded dramatically. The major step forward in creating specific disease models was the development of gene editing techniques, which allowed for targeted modification of the animal’s genome. In this review, we discuss the available tools for creating transgenic animal models, such as transgenesis methods, recombinases, and nucleases, including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR/Cas9 systems. We then focus specifically on the models of atherosclerosis, especially mouse models that greatly contributed to improving our understanding of the disease pathogenesis and we outline their characteristics and limitations. Key words: Animal models; Gene editing; Atherosclerosis Introduction Model organisms are widely used in biomedicine reliability of resulting models. Moreover, uncontrolled gene for studying the pathophysiology of human diseases and expression could affect multiple pathways in the animal developing novel therapeutic and diagnostic methods.
    [Show full text]
  • A Revolution Toward Gene-Editing Technology and Its Application to Crop Improvement
    International Journal of Molecular Sciences Review A Revolution toward Gene-Editing Technology and Its Application to Crop Improvement Sunny Ahmar 1, Sumbul Saeed 1, Muhammad Hafeez Ullah Khan 1 , Shahid Ullah Khan 1 , Freddy Mora-Poblete 2 , Muhammad Kamran 3 , Aroosha Faheem 4, Ambreen Maqsood 5 , Muhammad Rauf 6 , Saba Saleem 7, Woo-Jong Hong 8 and Ki-Hong Jung 8,* 1 College of Plant Sciences and Technology Huazhong Agricultural University, Wuhan 430070, China; [email protected] (S.A.); [email protected] (S.S.); [email protected] (M.H.U.K.); [email protected] (S.U.K.) 2 Institute of Biological Sciences, University of Talca, 2 Norte 685, Talca 3460000, Chile; [email protected] 3 Key Laboratory of Arable Land Conservation (Middle and Lower Reaches of Yangtze River), Ministry of Agriculture, College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, China; [email protected] 4 Sate Key Laboratory of Agricultural Microbiology and State Key Laboratory of Microbial Biosensor, College of Life Sciences Huazhong Agriculture University Wuhan, Wuhan 430070, China; [email protected] 5 Department of Plant Pathology, University College of Agriculture and Environmental Sciences, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan; [email protected] 6 National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad 38000, Pakistan; [email protected] 7 Department of Bioscience, COMSATS Institute of Information Technology, Islamabad 45550, Pakistan; [email protected] 8 Graduate School of Biotechnology & Crop Biotech Institute, Kyung Hee University, Yongin 17104, Korea; [email protected] * Correspondence: [email protected] Received: 14 July 2020; Accepted: 5 August 2020; Published: 7 August 2020 Abstract: Genome editing is a relevant, versatile, and preferred tool for crop improvement, as well as for functional genomics.
    [Show full text]
  • Designer Nucleases to Treat Malignant Cancers Driven by Viral Oncogenes Tristan A
    Scott and Morris Virol J (2021) 18:18 https://doi.org/10.1186/s12985-021-01488-1 REVIEW Open Access Designer nucleases to treat malignant cancers driven by viral oncogenes Tristan A. Scott* and Kevin V. Morris Abstract Viral oncogenic transformation of healthy cells into a malignant state is a well-established phenomenon but took decades from the discovery of tumor-associated viruses to their accepted and established roles in oncogenesis. Viruses cause ~ 15% of know cancers and represents a signifcant global health burden. Beyond simply causing cel- lular transformation into a malignant form, a number of these cancers are augmented by a subset of viral factors that signifcantly enhance the tumor phenotype and, in some cases, are locked in a state of oncogenic addiction, and sub- stantial research has elucidated the mechanisms in these cancers providing a rationale for targeted inactivation of the viral components as a treatment strategy. In many of these virus-associated cancers, the prognosis remains extremely poor, and novel drug approaches are urgently needed. Unlike non-specifc small-molecule drug screens or the broad- acting toxic efects of chemo- and radiation therapy, the age of designer nucleases permits a rational approach to inactivating disease-causing targets, allowing for permanent inactivation of viral elements to inhibit tumorigenesis with growing evidence to support their efcacy in this role. Although many challenges remain for the clinical applica- tion of designer nucleases towards viral oncogenes; the uniqueness and clear molecular mechanism of these targets, combined with the distinct advantages of specifc and permanent inactivation by nucleases, argues for their develop- ment as next-generation treatments for this aggressive group of cancers.
    [Show full text]
  • Therapeutic Genome Editing: Prospects and Challenges
    Therapeutic genome editing: prospects and challenges The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Cox, David Benjamin Turitz, Randall Jeffrey Platt, and Feng Zhang. "Therapeutic genome editing: prospects and challenges." Nature Medicine 21:2 (February 2015), pp.121-131. As Published http://dx.doi.org/10.1038/nm.3793 Publisher Springer Nature Version Author's final manuscript Citable link http://hdl.handle.net/1721.1/103783 Terms of Use Creative Commons Attribution-Noncommercial-Share Alike Detailed Terms http://creativecommons.org/licenses/by-nc-sa/4.0/ HHS Public Access Author manuscript Author Manuscript Author ManuscriptNat Med Author Manuscript. Author manuscript; Author Manuscript available in PMC 2015 July 06. Published in final edited form as: Nat Med. 2015 February ; 21(2): 121–131. doi:10.1038/nm.3793. Therapeutic Genome Editing: Prospects and Challenges David Benjamin Turitz Cox, Randall Jeffrey Platt, and Feng Zhang† Broad Institute of MIT and Harvard 7 Cambridge Center Cambridge, MA 02142, USA; McGovern Institute for Brain Research Department of Brain and Cognitive Sciences Department of Biological Engineering Massachusetts Institute of Technology Cambridge, MA 02139, USA Abstract Recent advances in the development of genome editing technologies based on programmable nucleases have significantly improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies.
    [Show full text]
  • Genome Editing with Zinc Finger Nuclease and Tale-Deaminase Fusions
    ARTICLE Received 13 May 2016 | Accepted 23 Sep 2016 | Published 2 Nov 2016 | Updated 9 Oct 2017 DOI: 10.1038/ncomms13330 OPEN Engineering and optimising deaminase fusions for genome editing Luhan Yang1,2,3,w, Adrian W. Briggs1,*, Wei Leong Chew1,2,*,w, Prashant Mali1, Marc Guell1,3,w, John Aach1, Daniel Bryan Goodman1,4, David Cox4, Yinan Kan1,3,w, Emal Lesha1,3,w, Venkataramanan Soundararajan1, Feng Zhang5,6,7 & George Church1,4,8 Precise editing is essential for biomedical research and gene therapy. Yet, homology-directed genome modification is limited by the requirements for genomic lesions, homology donors and the endogenous DNA repair machinery. Here we engineered programmable cytidine deaminases and test if we could introduce site-specific cytidine to thymidine transitions in the absence of targeted genomic lesions. Our programmable deaminases effectively convert specific cytidines to thymidines with 13% efficiency in Escherichia coli and 2.5% in human cells. However, off-target deaminations were detected more than 150 bp away from the target site. Moreover, whole genome sequencing revealed that edited bacterial cells did not harbour chromosomal abnormalities but demonstrated elevated global cytidine deamination at deaminase intrinsic binding sites. Therefore programmable deaminases represent a promising genome editing tool in prokaryotes and eukaryotes. Future engineering is required to overcome the processivity and the intrinsic DNA binding affinity of deaminases for safer therapeutic applications. 1 Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA. 2 Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts 02115, USA. 3 eGenesis Inc., 1 Kendal Square, Building 200, Cambridge Biolabs, Cambridge, Massachusetts 02139, USA.
    [Show full text]
  • Targeted Genome Modification in Mice Using Zinc-Finger Nucleases
    Copyright Ó 2010 by the Genetics Society of America DOI: 10.1534/genetics.110.117002 Targeted Genome Modification in Mice Using Zinc-Finger Nucleases Iara D. Carbery,* Diana Ji,* Anne Harrington,† Victoria Brown,* Edward J. Weinstein,* Lucy Liaw† and Xiaoxia Cui*,1 *Sigma Advanced Genetic Engineering Labs, Sigma-Aldrich Biotechnology, St. Louis, Missouri 63146 and †Maine Medical Center Research Institute, Scarborough, Maine 04074 Manuscript received March 26, 2010 Accepted for publication June 24, 2010 ABSTRACT Homologous recombination-based gene targeting using Mus musculus embryonic stem cells has greatly impacted biomedical research. This study presents a powerful new technology for more efficient and less time-consuming gene targeting in mice using embryonic injection of zinc-finger nucleases (ZFNs), which generate site-specific double strand breaks, leading to insertions or deletions via DNA repair by the nonhomologous end joining pathway. Three individual genes, multidrug resistant 1a (Mdr1a), jagged 1 (Jag1), and notch homolog 3 (Notch3), were targeted in FVB/N and C57BL/6 mice. Injection of ZFNs resulted in a range of specific gene deletions, from several nucleotides to .1000 bp in length, among 20– 75% of live births. Modified alleles were efficiently transmitted through the germline, and animals homozygous for targeted modifications were obtained in as little as 4 months. In addition, the technology can be adapted to any genetic background, eliminating the need for generations of backcrossing to achieve congenic animals. We also validated the functional disruption of Mdr1a and demonstrated that the ZFN-mediated modifications lead to true knockouts. We conclude that ZFN technology is an efficient and convenient alternative to conventional gene targeting and will greatly facilitate the rapid creation of mouse models and functional genomics research.
    [Show full text]
  • Autonomous Zinc-Finger Nuclease Pairs for Targeted Chromosomal Deletion
    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.
    [Show full text]
  • Advances in Zinc Finger Nuclease and Its Applications
    Review Gene and Gene Editing Copyright © 2015 American Scientific Publishers All rights reserved Vol. 1, 3–15, 2015 Printed in the United States of America www.aspbs.com/gge Advances in Zinc Finger Nuclease and Its Applications Li-Meng Tang1, Cui-Lan Zhou1, Zi-Fen Guo1,LiXiao2 ∗, and Anderly C. Chüeh3 1Institute of Pharmacy and Pharmacology, University of South China, Hengyang 421001, China 2Laboratory of Molecular Medicine, The Second Affiliated Hospital of Soochow University, Suzhou 215123, China 3Department of Medicine (Austin Health), The University of Melbourne, Parkville, VIC 3052, Australia Zinc finger nuclease (ZFN) is an artificially engineered hybrid protein consists of a series of zinc finger protein domains, fused to a cleavage domain of Fok I endonuclease. As a powerful molecular tool for gene editing, ZFN has been widely used for gene knock-in and knock-out in a variety of cell types and organisms. However, due to the limitations of its specificity, technological improvement for ZFN and its applications are much needed. This review summarizes the latest improvements of such technique for use in biology. KEYWORDS: Zinc Finger Nuclease, Gene Editing, Off-Target Effect. CONTENTS eukaryotic zinc finger-containing proteins (ZFPs) that Structure of ZFNS and Their Mechanism of Actions in exhibit an unique ability of recognizing and binding to Gene Editing . .IP: . 192.168.39.151 . .On: 3Sun,specific 26 Sep DNA 2021 sequences. 11:43:06 The series of zinc finger protein Copyright: American Scientific Publishers Applications and the Current Limitation of ZFNS . 6 domains is also termed zinc finger motifs or zinc finger Disease Modeling at Cellular Level or Delivered by Ingenta IntactOrganismalLevel.....................
    [Show full text]