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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...................... 6 arrays. Based on the conserved amino acids in the zinc Genome Modification of Organisms for finger domain, ZFPs and ZFNs can be categorized into Agriculture and Industry . 6 1 three major subtypes (C2H2,C4,andC6). Among these, HumanGeneTherapy....................... 7 C H is the classic type and the most widely used in the CurrentLimitationsofZFNs................... 8 2 2 AdvancesinZFNCloning........................ 8 artificially-engineered ZFNs. TFIIIA was the first identi- Optimization of the Zinc Finger Binding Domain . 8 fied C2H2 ZFP that consists of with zinc finger domain of Optimization of the Reporting and Screening System . 10 30 amino acids, in which the 8th and 13th amino acids Modification of the Cleaving Domain of Fok I . 10 of cysteine and the 26th and 30th amino acids of his- Optimization of ZFN Modification Technology . 11 tidine are highly conserved throughout evolution. These Optimization of Cell Transfection Technology . 11 Off-TargetEffectsofZFN..................... 11 conserved amino acids are responsible for the direct bind- 2 Acknowledgments............................. 12 ing to zinc ion, which critical in stabilizing the protein References.................................. 12 fold for DNA binding. This type of Cys2His2 (C2H2)ZFP typically binds to DNA in the form of monomer. By com- parion, the type of C ZFP has 4 cysteines in as the con- STRUCTURE OF ZFNS AND THEIR 4 MECHANISM OF ACTIONS IN GENE EDITING served amino acid, which binds to DNA in a form of dimer. Reversed repeats in target DNA sequence are recog- Zinc finger nuclease (ZFN) is a class of artificially- nized by homodimer, whereas direct repeats are bound by engineered nuclease that consists of a series of zinc finger heterodimer.3 The third type of C ZFP has the ability to protein domains fused with the cleavage domain of nucle- 6 bind to 2 zinc ions by six conserved cysteines, which are ase Fok I (Fig. 1). The sequence specificity and binding critical in binding to DNA either in the form of monomer affinity of ZFNs to the target DNA are determined by zinc or dimer.4–7 finger protein domains derived from naturally-occurring Due to the simplicity of using monomer in recogniz- ing and binding to DNA, the type of C2H2 ZFP is com- ∗Author to whom correspondence should be addressed. E-mail: [email protected] monly used in engineering ZFN. In particular, the most Received: 10 January 2014 ZFNs are engineered from the basic structure of naturally- Accepted: 6 May 2014 occurring mouse or human ZIF268. With regards to DNA Gene Gene Edit. 2015, Vol. 1, No. 1 2376-3949/2015/1/003/013 doi:10.1166/gge.2015.1010 3 Advances in Zinc Finger Nuclease and Its Applications Tang et al. binding ability, Fairall et al. analyzed the crystal structure + 6 position recognize 3 bases from the direction of 3 to of ZIF268 and identified the formation of hydrogen bonds 5 in the other DNA strand.9 Thus, a single zinc finger between the − 1, + 2, + 3, + 6 amino acids in the -helix protein can recognize upto 4 nucleotides for physiological of ZIF268 and the target DNA.8 The amino acids that are regulation. However, from the technological point of view, involved in the formation of hydrogen bonds with the DNA the string of 3 bases from 3 to 5 direction is easier to are theoretically responsible for the DNA sequence recog- handle in the zinc finger protein array. Thus, mutagenesis nition. It was suggested that the 2nd amino acid is able experiments that focus on the − 1∼+ 6 amino acids of the to recognize one base from the direction of 5 to 3 in a ZFP -helix was performed to screen for the most effi- single DNA strand, while the amino acids at − 1, + 3, and cient combination of amino acids for binding to a string Li-Meng Tang was born in 1989. Currently, She is studying for her Master’s degree in pharmacology at the Institute of Pharmacy and Pharmacology, University of South China, China. Her research interests are focused on gene diagnosis and the applications of engineered nucleases (including ZFN, TALEN and CRISPR/Cas9) in gene modifi- cation and gene therapy. Cui-Lan Zhou is a lecturer in the medical school at University of South China. Cur- rently she is pursuing her Ph.D. studies in Central South University under the supervi- sion of Professor Weijun Cai and Kai Li. She received her master’s degree in molecular pharmacology from University of South China in 2006. Her research areas of interests IP:are 192.168.39.151 molecular diagnostic On: andSun, gene 26 therapy.Sep 2021 11:43:06 Copyright: American Scientific Publishers Delivered by Ingenta Zi-Fen Guo received her B.S.M from Hengyang Medical College, China. She has since been teaching Pharmacology and Clinical Pharmacology Courses in the Department of pharmacology at University of South China, and currently she is an Associate Professor of pharmacology. Advised by Professor Duanfang Liao and Kai Li, she obtained her Ph.D. degree in Pathology and Pathophysiology from University of South China in 2011. Her research focuses on molecular diagnostic and individualized medication. Li Xiao is a Research Assistant in Molecular Medicine Center at the Second Affiliated Hospital of Soochow University. Currently she is pursuing her Ph.D. studies in Soochow University under the supervision of Professor Kai Li. She received her master’s degree in molecular pharmacology from University of South China in 2007. Her research areas of interests are molecular diagnostic and gene therapy. 4 Gene Gene Edit. 1, 3–15, 2015 Tang et al. Advances in Zinc Finger Nuclease and Its Applications Anderly C. Chüeh received his BBiomedSc degree from the University of Melbourne in Australia. During his undergraduate years from 2001 to 2002, he received significant research training from apoptosis pioneers Professors David L. Vaux and David C. S. Huang to study the mechanism of cell death at the Walter and Eliza Hall Institute for Medical Research. Dr. Chüeh subsequently move to the Murdoch Children’s Research Institute to complete his Honours and Ph.D. studies on the genetic and epigenetic regulation of centromeric chromatin between 2003 to 2008, under the supervision of internationally renowned chromosome biologists Professor KH Andy Choo and Dr. Lee H. Wong. From 2008 to 2013, he undertook his post-doctoral training with colorectal cancer expert Associate Professor John Mariadason at the Ludwig Institute for Cancer Research, where he investigated the genetic and epigenetic basis of colorectal tumour syndromes and their response to novel targeted therapy. Dr. Chüeh is currently a Research Fellow at The Walter and Eliza Hall Institute of Medical Research and an Honorary Fellow at the University of Melbourne in Australia. of 3 nucleotides. There are now engineered ZFPs available the Fok I cleavage domain executes the nuclease activity in recognizing all 3 string of GNN, most of ANN, some when two reversed domains form a dimmer. Fok I is an CNN, and a few TNN strings.10–12 This strategy makes endonuclease isolated from Flavobacterium okeanokoites. the design and assembly of artificial ZFN easier and more The recognition site of Fok I has an asymmetric recog- predictable. In addition to the genetically engineered ZFP nition sequence of 5-GGATG-3:5-CATCC-3.Thecut- based on the backbone of ZIF268, other naturally occur- ting site is distinct to the recognition site and is 9 bp 3 ring ZFPs in the human genome have been successfully downstream of GGATG and 13 bp upstream of CATCC.16 used in making ZFNs by Korean investigators.13 14 This feature of Fok I offer a unique opportunity of being To be a practically useful endonuclease for gene edit- used as a genetically-engineered nuclease because it con- ing in mammalian genome, recognition of a string of 18 sists of two components of separate recognizing and cut- base pair in DNA sequence is the minimum requirement.2 ting domains. As compared with the 5 bp recognition site Therefore, at least 6 zinc fingerIP: motifs 192.168.39.151 are needed to engi-On: Sun,of 26 wild Sep type 2021 Fok 11:43:06 I, engineered nucleases usually have a neer a novel ZFN.
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