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 recognizing 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 supervision 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. Since a ZFN consistsCopyright: of two monomers, American Scientificlonger recognition Publishers sequence. Thus, the spacer between the the minimal requirement of a monomer is a seriesDelivered of 3 zinc by 2Ingenta recognition elements is a potential factor that may reg- finger motifs. Typically, the 2 adjacent zinc finger motifs ulate nuclease activity and should to be evaluated. For are not directly linked to each other. Instead, a particu- this, Bibikova et al. investigated the contribution of spacer lar string of amino acids such as the typical sequences of length to the enzymatic activities of ZFNs.17 When the TGEKP is commonly used between as the spacer between separation between the 2 recognition elements was 4 bp, 2 zinc finger motifs.15 It was initially believed that the the enzymatic activity was almost completely abolished. zinc finger motifs may be serially linked to form a long Increasing the length of the spacer sequence showed a string without the losing their DNA binding ability. How- bimodal effect, with the highest enzymatic activity using ever, through series of experimentation, we have now real- 8 bp spacer and the second highest activity contributed by ized that the highest binding ability is only maintained in a spacer of 16 bp. strings containing 3 to 4 zinc finger motifs. The development of ZFNs opens a new avenue for gene The ZFN functions as a dimer, recognizing each single- editing with direct application in gene therapy. In con- strand DNA sites by each of the monomer. The zinc fin- trast to conventional gene knockout using spontaneous ger motifs are responsible for the DNA recognition, while homologous recombination, DNA repair mechanisms

Figure 1. Structure of Zinc finger nuclease. Each monomer is composed of ZFA and Fok I nuclease, it generally works when two monomers form a dimer, F1–F6 are zinc fingers, each zinc finger is connected through the linker. + 6, + 3, and − 1amino acid of each zinc finger bind DNA.

Gene Gene Edit. 1, 3–15, 2015 5 Advances in Zinc Finger Nuclease and Its Applications Tang et al.

The major applications of gene editing using ZFNs can be divided into 3 major categories: (1) disease modeling at cellular level or intact organismal level; (2) genome modiﬁcation of economically interested species for agriculture and industry; and (3) human gene therapy. A short summary of these exam- ples is listed in the Table I.

Disease Modeling at Cellular Level or Intact Organismal Level ZFNs mediated gene editing has been widely used in the modification of many types of cells, including leukemia 33 34 Figure 2. Two cell repair mechanisms: NHEJ and HR. cell line K562, induced pluripotent stem cell (iPS), and embryonic stem cells.35 Genetic engineering approaches such as traditional gene knockout and transgenic tech- are greatly activated following double-stranded breaks nologies and the latest ZFN-associated gene editing are induced by ZFNs. Subsequent to the formation of extremely powerful in the development of animal mod- double-stranded breaks, both non-homologous end-joining els for human diseases. Conventional technologies based (NHEJ) and homologous-directed repair (HR) machiner- on spontaneous homogenous or heterogeneous recombina- ies are recruited for genomic repair (Fig. 2). NHEJ is tion have led the production of a suite of useful geneti- a DNA template independent mechanism by which a cally modified animals. In medical research, both mouse series of proteins act to join the break ends through lig- and rat disease models are frequently used tools. How- ation. In contrast, HR is DNA template dependent mech- ever, the difficulty in obtaining rat embryonic stem (ES) anism that allows for the integration of external DNA 18 cells restrains the development of rat disease models via fragments into the host genome. It was reported that traditional methods. In 2009, Geurts et al.29 reported a the HR efficiency increases up to several thousand fold successful gene knockout in rat by injecting ZFN into fer- IP: 192.168.39.151 On: Sun, 26 Sep 2021 11:43:06 with presence of the double strand breaks induced by tilized egg, which paved a new way for the production ZFN.19 Copyright: American Scientific Publishers Delivered by ofIngenta genetically engineered animals. DNA microinjection of The high efficiency of gene editing after the produc- fertilized eggs, coupled with ZFN technology, provides a tion of double-stranded breaks open up the opportunity to possible route to construct genetically-modified models for several kinds of gene editing, including both gene knock- the species by which ES cells cannot be obtained, which in and gene knock-out. Gene knock-in can be used to also greatly shorten the time required for experimentation. ‘fix’ abnormal or mutated gene and to introduce external ‘gene pieces’ such as reporter genes by homologous Genome Modification of Organisms for recombination.20–22 By comparison, NHEJ is a template Agriculture and Industry independent mechanism that is more efficient than homol- There have been multiple recent successes in modify- ogous recombination. Depending on the gene target to be ing the genome of organisms for agriculutre and indus- edited, NHEJ can work to either activate or inactivate an 36 internal gene of interests. trial uses. Shukla et al. has successfully employed the ZFN technology to produce a herbicide-resistant and phytate levels-reduced, ‘double-advantage’ corn, while APPLICATIONS AND THE CURRENT Townsend et al.28 has produced a herbicide-resistant LIMITATION OF ZFNS tobacco plant using similar approach. These 2 landmark ZFN is the first engineered endonuclease to be used for studies, published in Nature, highlighted the great potential gene editing. In the past few years, ZFNs have been widely of using ZFN technology in producing transgenic plants used in gene editing of specific targets for gene knock- for agriculture. Currently, genetical-engineering domes- out, gene integration, and gene replacement. The latter two ticated animals are still a challenge. Galli et al.37 has can be broadly considered as gene knock-ins. As one of recently employed the microinjection ZFN and nuclear the pioneer application, the ‘yellow’ gene was successfully transfer technology to successfully cloned pigs with EGFP knock-outed in Drosophila by ZFN via NHEJ.20 There are sequences.38 This provides a simple and efficient way for- an ever-increasing number of model organisms in which ward for gene editing in livestock. As pigs and other genes are edited by ZFNs or other similar types of engi- animals can be readily used for organ transplantation, neered endonucleases. As tabulated in Table I, some of the ZFN technology may be utilized for cloning genetically- species including Drosophila, zebrafish, C. elegans, mouse, modified pig with reduced immune response-a future appli- rat, and pig.23–32 cation that can be considered for organ transplantation.

6 Gene Gene Edit. 1, 3–15, 2015 Tang et al. Advances in Zinc Finger Nuclease and Its Applications

Table I. Applications of ZFNs in different species.

Applications Species Gene Modiﬁcation methods References

Disease modeling Drosophila melanogaster Yellow NHEJ [20] at cellular level Rosy NHEJ, HR-mutation [23] or intact animal level Brown NHEJ [23] Danio rerio slc24a5 (golden) NHEJ [24] ntl NHEJ [24] kdrl (kdr, ﬂk) NHEJ [25] Caenorhabditis elegans Synthetic QQR target NHEJ, HR [27] Mouse Mdr1a NHEJ [31] Jag1 NHEJ [31] Notch3 NHEJ [31] Rat Il2rg NHEJ [30] Rab38 NHEJ [29] IgM NHEJ [29] Genome modiﬁcation of Arabidopsis ADH1 NHEJ [26] economically interested TT4 NHEJ [26] species for agriculture Nicotiana tabacum SuRA NHEJ [28] and industry SuRB NHEJ, HR-mutation [28] Zea mays Ipk1 NHEJ, HR-insertion [36] Zp15 NHEJ, HR-insertion [36] pig Exogenous eGFP unknown [32] Human gene therapy Homo sapiens CCR5 NHEJ [39] CXCR4 NHEJ [42] IL2RG HR-insertion [46] F9 HR-insertion [47] Ush1c HR-mutation [48] -synuclein HR-mutation [49] AAVS1 NHEJ, HR-insertion [53]

IP: 192.168.39.151 On: Sun, 26 Sep 2021 11:43:06 Human Gene Therapy Copyright: American Scientificuse of gene Publishers therapy to cure AIDS/HIV will no longer be a The use of ZFN technology to generate knockoutDelivered and gene by dream.Ingenta replacement could potentially be a very promising method- In addition to AIDS, hepatitis B is another human dis- ology for gene therapy, if under the premise of possibly ease that has been targeted by novel ZFN technology. reducing the toxicity. Investigations on ZFN-related gene Using designer ZFN targetting to hepatitis B virus, Cradick therapy are currently underway and can be divided into 2 et al.45 performed proof-of-principle experiments demon- research trends: gene knockout and gene repair. The devel- strating the specific killing of hepatitis B virus to certain opment of treatment options of HIV using ZFN technology extent, which provides a new avenue for the treatment of belongs to gene knockout studies. HIV enters the body hepatitis B and other similar diseases. via the CCR5 receptor. Deleting a segment of 32 bp in The repair of dysfunctional genes for ‘gene correction’ the CCR5 gene can make T cells exhibit anti-HIV activ- using ZFN is largely limited to single gene disorders. The ities, and the use of ZFN technology can theoretically be introduction of ZFN generates DSB and cellular repair employed to edit the CCR5 gene in T cells to provide mechanisms using homologous recombination can theo- potential cure for AIDS. Number in vitro studies have now retically serve the purpose of repairing dysfective genes. been performed and demonstrated that this approach is Scientists have now successfully applied this method for quite promising.39–41 Yuan et al.42 has recently reported in vitro ‘gene correction’ of a variety of human dis- that in addition to modifying CCR5, knockout of CXCR4 eases, including X-linked severe combined immunodefi- gene can make mouse cells resistant to HIV. By com- ciency disease,46 hemophilia B,47 Usher syndrome48 and parison, Didigu et al.43 generated a double knockout of so on. Soldner et al.49 was the first group to modify a CCR5 and CXCR4 cell line using 2 designer ZFNs and single disease-causing mutation in human stem cells with achieved a significant enhancement of resistance to HIV ZFN without causing changes any other part of the stem infection. Holt et al.41 has recently provide compelling evi- cell genome. The researchers carefully inserted or removed dence supporting the notions that HIV can be clinically a single base pair in the Parkinson’s disease-causing cured by ZFN transformed autologous hematopoietic stem -synuclein (alpha-synuclein) gene in induced pluripotent cells.44 Excitingly, ZFN-based drugs developed by Sang- stem (iPS) cells. This approach have the ablity to correct amo for the treatment of AIDS has recently entered the for point mutations that were present in the patient’s iPS, Phase 1 clinical trial in humans. Based on these proof-of- and may be readily applicable for the development of treat- principle studies, it is believed that in the near future, the ment strategies for other single gene disorders. For genetic

Gene Gene Edit. 1, 3–15, 2015 7 Advances in Zinc Finger Nuclease and Its Applications Tang et al. diseases that are caused by the constitutional deletion of a cloning methods. Pruett-Miller et al. compared Bacterial large DNA fragment such as -thalassemia, it is difficult 2-Hybrid (B2H) selection-based method with a pair made to use ZFN for gene repair via the HR mechanism. As using modular-assembly, which results in better output, lentivirus-mediated gene transfer may cause cytotoxicity, more efficient and less toxicity when compared with the many recent studies focus on integration via the AAVS1 B2H strategy.54 Third, proper Fok I domain need to be gene locus,50–52 AAVS1 is a locus in PPP1R12C gene on chosen and assembled to ZFN. Typically, wild-type Fok I human chromosome 19. It was recently demonstrated that cleaving domain was used in this process. As ZFP homod- the DNA can be faithfully integrated into this site with- imer showed a higher off-target effect than that of the out affect expression of the surounding genes,53 hence the heterodimer, the recent trends have been using geneti- cytotoxicity is minimized via this approach. Therefore, cally modified Fok I domain for the development of ZFN. AAVS1 locus is potentially a good target site for ZFN- Depending on the application (gene repair, knock-in or based treatment of genetic diseases. knock-out), eitherhomologous recombination (HR) or non- homologous ending joining (NHEJ) will be selected as the Current Limitations of ZFNs mechanism of gene editing by the biological system. Despite the success in the use of ZFN technology in gene editing, there are also many obstacles that severely limited Optimization of the Zinc Finger Binding Domain its use for wider application. Firstly, the selection of tar- Zinc finger arrays assembled from single zinc finger with get gene sequences for ZFN binding with high specificity high affinity does not necessarily have high affinity as and affinity is a major challenge with the target sequence a whole. Zinc finger binding domain constructs exert 3 triple each combination of sub-assembled zinc finger but context-dependent cellular effects, therefore engineered did not specifically identify the sequence 9 bp, which adds zinc fingers must be filtered after the assembly for the great difficulty in ZFP screening and assemblying. selection of zinc finger bind domain with the strongest Furthermore, how to select zinc finger frame, the num- affinity to target DNA. There are currently 4 frequently ber of zinc finger and the polypeptides connected ZFP used assembling method in the literature including Mod- zinc fingers to optimize the ZNF’s specificity and affinity ule Assembly (MA), OPEN (Oligomerized pool engi- must be considered. Using experience to infer the ZFN’s neering) scheme,55 Wolfe’s scheme,24 a context-dependent effect is undesirable, because its effects may vary greatly assembly (CoDA) scheme.56 MA program is simple, fast in different cells or different species.IP: 192.168.39.151 Sangamo’s monopoly On: Sun,and 26 Sep is not 2021 limited 11:43:06 to zinc finger DNA sequences and 13 severely limits the widespread applicationCopyright: of ZFN American technol- Scientificframework. PublishersHowever it was recently reported a very ogy. ZFN off-target problem somewhat limitedDelivered its appli- by lowIngenta probability (6∼30%) of success to generate active 57 58 cation, cytotoxicity caused by non-specific cleavage may ZFN. OPEN program seems to be able to generate cause great harm in cells, embryos or individuals, espe- much higher success than the MA in ZFN development, cially in gene therapy. A large number of studies have but the OPEN program is complex, time consuming, and is shown that, when the DSB in the genome of the cells limited to all GNN and a small part of TNN. For example, if zinc finger array is composed of 3 zinc fingers, the prob- occurs, cells tend to use NHEJ repair mechanism but not HR mechanism, which increases the cytotoxicity while not ability of 5 -GNNGNNGNN-3 is less than 4%, as a result serve the purpose of repair gene replacement. And when the DNA sequence that can be identified by the two left 59 using ZFN technology in the clinical setting, whether ZFN and right arrays is as low as 0.16%, thus OPEN program injection causes the body to produce an immune response, can only configure 3 zinc finger arrays. By comparison as well as on the human body in addition to the purpose of to OPEN, Wolfe’s program has the same advantages and treatment will cause any side effects? We must face these disadvantages, but the process development using Wolfe’s 60 ZFN application problems. So in recent years, to solve the program is much simpler. Similarly to other programs, above problems, the scientists have done a lot of research CoDA scheme also perform direct assemblely of ZFPs, to improve ZFN technology. but due to the fact that CoDA’s zinc finger design is based on the OPEN program, it has significant limitations. Each of the four programs have their own advan- ADVANCES IN ZFN CLONING tages and limitations. When the target sequence is mostly Gene editing using ZFN is a multiple-step process. GNN, OPEN, Wolfe’s, and the CoDA programs will be In order to obtain efficient gene editing, optimal sequences the ideal platform of choice for ZFN development because first need to be selected based on surrounding sequences the technology is more mature and processes are effi- of the target genes of interests and then a list of the cor- cient. However, due to the complexity and time-consuming responding ZFP arrays are cloned. Second, DNA bind- nature of using OPEN and other related methodologies, ing ability of the zinc finger binding domains need to be in recent years, many researchers have focused on the tested before assembling into ZFN as the DNA binding improvement of the module assembly program. As early ability is crucial for possessing efficient enzymatic activ- as 2001, Isalan et al.61 developed a program different ities. Different affinity assays can be used for different from the 4 above-mentioned ZFA construction scheme or

8 Gene Gene Edit. 1, 3–15, 2015 Tang et al. Advances in Zinc Finger Nuclease and Its Applications programs. This newly developed method is based on 2 pre- ZFP. The usefulness of this improvement was confirmed prepared zinc finger phage display libraries #12 and #23. in many recent studies.65 66 Despite this, the correlation Separately generated highaffinity two-module (2F) ZFA between increase activity/specificity and ZFP number is from these libraries were subsequently assemble into a 3 not linear, this is because that a few ZFN decreases effi- finger Zinc finger arrays. It may seem somewhat similar ciency when the number of zinc finger increases. Based on to CoDA, but CoDA has only 1 common F2 zinc fin- the foundation of these principles, the authors developed ger. The principle of this method is as follows: The zinc a ‘B-score’ scoring system that facilitates the wide-use of finger in libraries #12 identify 3-HIJKLMGGCG-5,the the direct module assembly method. However, the scor- zinc finger in library #23 identify 3-GCGGMNOPQ-5 as ing system is far from perfect and significant improvement the target sequence HIJKLM and MNOPQ in the target needs to be performed in order for obtain precise predic- sequences are random nucleotides and shared 1 in base tion of ZFN activity with pre-screening in the near future. pair common designated as ‘M’. Based on the zinc fin- By altering ZFN’s intrinsic backbone, ZFN techniques gers generated from the 2 libraries, the ‘M’ base pair of can also be improved. ZFNs are commonly and classi- the zinc finger nucleotide are removed and subsequently cally build from ZIF268 backbone skeleton. Recently Bae synthesized into the 3F zinc finger arrays, which now con- et al.13 screened 56 kinds of natural zinc finger encoded tains 9 bp with the sequence of “HIJKLMNOPQ.” This by the human genome and used them directly to con- methodology is not only simple, but more importantly, it struct ZFN. As a validation study, Kim et al.14 used these makes the sequence recognition of zinc finger no longer natural zinc fingers to construct ZFN and demonstrated restricted to any triplet codons. Gupta et al.62 developed enhanced activities when compared with one derived from a ZFN construction scheme named “finger stitching,” and the ZIF268 skeleton. Thus, naturally occuring zinc fingers performed a full validation study.63 The method is based of the human genome are perhaps the better backbone of on 2F-MA, which almost exclusively recognize the NG choice for constructing ZFNs. junction of ‘GNN-GNN.’ “Finger stitching” can identify 2 The linker sequences connecting between the invididual zinc finger random joints of the 2F module. The recogni- zinc fingers also have an impact on ZFN activity and speci- tion sequence is ‘GRN-NYG,’ wherein R represents A or ficity. Classical linker sequence between the zinc fingers is G and Y represents C or T. This method can efficiently TGEKP. It was recently reported that the additional of Ser- identify zinc finger DNA sequences with 3-fold increase ine to the TGEKP (i.e., “TGSQKP”) sequence significantly 62 67 in the probability of successfulIP: design, 192.168.39.151 and is not limited On: Sun,increases 26 Sep 2021 the activity 11:43:06 of the engineered ZFN. How- to 3F ZFA. 2013, Bhakta et al.64 extendedCopyright: the direct American mod- Scientificever, further Publishers validation experiments are required to confirm ule assembly method, where they use a “drop-outDelivered linker” by whetherIngenta ZFN’s activity can be enhanced by increasing the method (Fig. 3) to study the activity of ZFAs configured length of the linker as there is one study presents the con- 64 68 in a form of 3–6 zinc fingers. The result of this improved trary results. Wilson et al. have recently studied how method is a significant increase in ZFN activity and speci- the different types of linker length and spacers affects the ficity as correlated with the increase of the number of ZFN activity. They found 2 to 4 amino acid linker have the highest efficiency of the 5 to 6 bp spacer sequence, while 5 amino acids linker length have the highest efficiency of the 6 to 7 bp spacer sequence. Similarly, Anand et al.69 have recently demonstrated that an increase in the linker sequence between two zinc fingers makes the 2 zinc fingers skip a maximum length of 10 bp sequence. Together, these results highlighted the importance of amino acid linker in regulating ZFN activities and specificities. The construction of ZFN can also be produced from a number of new methods. Latest research70 indicates that a ZFN monomer and TALEN (transcription activator like effector nuclease) monomer can form a hybrid nucleic acid enzyme, which enhanced specificity in the performance of the different targets. These hybrids can be produced in different cell types, including human induced pluripotent stem (iPS). Yusuke et al.71 invented a sandwiched ZFN (Fig. 4), composed of 2 ZFN domains and a sandwiched Staphylococcus aureus nuclease (SNase). This novel ZFN- enzyme can cut DNA in the form of monomers. It is Figure 3. Drop-out linker. Hind III, Bsm I etc. are the restriction enzyme sites, firstly synthesis of a 6F zinc finger arrays reported that the advantages of this enzyme is its ability then excised by restriction enzyme to generate 5F, 4F, 3F zinc indistinguishing DNA substrate cleavage products and to finger arrays. improve the efficiency of ZFN. In addition, Fujii et al.72

Gene Gene Edit. 1, 3–15, 2015 9 Advances in Zinc Finger Nuclease and Its Applications Tang et al.

a high active ZFN monomer could assemble a new ZFN that can produce biological activities,74 in order to save these monomers of low activity, Zhang et al.75 was the first group to invent a reporting system for high sensitivity detection of low activity ZFN. They assembled ZFN from highly active monomers and monomers with low activities. They demonstrated that the engineered ZFNs derived Figure 4. Sandwiched ZFN. SNase is a Sandwiched single from this reporting systems function properly and that the Staphylococcus aureus nuclease, the zigzag line is the linked approach is economical. This new method also produces polypeptide. lower ZFN activity and reduces the cytotoxicity.76 have invented a new construct method of ZFN, named Modification of the Cleaving Domain of Fok I OLTA (OverLap extension PCR and TA-cloning), in which In recent years, there have been a number of studies the zinc finger DNA encoding fragment was synthesized that investigate on effects of modifying the Fok I cleav- by PCR. A short sequence primer homologous to the DNA age domain to improve ZFN’s specificity. DNA shear- recognition helix domain is responsible binding to ZFP, ing requires 2 Fok I units for dimerization and at least while different primers encodes different recognition helix one to bind DNA. Dimerization is a process indepen- sequences. The 4–6 zinc fingers were synthesized by over- dent of DNA shearing. Heterodimers, homodimers and lap extension PCR and cloned into a vector with nuclease monomers of Fok I all exhibit DNA shearing activi- using TA cloning. The advantage of this new approach is ties, with different recognition sequences and non-specific that to the making of the construct of ZFN is easy, quick behavior that may results in increases in ZFN toxicity.77 and cheap, however the drawback is obvious. If not per- To improve ZFN specificity and reduce the toxicity, dimer- form properly, PCR often results in high base mismatch, ization region of Fok I is commonly modified to reduce and that a single base mismatch could results in detrimen- their homo-dimerization activity. ZFN pair constructed tal effects in the activity of ZFN. from two variants, only the hetero-dimerization induces specific cleavage, while homo-dimerization induces low Optimization of the Reporting and cleavage activity. Following this principle, Sangamo syn- Screening System IP: 192.168.39.151 On: Sun,thesised 26 Sep a2021 mutant 11:43:06 Fok I with two variants, Q486E: I499L In addition to direct module assemblyCopyright: method, American OPEN, Scientificand E490K: Publishers I538K (also called EL_KK), each having 10- CoDA and other methods all required to pre-screeningDelivered for by foldIngenta reduction in cytotoxicity when compared to the wild- ZFN development. Various bioinformatic programs have type Fok I. In 2007, Szczepek et al.78 synthesised a mutant been developed for screening and testing for newly derived Fok I, in which the residue pair of the dimerization region ZFN. Typically, a ZFA library is first built, then merged was mutated by site-direct mutagenesis. The two variants into a transcriptional activation domain to form a zinc R487D and D483R (also named DD and RR) are now finger-activating factor (also abbreviated as ZFA). Subse- commonly used in ZFN technology. Similarly, Cathomen’s quent to this, a reporter gene expression system carrying laboratory generated a pair of variants having 2 Fok I the target site is used to detect the intensity of the reporter mutants D483R: I538V and R487D: I499A (also called gene expression to filter select highly active ZFAs. Tradi- RV_DA). These mutants have reduced some cytotoxic- tional reporting and screening systems are Phage display, ity when compared to wild-type. Based on the work of yeast two-hybrid (Y2H), bacterial two-hybrid (B2H), bac- Sangamo and Szczepek, Ramalingam et al.79 synthesised terial one-hybrid (B1H), and so on. However, these screen- 2 additional variants, REL_DKK and RELV_DKAK, and ing systems are laborious and time-consuming, therefore a new variant, the Fok I_Sts I. The principle behind the researchers have focused on developing new screening sys- design was the use of partial sequence of Sts I restriction tem to improve ZFN technology. Wang et al.73 invented a enzymes to replace dimerization domain of Fok I. In these novel system, which can simultaneously screen and val- new variants, when REL_DKK was assembled with a idate ZFN activity. They used yeast AH109 to directly ZFA of 3–4 fingers, the authors demonstrated a remark- obtain active ZFNs. The system is based on OPEN and able ability to reduce toxicity in human cells. Further- Gal4 reporting system, with a special reporter plasmid con- more, Guo et al.65 used directed evolution approach at a taining a 30-bp Gal4 homologous arm and a ZFN target. dimerization region of Fok I to generate a hyper-activation ZFN binds to the target and leads to DSB, which is subse- variant, Sharkey. Compared with conventional techniques, quently repaired by single-strand annealing (SSA) mecha- ‘Sharkey’ exhibited 15 times higher ZFN cleavage activ- nism. Although based on OPEN, this system can produce ity, and is compatible with a variety of old and new ZFN up to 24-bp specific recognition of ZFN and is capable of skeletons.80 In 2012, Schierling et al.81 made a simple generating active ZFN or verify the constructed ZFN activ- change of the Fok I endonuclease to PvuII and built a new ity in a time efficient manner. Reported that a low-active ZFN. Direct experiments performed the authors proved ZFN monomer which can not produce ZFN activity and that ZF-PvuII cleavage of the target is 1000 fold more

10 Gene Gene Edit. 1, 3–15, 2015 Tang et al. Advances in Zinc Finger Nuclease and Its Applications efficient than cleavage for the non-target sites. Importantly, required to exposure the cells to ZFNs (and therefore to ZF-PvuII treatment does not produce any excess enzyme minize off-target effects), but also avoid the cells to be activities on the substrate, nor produced any non-specific exposed to any other genetic material that is not part of cleavage in long time culture. This novel ZFN variant may the knockin gene. In addition, it is was recently reported be a valid alternative to existing ZFNs. that directly transfering the ZFN-encoding DNA fragment in the cells, rather than using plasmids as the vector, Optimization of ZFN Modification Technology avoids off-target homologous recombination and reduce NHEJ and HR are the 2 major mechanisms by which ZFN cytotoxicity.90 introduce gene editing. As previously mentioned, NHEJ In addition to modifying the ZFN itself, optimization is the more favorable mechanism over HR in cell. This for DNA homology of the donor sequence is also impor- processes a problem as NHEJ is more prone to introduce tant. The donor sequence usually need to be about 750 bp novel mutations. So, how to avoid NHEJ mechanism at on each arm to generate HR. Salvatore et al.83 provides the time of HR has become the central in the improve- a new donor, only about 50 bp in length. When the ment of ZFN technology. Ramirez et al.82 introduced a new donor sequence was designed to contain a single-stranded modification method of ZFN, through inactivating Fok I 5 protruding that is complementary with ZFN notch, restriction enzyme activity in a monomer of a ZFN dimer the rate of integration of the exogenous genes can be to generate ZFN cutout (ZFNickases), and then produce greatly enhanced. This is because the mechanisms of cell DNA single-stranded break (SSB) in the target sequence. are using NHEJ instead of HR and this approach sim- SSB incision may induce HR but results in a lower fre- plifies the design of the donor DNA. It has also been quency than conventional DSB approach. The advantage is recently reported91 92 that single-stranded oligonucleotide that it can avoid NHEJ, thus greatly reduce the degree of (ssODN) can be used as an alternative to traditional cytotoxicity. Salvatore et al.83 used 2 ZFNs simultaneously double-stranded donor DNA, These studies demonstrated to cut the target sequence to generate 2 DSBs, and then that ssODN improves ZFN activitiy and increases the fre- integrated the donor DNA sequence by NHEJ mechanisms. quency of HR mechanisms within the cell. This methodology led to approximately 50% of success- Interestingly, the biologic activity of ZFNs can be mod- ful deletion events for the inserted exogenous gene. This ified by using a variety of small molecules as well. method hase allowed the improvement in efficiency for the DNA end-processing enzyme was reported to increase 93 ZFN technology, but it may alsoIP: increase 192.168.39.151 in cytotoxicity. On: Sun,the 26 geneSep 2021 disruption 11:43:06 effects elicited by ZFNs. Further- Copyright: American Scientificmore, thePublishers addition of proteasome inhibitors such as Optimization of Cell Transfection TechnologyDelivered by MG132Ingenta can increase the stability of ZFNs in gene edit- 96 Typically, the gene encoding ZFN is introduced into the ing experiments. Other interventions such as increasing cell via DNA plasmid, viral vectors, or in vitro transcrip- the transduction efficiency and changing the cell culture 97 98 tion of the mRNA.84 However, the reagents that are used conditions may also enhance the gene editing effects. to transfect DNA plasmid or the mRNA by electricical Non-toxic cell cycle modulator Indirubin was reported in 97 or liposomal transfer method may be toxic to the cell, augmenting AAV transduction. In addition, positively depending of the cell type. This is a major limitation of the gene-edited cells can be significantly enriched by magnetic ZFN technology that needs to be further optimized.85 The separation, co-expression of florescence reporter gene, or 94 95 use of viral vectors is also another limiting factor because selection using antibiotic resistant gene. they are difficult to assemble and could cause potential immunogenicity issues. Recently, adeno-associated virus Off-Target Effects of ZFN (AAV) has been proven to be safe in humans and does Despite much success in the development of ZFN technol- not produce major side effects as demonstrated by multi- ogy, off-target effects may contribute to ‘genetic toxicity’ ple clinical trials. AAV is a promising ZFN vector. They and side effects when using ZFNs to study the normal have already been used to enhance HR and drive ZFN- physiology of cells and organisms. To circumvent this mediated gene correction in vivo.86 Efficient AAV assem- problem, two strategies can be employed to minimize bly occurs only in expression cassette that is less than 4.2 potential off-target effects. The first strategy is to perform kb; however the insert size is large enough to accommo- in vitro selection using a partly degraded library (based date 2 ZFN monomers and an engineered construct donor. on the expected ZFN cut sites).99 The second option is It has been reported that the use of AAV scheme demon- to identify possible off-targets at the genomic level based strated to be feasible and improves the ZFN technology.87 on IDLV DNAs.100 However, due to the limitations on Integration-defective lentiviral vector is (IDLV) is another the sensitivity of the proposed methodologies described attractive alternative. ZFNs can be transfected into the above, many off-targets may still be missed.101 As a result, multiple tolerated cell types.88 Recently, there is a report Sander et al. suggested that a unbiased bioinformatics anal- on a new form of transfection, where purified ZFN pro- ysis should be applied to recover wider spectrum of the teins can directly introduced into the cell to cause a genetic off-targets, using specific programs such as ZiFiT,102 Zinc modification.89 This method not only by shorten the time Finer Tools,103 and ZFN-Site.104

Gene Gene Edit. 1, 3–15, 2015 11 Advances in Zinc Finger Nuclease and Its Applications Tang et al.

In addition to the in silico analysis, novel experimental the construction of artificial transcription factors. Nat. Biotechnol. approaches are urgently needed to screen for genome-wide 21, 275–80. off-target effects. Both beta-Gal-based assays and the use 13. Kim, H. J., Lee, H. J., Kim, H., Cho, S. W., and Kim, J. S. (2009). Targeted genome editing in human cells with zinc finger nucleases of antibiotic resistance genes have been widely performed constructed via modular assembly. Genome Res. 19, 1279–88. in evaluation of the enzymatic activity of engineered nucle- 14. Peisach, E. and Pabo, C. O. (2003). Constraints for zinc finger ases. They are expected to be useful in the development of linker design as inferred from X-ray crystal structure of tandem more sensitive technologies as screen for off-target effects Zif268-DNA complexes. J. Mol. Biol. 330, 1–7. by further optimization of there reporter genes or some 15. Looney, M. C., Moran, L. S., Jack, W. E., Feehery, G. R., Benner, J. S., Slatko, B. E., and Wilson, G. G. (1989). Nucleotide other new reporter system. sequence of the FokI restriction-modification system: Separate strand-specificity domains in the methyltransferase. Gene 80, Acknowledgments: We acknowledge the financial sup- 193–208. ports of the Chinese National 863 Major Grant (No. 16. Bibikova, M., Carroll, D., Segal, D. J., Trautman, J. K., Smith, J., 2012AA020905), the National Natural Science Foundation Kim, Y. G., and Chandrasegaran, S. (2001). Stimulation of homologous recombination through targeted cleavage by chimeric nucle- of China (No. 81301267), the Priority Academic Program ases. Mol. Cell. Biol. 21, 289–97. Development of Jiangsu Higher Education Institutions, the 17. Capecchi, M. R. (1989). Altering the genome by homologous Innovation Project of Jiangsu Graduate Education (No. recombination. Science 244, 1288–92. CXZZ11_0117) and Jiangsu Province’s clinical medical 18. Klug, A. (2010). The discovery of zinc fingers and their appli- science and technology program (No. BL2013016). cations in gene regulation and genome manipulation. Annu. Rev. Biochem. 79, 213–31. 19. Bibikova, M., Golic, M., Golic, K. G., and Carroll, D. (2002). Tar- REFERENCES geted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161, 1169–75. 1. MacPherson, S., Larochelle, M., and Turcotte, B. (2006). A fun- 20. Orlando, S. J., Santiago, Y., DeKelver, R. C., Freyvert, Y., gal family of transcriptional regulators: The zinc cluster proteins. Boydston, E. A., Moehle, E. A., Choi, V. M., Gopalan, S. M., Microbiol. Mol. Biol Rev. 70, 583–604. Lou, J. F., Li, J., Miller, J. C., Holmes, M. C., Gregory, P. D., 2. Zhong, Q. and Zhao, S. H. (2011). [The mechanism and application Urnov, F. D., and Cost, G. J. (2010). Zinc-finger nuclease-driven of zinc finger nucleases]. Yi Chuan. 33, 123–30. targeted integration into mammalian genomes using donors with 3. Perez-Pinera, P., Ousterout, D. G., and Gersbach, C. A. (2012). limited chromosomal homology. Nucleic Acids Res. 38, e152. Advances in targeted genome editing. Curr. Opin. Chem. Biol. 21. Kakarougkas, A. and Jeggo, P. A. (2014). DNA DSB repair path- 16, 268–77. way choice: An orchestrated handover mechanism. Br. J. Radiol. 4. Lohr, D., Venkov, P., and Zlatanova,IP:J. 192.168.39.151 (1995). Transcriptional On: reg- Sun, 26 Sep87, 20130685. 2021 11:43:06 ulation in the yeast GAL gene family: ACopyright: complex genetic American network. Scientific22. Beumer, Publishers K., Bhattacharyya, G., Bibikova, M., Trautman, J. K., and FASEB J. 9, 777–87. Delivered by IngentaCarroll, D. (2006). Efficient gene targeting in Drosophila with zinc- 5. Schjerling, P. and Holmberg, S. (1996). Comparative amino acid finger nucleases. Genetics 172, 2391–403. sequence analysis of the C6 zinc cluster family of transcriptional 23. Doyon, Y., McCammon, J. M., Miller, J. C., Faraji, F., Ngo, C., regulators. Nucleic Acids Res. 24, 4599–607. Katibah, G. E., Amora, R., Hocking, T. D., Zhang, L., Rebar, E. J., 6. Todd, R. B. and Andrianopoulos, A. (1997). Evolution of a fungal Gregory, P. D., Urnov, F. D., and Amacher, S. L. (2008). Herita- regulatory gene family: The Zn(II)2Cys6 binuclear cluster DNA ble targeted gene disruption in zebrafish using designed zinc-finger binding motif. Fungal Genet Biol. 21, 388–405. nucleases. Nat. Biotechnol. 26, 702–8. 7. Fairall, L., Schwabe, J. W., Chapman, L., Finch, J. T., and 24. Meng, X., Noyes, M. B., Zhu, L. J., Lawson, N. D., and Wolfe, Rhodes, D. (1993). The crystal structure of a two zinc-finger pep- S. A. (2008). Targeted gene inactivation in zebrafish using engi- tide reveals an extension to the rules for zinc-finger/DNA recogni- neered zinc-finger nucleases. Nat. Biotechnol. 26, 695–701. tion. Nature 366, 483–7. 25. Zhang, F., Maeder, M. L., Unger-Wallace, E., Hoshaw, J. P., Reyon, 8. Isalan, M., Klug, A., and Choo, Y. (1998). Comprehensive DNA D., Christian, M., Li, X., Pierick, C. J., Dobbs, D., Peterson, T., recognition through concerted interactions from adjacent zinc fin- Joung, J. K., and Voytas, D. F. (2010). High frequency targeted gers. Biochemistry 37, 12026–33. mutagenesis in Arabidopsis thaliana using zinc finger nucleases. 9. Dreier, B., Beerli, R. R., Segal, D. J., and Flippin, J. D. (2001). Proc. Natl. Acad. Sci. USA 107, 12028–33. Barbas CF 3rd. Development of zinc finger domains for recogni- 26. Morton, J., Davis, M. W., Jorgensen, E. M., and Carroll, D. (2006). tion of the 5 -ANN-3 family of DNA sequences and their use in Induction and repair of zinc-finger nuclease-targeted double-strand the construction of artificial transcription factors. J. Biol. Chem. breaks in Caenorhabditis elegans somatic cells. Proc. Natl. Acad 276, 29466–78. Sci. USA 103, 16370–5. 10. Dreier, B., Fuller, R. P., Segal, D. J., Lund, C. V., Blancafort, P., 27. Townsend, J. A., Wright, D. A., Winfrey, R. J., Fu, F., Maeder, Huber, A., Koksch, B., and Barbas, C. F. 3rd. (2005). Development M. L., Joung, J. K., and Voytas, D. F. (2009). High-frequency of zinc finger domains for recognition of the 5 -CNN-3 family modification of plant genes using engineered zinc-finger nucleases. DNA sequences and their use in the construction of artificial tran- Nature 459, 442–5. scription factors. J. Biol. Chem. 280, 35588–97. 28. Geurts, A. M., Cost, G. J., Freyvert, Y., Zeitler, B., Miller, J. C., 11. Segal, D. J., Dreier, B., and Beerli, R. R. (1999). Barbas CF 3rd. Choi, V. M., Jenkins, S. S., Wood, A., Cui, X., Meng, X., Toward controlling gene expression at will: Selection and design of Vincent, A., Lam, S., Michalkiewicz, M., Schilling, R. Foeckler, J., zinc finger domains recognizing each of the 5-GNN-3 DNA target Kalloway, S., Weiler, H., Menoret, S., Anegon, I., Davis, G. D., sequences. Proc. Natl. Acad Sci. USA 96, 2758–63. Zhang,L.,Rebar,E.J.,Gregory,P.D.,Urnov,F.D.,Jacob,H.J., 12. Bae, K. H., Kwon, Y. D., Shin, H. C., Hwang, M. S., Ryu, E. H., and Buelow, R. (2009). Knockout rats via embryo microinjection Park,K.S.,Yang,H.Y.,Lee,D.K.,Lee,Y.,Park,J.,Kwon,H.S., of zinc-finger nucleases. Science 325, 433. Kim, H. W., Yeh, B. I., Lee, H. W., Sohn, S. H., Yoon, J., Seol, W., 29. Mashimo, T., Takizawa, A., Voigt, B., Yoshimi, K., Hiai, H., and Kim, J. S. (2003). Human zinc fingers as building blocks in Kuramoto, T., and Serikawa, T. (2010). Generation of knockout rats

12 Gene Gene Edit. 1, 3–15, 2015 Tang et al. Advances in Zinc Finger Nuclease and Its Applications

with X-linked severe combined immunodeficiency (X-SCID) using 43. Zhang, J., Li, A. S., Kou, X., Xiao, L., Li, P., He, N., and Li, zinc-finger nucleases. PLoS ONE 5, e8870. K. (2012). Why CCR5 is chosen as the target for stem cell gene 30. Carbery, I. D., Ji, D., Harrington, A., Brown, V., Weinstein, E. J., therapy for HIV infection. J Nanosci Nanotechnol 12, 2045–8. Liaw, L., and Cui, X. (2010). Targeted genome modification in mice 44. Cradick, T. J., Keck, K., Bradshaw, S., Jamieson, A. C., and using zinc-finger nucleases. Genetics 186, 451–9. McCaffrey, A. P. (2010). Zinc-finger nucleases as a novel thera- 31. Whyte, J. J., Zhao, J., Wells, K. D., Samuel, M. S., Whitworth, peutic strategy for targeting hepatitis B virus DNAs. Mol. Ther. 18, K. M., Walters, E. M., Laughlin, M. H., and Prather, R. S. (2011). 947–54. Gene targeting with zinc finger nucleases to produce cloned eGFP 45. Urnov, F. D., Miller, J. C., Lee, Y. L., Beausejour, C. M., Rock, knockout pigs. Mol. Reprod. Dev. 78, 2. J. M., Augustus, S., Jamieson, A. C., Porteus, M. H., Gregory, P. D., 32. Moehle, E. A., Rock, J. M., Lee, Y. L., Jouvenot, Y., DeKelver, and Holmes, M. C. (2005). Highly efficient endogenous human R. C., Gregory, P. D., Urnov, F. D., and Holmes, M. C. (2007). Tar- gene correction using designed zinc-finger nucleases. Nature 435, geted gene addition into a specified location in the human genome 646–51. using designed zinc finger nucleases. Proc. Natl. Acad. Sci. USA 46. Li, H., Haurigot, V., Doyon, Y., Li, T., Wong, S. Y., Bhagwat, A. S., 104, 3055–60. Malani, N., Anguela, X. M., Sharma, R., Ivanciu, L., Murphy, S. L., 33. Hockemeyer, D., Soldner, F., Beard, C., Gao, Q., Mitalipova, M., Finn, J. D., Khazi, F. R., Zhou, S., Paschon, D. E., Rebar, E. J., DeKelver, R. C., Katibah, G. E., Amora, R., Boydston, E. A., Bushman, F. D., Gregory, P. D., Holmes, M. C., and High, K. A. Zeitler, B., Meng, X., Miller, J. C., Zhang, L., Rebar, E. J., Gregory, (2011). In vivo genome editing restores haemostasis in a mouse P. D., Urnov, F. D., and Jaenisch, R. (2009). Efficient targeting of model of haemophilia. Nature 475, 217–21. expressed and silent genes in human ESCs and iPSCs using zinc- 47. Overlack, N., Goldmann, T., and Wolfrum, U., Nagel-Wolfrum, K. finger nucleases. Nat. Biotechnol. 27, 851–7. (2012). Gene repair of an Usher syndrome causing mutation by 34. Zou, J., Maeder, M. L., Mali, P., Pruett-Miller, S. M., Thibodeau- zinc-finger nuclease mediated homologous recombination. Invest Beganny, S., Chou, B. K., Chen, G., Ye, Z., Park, I. H., Daley, Ophthalmol. Vis. Sci. 53, 4140–6. G. Q., Porteus, M. H., Joung, J. K., and Cheng, L. (2009). Gene 48. Ren, B., Robert, F., Wyrick, J. J., Aparicio, O., Jennings, E. G., targeting of a disease-related gene in human induced pluripotent Simon, I., Zeitlinger, J., Schreiber, J., Hannett, N., Kanin, E., stem and embryonic stem cells. Cell Stem Cell 5, 97–110. Volkert, T. L., Wilson, C. J., Bell, S. P., and Young, R. A. (2000). 35. Shukla, V. K., Doyon, Y., Miller, J. C., DeKelver, R. C., Moehle, Genome-wide location and function of DNA binding proteins. Sci- E. A., Worden, S. E., Mitchell, J. C., Arnold, N. L., Gopalan, S., ence 290, 2306–9. Meng, X., Choi, V. M., Rock, J. M., Wu, Y. Y., Katibah, 49. Soldner, F., Laganiere, J., Cheng, A. W., Hockemeyer, D., Gao, Q., G. E., Zhifang, G., McCaskill, D., Simpson, M. A., Blakeslee, B., Alagappan, R., Khurana, V., Golbe, L. I., Myers, R. H., Lindquist, Greenwalt, S. A., Butler, H. J., Hinkley, S. J., Zhang, L., Rebar, S., Zhang, L., Guschin, D., Fong, L. K., Vu, B. J., Meng, X., Urnov, E. J., Gregory, P. D., and Urnov, F. D. (2009). Precise genome mod- F. D., Rebar, E. J., Gregory, P. D., Zhang, H. S., and Jaenisch, ification in the crop species Zea mays using zinc-finger nucleases. R. (2011). Generation of isogenic pluripotent stem cells differing Nature 459, 437–41. exclusively at two early onset Parkinson point mutations. Cell 146, 36. Galli, C., Perota, A., Brunetti, D.,IP: Lagutina, 192.168.39.151 I., Lazzari, G., On: and Sun, 26 Sep318–31. 2021 11:43:06 Lucchini, F. (2010). Genetic engineeringCopyright: including supersedingAmerican Scientific50. Chang, Publishers C. J. and Bouhassira, E. E. (2012). Zinc-finger nuclease- microinjection: New ways to make GM pigs. XenotransplantationDelivered by Ingentamediated correction of alpha-thalassemia in iPS cells. Blood 120, 17, 397–410. 3906–14. 37. Watanabe, M., Umeyama, K., Matsunari, H., Takayanagi, S., 51. Phang,R.Z.,Tay,F.C.,Goh,S.L.,Lau,C.H.,Zhu,H.,Tan,W.K., Haruyama, E., Nakano, K., Fujiwara, T., Ikezawa, Y., Nakauchi, H., Liang,Q.,Chen,C.,Du,S.,Li,Z.,Tay,J.C.,Wu,C.,Zeng,J., and Nagashima, H. (2010). Knockout of exogenous EGFP gene in Fan, W., Toh, H. C., and Wang, S. (2013). Zinc finger nuclease- porcine somatic cells using zinc-finger nucleases. Biochem. Bio- expressing baculoviral vectors mediate targeted genome integration phys. Res. Commun. 402, 14–8. of reprogramming factor genes to facilitate the generation of human 38. Perez, E. E., Wang, J., Miller, J. C., Jouvenot, Y., Kim, K. A., Liu, induced pluripotent stem cells. Stem Cells Transl Med 2, 935–45. O., Wang, N., Lee, G., Bartsevich, V. V., Lee, Y. L., Guschin, D. Y., 52. Coluccio, A., Miselli, F., Lombardo, A., Marconi, A., Malagoli, Rupniewski, I., Waite, A. J., Carpenito, C., Carroll, R. G., Orange, T. G., Goncalves, M. A., Pincelli, C., Maruggi, G., Del, R. M., J. S., Urnov, F. D., Rebar, E. J., Ando, D., Gregory, P. D., Riley, Naldini, L., Larcher, F., Mavilio, F., and Recchia, A. (2013). J. L., Holmes, M. C., and June, C. H. (2008). Establishment of Targeted gene addition in human epithelial stem cells by zinc- HIV-1 resistance in CD4+ T cells by genome editing using zinc- finger nuclease-mediated homologous recombination. Mol. Ther. finger nucleases. Nat. Biotechnol. 26, 808–16. 21, 1695–704. 39. Kandavelou, K., Ramalingam, S., London, V., Mani, M., Wu, J., 53. DeKelver, R. C., Choi, V. M., Moehle, E. A., Paschon, D. E., Alexeev, V., Civin, C. I., and Chandrasegaran, S. (2009). Targeted Hockemeyer, D., Meijsing, S. H., Sancak, Y., Cui, X., Steine, E. J., manipulation of mammalian genomes using designed zinc finger Miller, J. C., Tam, P., Bartsevich, V. V., Meng, X., Rupniewski, I., nucleases. Biochem. Biophys. Res. Commun. 388, 56–61. Gopalan, S. M., Sun, H. C., Pitz, K. J., Rock, J. M., Zhang, L., 40. Holt, N., Wang, J., Kim, K., Friedman, G., Wang, X., Taupin, V., Davis, G. D., Rebar, E. J., Cheeseman, I. M., Yamamoto, K. R., Crooks, G. M., Kohn, D. B., Gregory, P. D., Holmes, M. C., and Sabatini, D. M., Jaenisch, R., Gregory, P. D., and Urnov, F. D. Cannon, P. M. (2010). Human hematopoietic stem/progenitor cells (2010). Functional genomics, proteomics, and regulatory DNA modified by zinc-finger nucleases targeted to CCR5 control HIV-1 analysis in isogenic settings using zinc finger nuclease-driven trans- in vivo. Nat. Biotechnol. 28, 839–47. genesis into a safe harbor locus in the human genome. Genome 41. Yuan,J.,Wang,J.,Crain,K.,Fearns,C.,Kim,K.A.,Hua,K.L., Res. 20, 1133–42. Gregory, P. D., Holmes, M. C., and Torbett, B. E. (2012). Zinc- 54. Pruett-Miller, S. M., Connelly, J. P., Maeder, M. L., Joung, J. K., finger nuclease editing of human cxcr4 promotes HIV-1 CD4(+ T and Porteus, M. H. (2008). Comparison of zinc finger nucleases for cell resistance and enrichment. Mol. Ther. 20, 849–59. use in gene targeting in mammalian cells. Mol. Ther. 16, 707–17. 42. Didigu, C. A., Wilen, C. B., Wang, J., Duong, J., Secreto, A. J., 55. Maeder, M. L., Thibodeau-Beganny, S., Osiak, A., Wright, D. A., Danet-Desnoyers, G. A., Riley, J. L., Gregory, P. D., June, C. H., Anthony, R. M., Eichtinger, M., Jiang, T., Foley, J. E., Winfrey, Holmes, M. C., and Doms, R. W. (2014). Simultaneous zinc-finger R. J., Townsend, J. A., Unger-Wallace, E., Sander, J. D., Muller- nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects Lerch, F., Fu, F., Pearlberg, J., Gobel, C., Dassie, J. P., Pruett- CD4+ T cells from HIV-1 infection. Blood 123, 61–9. Miller, S. M., Porteus, M. H., Sgroi, D. C., Iafrate, A. J., Dobbs, D.,

Gene Gene Edit. 1, 3–15, 2015 13 Advances in Zinc Finger Nuclease and Its Applications Tang et al.

McCray, P. B. Jr, Cathomen, T., Voytas, D. F., and Joung, J. K. DNA by sandwiched zinc-finger nuclease. Nucleic Acids Symp. Ser. (2008). Rapid “open-source” engineering of customized zinc-finger (Oxf). 279–80. nucleases for highly efficient gene modification. Mol. Cell 31, 72. Fujii, W., Kano, K., Sugiura, K., and Naito, K. (2013). Repeatable 294–301. construction method for engineered zinc finger nuclease based on 56. Sander, J. D., Dahlborg, E. J., Goodwin, M. J., Cade, L., Zhang, overlap extension PCR and TA-cloning. PLoS One 8, e59801. F., Cifuentes, D., Curtin, S. J., Blackburn, J. S., Thibodeau- 73. Wang, L., Lin, J., Zhang, T., Xu, K., Ren, C., and Zhang, Z. Beganny, S., Qi, Y., Pierick, C. J., Hoffman, E., Maeder, M. L., (2013). Simultaneous screening and validation of effective zinc fin- Khayter, C., Reyon, D., Dobbs, D., Langenau, D. M., Stupar, R. M., ger nucleases in yeast. PLoS One 8, e64687. Giraldez, A. J., Voytas, D. F., Peterson, R. T., Yeh, J. R., and 74. Herrmann, F., Garriga-Canut, M., Baumstark, R., Fajardo-Sanchez, Joung, J. K. (2011). Selection-free zinc-finger-nuclease engineering E., Cotterell, J., Minoche, A., Himmelbauer, H., and Isalan, M. by context-dependent assembly (CoDA). Nat. Methods 8, 67–9. (2011). p53 Gene repair with zinc finger nucleases optimised by 57. Ramirez, C. L., Foley, J. E., Wright, D. A., Muller-Lerch, F., yeast 1-hybrid and validated by Solexa sequencing. PLoS ONE 6, Rahman, S. H., Cornu, T. I., Winfrey, R. J., Sander, J. D., Fu, F., e20913. Townsend, J. A., Cathomen, T., Voytas, D. F., and Joung, J. K. 75. Zhang, W., Guo, Y., Zhang, C., Ji, H., Meng, W., Wang, D., Li, X., (2008). Unexpected failure rates for modular assembly of engi- Mao, Q., and Xia, H. (2012). Rescue the failed half-ZFN by a neered zinc fingers. Nat. Methods 5, 374–5. sensitive mammalian cell-based luciferase reporter system. PLoS 58. Kim, S. and Kim, J. S. (2011). Targeted genome engineering via ONE 7, e45169. zinc finger nucleases. Plant Biotechnol. Rep. 5, 9–17. 76. Pattanayak, V., Ramirez, C. L., Joung, J. K., and Liu, D. R. (2011). 59. Bhakta, M. S., Henry, I. M., Ousterout, D. G., Das, K. T., Revealing off-target cleavage specificities of zinc-finger nucleases Lockwood, S. H., Meckler, J. F., Wallen, M. C., Zykovich, A., by in vitro selection. Nat. Methods 8, 765–70. Yu, Y., Leo, H., Xu, L., Gersbach, C. A., and Segal, D. J. (2013). 77. Moore, F. E., Reyon, D., Sander, J. D., Martinez, S. A., Blackburn, Highly active zinc-finger nucleases by extended modular assembly. J. S., Khayter, C., Ramirez, C. L., Joung, J. K., and Langenau, Genome Res. 23, 530–8. D. M. (2012). Improved somatic mutagenesis in zebrafish using 60. Xiao, A., Hu, Y. Y., Wang, W. Y., Yang, Z. P., Wang, Z. X., Huang, transcription activator-like effector nucleases (TALENs). PLoS P., Tong, X. J., Zhang, B., and Lin, S. (2011). [Progress in zinc ONE 7, e37877. finger nuclease engineering for targeted genome modification]. Yi 78. Szczepek, M., Brondani, V., Buchel, J., Serrano, L., Segal, D. J., Chuan 33, 665–83. and Cathomen, T. (2007). Structure-based redesign of the dimer- 61. Isalan, M., Klug, A., and Choo, Y. (2001). A rapid, generally appli- ization interface reduces the toxicity of zinc-finger nucleases. Nat. cable method to engineer zinc fingers illustrated by targeting the Biotechnol. 25, 786–93. HIV-1 promoter. Nat. Biotechnol. 19, 656–60. 79. Ramalingam, S., Kandavelou, K., Rajenderan, R., and 62. Gupta, A., Christensen, R. G., Rayla, A. L., Lakshmanan, A., Chandrasegaran, S. (2011). Creating designed zinc-finger nucleases Stormo, G. D., and Wolfe, S. A. (2012). An optimized two-finger with minimal cytotoxicity. J. Mol. Biol. 405, 630–41. archive for ZFN-mediated gene targeting.IP: 192.168.39.151Nat. Methods 9, 588–90. On: Sun, 2680. SepDoyon, 2021 Y., Vo, 11:43:06 T. D., Mendel, M. C., Greenberg, S. G., Wang, J., 63. Zhu, C., Gupta, A., Hall, V. L., Rayla,Copyright: A. L., Christensen, American R. G., ScientificXia, Publishers D. F., Miller, J. C., Urnov, F. D., Gregory, P. D., and Dake, B., Lakshmanan, A., Kuperwasser, C., Stormo,Delivered G. D., and by IngentaHolmes, M. C. (2011). Enhancing zinc-finger-nuclease activity with Wolfe, S. A. (2013). Using defined finger-finger interfaces as units improved obligate heterodimeric architectures. Nat. Methods 8, of assembly for constructing zinc-finger nucleases. Nucleic Acids 74–9. Res. 41, 2455–65. 81. Schierling, B., Dannemann, N., Gabsalilow, L., Wende, W., 64. Bhakta, M. S., Henry, I. M., Ousterout, D. G., Das, K. T., Cathomen, T., and Pingoud, A. (2012). A novel zinc-finger nuclease Lockwood, S. H., Meckler, J. F., Wallen, M. C., Zykovich, A., platform with a sequence-specific cleavage module. Nucleic Acids Yu, Y., Leo, H., Xu, L., Gersbach, C. A., and Segal, D. J. (2013). Res. 40, 2623–38. Highly active zinc-finger nucleases by extended modular assembly. 82. Ramirez, C. L., Certo, M. T., Mussolino, C., Goodwin, M. J., Genome Res. 23, 530–8. Cradick, T. J., McCaffrey, A. P., Cathomen, T., Scharenberg, A. M., 65. Guo, J. and Gaj, T. (2010). Barbas CF 3rd. Directed evolution of an and Joung, J. K. (2012). Engineered zinc finger nickases induce enhanced and highly efficient FokI cleavage domain for zinc finger homology-directed repair with reduced mutagenic effects. Nucleic nucleases. J. Mol. Biol. 400, 96–107. Acids Res. 40, 5560–8. 66. Sood, R., Carrington, B., Bishop, K., Jones, M., Rissone, A., 83. Orlando, S. J., Santiago, Y., DeKelver, R. C., Freyvert, Y., Candotti, F., Chandrasekharappa, S. C., and Liu, P. (2013). Effi- Boydston, E. A., Moehle, E. A., Choi, V. M., Gopalan, S. M., cient methods for targeted mutagenesis in zebrafish using zinc- Lou, J. F., Li, J., Miller, J. C., Holmes, M. C., Gregory, P. D., finger nucleases: data from targeting of nine genes using CompoZr Urnov, F. D., and Cost, G. J. (2010). Zinc-finger nuclease-driven or CoDA ZFNs. PLoS ONE 8, e57239. targeted integration into mammalian genomes using donors with 67. Moore, M., Klug, A., and Choo, Y. (2001). Improved DNA binding limited chromosomal homology. Nucleic Acids Res. 38, e152. specificity from polyzinc finger peptides by using strings of two- 84. Watanabe, M., Nakano, K., Matsunari, H., Matsuda, T., Maehara, finger units. Proc. Natl. Acad Sci. USA 98, 1437–41. M., Kanai, T., Kobayashi, M., Matsumura, Y., Sakai, R., 68. Wilson, K. A., McEwen, A. E., Pruett-Miller, S. M., Zhang, J., Kuramoto, M., Hayashida, G., Asano, Y., Takayanagi, S., Arai, Y., Kildebeck, E. J., and Porteus, M. H. (2013). Expanding the Reper- Umeyama, K., Nagaya, M., Hanazono, Y., and Nagashima, H. toire of Target Sites for Zinc Finger Nuclease-mediated Genome (2013). Generation of interleukin-2 receptor gamma gene knock- Modification. Mol. Ther. Nucleic Acids 2, e88. out pigs from somatic cells genetically modified by zinc finger 69. Anand, P., Schug, A., and Wenzel, W. (2013). Structure based nuclease-encoding mRNA. PLoS ONE 8, e76478. design of protein linkers for zinc finger nuclease. FEBS Lett. 587, 85. Gaj, T. and Gersbach, C. A. (2013). Barbas CF 3rd. ZFN, TALEN, 3231–5. and CRISPR/Cas-based methods for genome engineering. Trends 70. Yan, W., Smith, C., and Cheng, L. (2013). Expanded activity of Biotechnol. 31, 397–405. dimer nucleases by combining ZFN and TALEN for genome edit- 86. Li, H., Haurigot, V., Doyon, Y., Li, T., Wong, S. Y., Bhagwat, A. S., ing. Sci. Rep. 3, 2376. Malani, N., Anguela, X. M., Sharma, R., Ivanciu, L., Murphy, S. L., 71. Mineta, Y., Okamoto, T., Takenaka, K., Doi, N., Aoyama, Y., Finn, J. D., Khazi, F. R., Zhou, S., Paschon, D. E., Rebar, E. J., and Sera, T. (2009). Multiple-turnover cleavage of double-stranded Bushman, F. D., Gregory, P. D., Holmes, M. C., and High, K. A.

14 Gene Gene Edit. 1, 3–15, 2015 Tang et al. Advances in Zinc Finger Nuclease and Its Applications

(2011). In vivo genome editing restores haemostasis in a mouse 95. Kim,H.,Kim,M.S.,Wee,G.,Lee,C.I.,Kim,H.,andKim,J.S. model of haemophilia. Nature 475, 217–21. (2013). Magnetic separation and antibiotics selection enable enrich- 87. Ellis, B. L., Hirsch, M. L., Porter, S. N., Samulski, R. J., and ment of cells with ZFN/TALEN-induced mutations. PLoS One 8, Porteus, M. H. (2013). Zinc-finger nuclease-mediated gene cor- e56476. rection using single AAV vector transduction and enhancement 96. Ramakrishna, S., Kim, Y. H., and Kim, H. (2013). Stability of zinc by food and drug administration-approved drugs. Gene Ther. 20, finger nuclease protein is enhanced by the proteasome inhibitor 35–42. MG132. PLoS One 8, e54282. 88. Lombardo, A., Genovese, P., Beausejour, C. M., Colleoni, S., Lee, 97. Rahman, S. H., Bobis-Wozowicz, S., Chatterjee, D., Gellhaus, K., Y. L., Kim, K. A., Ando, D., Urnov, F. D., Galli, C., Gregory, P. D., Pars, K., Heilbronn, R., Jacobs, R., and Cathomen, T. (2013). The Holmes, M. C., and Naldini, L. (2007). Gene editing in human stem nontoxic cell cycle modulator indirubin augments transduction of cells using zinc finger nucleases and integrase-defective lentiviral adeno-associated viral vectors and zinc-finger nuclease-mediated vector delivery. Nat. Biotechnol. 25, 1298–306. gene targeting. Hum. Gene Ther. 24, 67–77. 89. Gaj, T., Guo, J., Kato, Y., and Sirk, S. J., (2012). Barbas CF 3rd. 98. Doyon, Y., Choi, V. M., Xia, D. F., Vo, T. D., Gregory, P. D., and Targeted gene knockout by direct delivery of zinc-finger nuclease Holmes, M. C. (2010). Transient cold shock enhances zinc-finger proteins. Nat. Methods 9, 805–7. nuclease-mediated gene disruption. Nat. Methods 7, 459–60. 90. Zhang, X., Li, H., Mao, Y., Li, Z., Wang, R., Guo, T., Jin, L., Song, 99. Pattanayak, V., Ramirez, C. L., Joung, J. K., and Liu, D. R. (2011). R., Xu, W., Zhou, N., Zhang, Y., Hu, R., Wang, X., Huang, H., Revealing off-target cleavage specificities of zinc-finger nucleases Lei, Z., Niu, G., Irwin, D. M., and Tan, H. (2013). An over by in vitro selection. Nat. Methods 8, 765–70. expression APP model for anti-Alzheimer disease drug screen- 100. Gabriel, R., Lombardo, A., Arens, A., Miller, J. C., Genovese, ing created by zinc finger nuclease technology. PLoS ONE 8, P., Kaeppel, C., Nowrouzi, A., Bartholomae, C. C., Wang, J., e75493. Friedman, G., Holmes, M. C., Gregory, P. D., Glimm, H., 91. Shen, B., Zhang, X., Du Y, Wang, J., Gong, J., Zhang, X., Schmidt, M., Naldini, L., and von Kalle, C. (2011). An unbi- Tate, P. H., Li, H., Huang, X., and Zhang, W. (2013). Efficient ased genome-wide analysis of zinc-finger nuclease specificity. Nat. knockin mouse generation by ssDNA oligonucleotides and zinc- Biotechnol. 29, 816–23. finger nuclease assisted homologous recombination in zygotes. 101. Sander, J. D., Ramirez, C. L., Linder, S. J., Pattanayak, V., Shoresh, PLoS ONE 8, e77696. N., Ku, M., Foden, J. A., Reyon, D., Bernstein, B. E., Liu, D. R., 92. Beumer, K. J., Trautman, J. K., Mukherjee, K., and Carroll, D. and Joung, J. K. (2013). In silico abstraction of zinc finger nuclease (2013). Donor DNA utilization during gene targeting with zinc- cleavage profiles reveals an expanded landscape of off-target sites. finger nucleases. G3 (Bethesda) 3, 657–64. Nucleic Acids Res. 41, e181. 93. Certo, M. T., Gwiazda, K. S., Kuhar, R., Sather, B., Curinga, G., 102. Sander, J. D., Maeder, M. L., Reyon, D., Voytas, D. F., Joung, J. K., Mandt, T., Brault, M., Lambert, A. R., Baxter, S. K., Jacoby, K., and Dobbs, D. (2010). ZiFiT (Zinc Finger Targeter): an updated Ryu, B. Y., Kiem, H. P., Gouble, A., Paques, F., Rawlings, D. J., zinc finger engineering tool. Nucleic Acids Res. 38, W462–8. and Scharenberg, A. M. (2012). CouplingIP: 192.168.39.151 endonucleases with On: DNA Sun,103. 26 SepMandell, 2021 J. G. 11:43:06 (2006). Barbas CF 3rd. Zinc Finger Tools: Cus- end-processing enzymes to drive gene disruption.Copyright:Nat. MethodsAmerican9, Scientifictom Publishers DNA-binding domains for transcription factors and nucleases. 973–5. Delivered by IngentaNucleic Acids Res. 34, W516–23. 94. Kim, H., Um, E., Cho, S. R., Jung, C., Kim, H., and Kim, J. S. 104. Cradick, T. J., Ambrosini, G., Iseli, C., Bucher, P., and McCaffrey, (2011). Surrogate reporters for enrichment of cells with nuclease- A. P. (2011). ZFN-site searches genomes for zinc finger nuclease induced mutations. Nat. Methods 8, 941–3. target sites and off-target sites. BMC Bioinformatics 12, 152.

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