Genome engineering with targetable nucleases.

Current technology enables the production of highly specific genome modifications with excellent efficiency and specificity. Key to this capability are targetable DNA cleavage reagents and cellular DNA repair pathways. The break made by these reagents can produce localized sequence changes through inaccurate nonhomologous end joining (NHEJ), often leading to gene inactivation. Alternatively, user-provided DNA can be used as a template for repair by homologous recombination (HR), leading to the introduction of desired sequence changes. This review describes three classes of targetable cleavage reagents: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas RNA-guided nucleases (RGNs). As a group, these reagents have been successfully used to modify genomic sequences in a wide variety of cells and organisms, including humans. This review discusses the properties, advantages, and limitations of each system, as well as the specific considerations required for their use in different biological systems.

[1]  Dana Carroll,et al.  Reply to “Genome editing with modularly assembled zinc-finger nucleases” , 2010, Nature Methods.

[2]  Susan R. Wente,et al.  Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system , 2013, Proceedings of the National Academy of Sciences.

[3]  Feng Zhang,et al.  Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA , 2014, Cell.

[4]  Chris P Ponting,et al.  Highly Efficient Targeted Mutagenesis of Drosophila with the CRISPR/Cas9 System , 2014, Cell reports.

[5]  H. Heuer,et al.  Breaking the DNA-binding code of Ralstonia solanacearum TAL effectors provides new possibilities to generate plant resistance genes against bacterial wilt disease. , 2013, The New phytologist.

[6]  P. Gregory,et al.  Genomic Editing of the HIV-1 Coreceptor CCR5 in Adult Hematopoietic Stem and Progenitor Cells Using Zinc Finger Nucleases , 2013, Molecular therapy : the journal of the American Society of Gene Therapy.

[7]  T. Cathomen,et al.  Adding fingers to an engineered zinc finger nuclease can reduce activity. , 2011, Biochemistry.

[8]  Pilar Blancafort,et al.  Designing Transcription Factor Architectures for Drug Discovery , 2004, Molecular Pharmacology.

[9]  Ryo Takeuchi,et al.  Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease , 2009, Proceedings of the National Academy of Sciences.

[10]  S. Boulton,et al.  The choice in meiosis – defining the factors that influence crossover or non-crossover formation , 2011, Journal of Cell Science.

[11]  D. Segal,et al.  Genome engineering at the dawn of the golden age. , 2013, Annual review of genomics and human genetics.

[12]  J. Keith Joung,et al.  Targeted gene disruption in somatic zebrafish cells using engineered TALENs , 2011, Nature Biotechnology.

[13]  Mario R. Capecchi,et al.  Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century , 2005, Nature Reviews Genetics.

[14]  D. Voytas,et al.  Comparing Zinc Finger Nucleases and Transcription Activator-Like Effector Nucleases for Gene Targeting in Drosophila , 2013, G3: Genes, Genomes, Genetics.

[15]  David J Segal,et al.  Restricted spacer tolerance of a zinc finger nuclease with a six amino acid linker. , 2009, Bioorganic & medicinal chemistry letters.

[16]  Daniel F. Voytas,et al.  Transcription Activator-Like Effector Nucleases Enable Efficient Plant Genome Engineering1[W][OA] , 2012, Plant Physiology.

[17]  Dana Carroll,et al.  Targeted mutagenesis using zinc-finger nucleases in Arabidopsis. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[18]  R. Jiao,et al.  Efficient and specific modifications of the Drosophila genome by means of an easy TALEN strategy. , 2012, Journal of genetics and genomics = Yi chuan xue bao.

[19]  G. Stormo,et al.  Program in Gene Function and Expression Publications and Presentations Program in Gene Function and Expression 1-8-2013 Using defined finger-finger interfaces as units of assembly for constructing zinc-finger nucleases , 2014 .

[20]  D. Carroll.  Staying on target with CRISPR-Cas , 2013, Nature Biotechnology.

[21]  T. Shibata,et al.  Targeted mutagenesis in the sea urchin embryo using zinc‐finger nucleases , 2010, Genes to cells : devoted to molecular & cellular mechanisms.

[22]  P. Glazer,et al.  Repair of DNA lesions associated with triplex‐forming oligonucleotides , 2009, Molecular carcinogenesis.

[23]  W. Wurst,et al.  Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases , 2010, Proceedings of the National Academy of Sciences.

[24]  J. Joung,et al.  Profiling the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a rapid cell-based method , 2007, Nucleic acids research.

[25]  George M. Church,et al.  Multiplex and homologous recombination–mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9 , 2013, Nature Biotechnology.

[26]  C. Pabo,et al.  Design and selection of novel Cys2His2 zinc finger proteins. , 2001, Annual review of biochemistry.

[27]  Yolanda Santiago,et al.  Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases , 2011, Proceedings of the National Academy of Sciences.

[28]  J. Keith Joung,et al.  Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs , 2012, Nucleic acids research.

[29]  S. Rafii,et al.  Distinct Factors Control Histone Variant H3.3 Localization at Specific Genomic Regions , 2010, Cell.

[30]  Jeremy M. Berg,et al.  A consensus zinc finger peptide: design, high-affinity metal binding, a pH-dependent structure, and a His to Cys sequence variant , 1991 .

[31]  Y. Rong,et al.  Gene targeting by homologous recombination in Drosophila. , 2000, Science.

[32]  Bo Zhang,et al.  EENdb: a database and knowledge base of ZFNs and TALENs for endonuclease engineering , 2012, Nucleic Acids Res..

[33]  David Baltimore,et al.  Chimeric Nucleases Stimulate Gene Targeting in Human Cells , 2003, Science.

[34]  George M. Church,et al.  Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems , 2013, Nucleic acids research.

[35]  Guodong Huang,et al.  Site-specific gene targeting using transcription activator-like effector (TALE)-based nuclease in Brassica oleracea. , 2013, Journal of integrative plant biology.

[36]  Daniel F. Voytas,et al.  Targeting G with TAL Effectors: A Comparison of Activities of TALENs Constructed with NN and NK Repeat Variable Di-Residues , 2012, PloS one.

[37]  Luke A. Gilbert,et al.  Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression , 2013, Cell.

[38]  S. Boulton,et al.  Playing the end game: DNA double-strand break repair pathway choice. , 2012, Molecular cell.

[39]  Shuo Lin,et al.  TALEN-mediated precise genome modification by homologous recombination in zebrafish , 2013, Nature Methods.

[40]  A. Pingoud,et al.  Site- and strand-specific nicking of DNA by fusion proteins derived from MutH and I-SceI or TALE repeats , 2013, Nucleic acids research.

[41]  Bob Goldstein,et al.  Engineering the Caenorhabditis elegans Genome Using Cas9-Triggered Homologous Recombination , 2013, Nature Methods.

[42]  Charles A Gersbach,et al.  Reading Frame Correction by Targeted Genome Editing Restores Dystrophin Expression in Cells From Duchenne Muscular Dystrophy Patients , 2013, Molecular therapy : the journal of the American Society of Gene Therapy.

[43]  Ning Li,et al.  Highly efficient modification of beta-lactoglobulin (BLG) gene via zinc-finger nucleases in cattle , 2011, Cell Research.

[44]  Carlos F. Barbas,et al.  A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells , 2013, Nucleic acids research.

[45]  J. Haber,et al.  Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae. , 1989, Genetics.

[46]  Gang Bao,et al.  CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity , 2013, Nucleic acids research.

[47]  Toni Cathomen,et al.  Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases , 2007, Nature Biotechnology.

[48]  D. Carroll,et al.  Genetic Analysis of Zinc-Finger Nuclease-Induced Gene Targeting in Drosophila , 2009, Genetics.

[49]  S. Wolfe,et al.  Targeted chromosomal deletions and inversions in zebrafish , 2013, Genome research.

[50]  M. Noyes,et al.  Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases , 2008, Nature Biotechnology.

[51]  Dana Carroll,et al.  Stimulation of Homologous Recombination through Targeted Cleavage by Chimeric Nucleases , 2001, Molecular and Cellular Biology.

[52]  I. Katic,et al.  Targeted Heritable Mutation and Gene Conversion by Cas9-CRISPR in Caenorhabditis elegans , 2013, Genetics.

[53]  P. Dervan,et al.  Programmable oligomers for minor groove DNA recognition. , 2006, Journal of the American Chemical Society.

[54]  J. Keith Joung,et al.  High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells , 2013, Nature Biotechnology.

[55]  Carl O. Pabo,et al.  A General Strategy for Selecting High-Affinity Zinc Finger Proteins for Diverse DNA Target Sites , 1997, Science.

[56]  J. Keith Joung,et al.  Improving CRISPR-Cas nuclease specificity using truncated guide RNAs , 2014, Nature Biotechnology.

[57]  P. Sternberg,et al.  Transgene-Free Genome Editing in Caenorhabditis elegans Using CRISPR-Cas , 2013, Genetics.

[58]  Daniel F. Voytas,et al.  Efficient TALEN-mediated gene knockout in livestock , 2012, Proceedings of the National Academy of Sciences.

[59]  A Klug,et al.  Toward a code for the interactions of zinc fingers with DNA: selection of randomized fingers displayed on phage. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[60]  Ying Lin,et al.  Highly Efficient and Specific Genome Editing in Silkworm Using Custom TALENs , 2012, PloS one.

[61]  Pilar Blancafort,et al.  Development of Zinc Finger Domains for Recognition of the 5′-CNN-3′ Family DNA Sequences and Their Use in the Construction of Artificial Transcription Factors* , 2005, Journal of Biological Chemistry.

[62]  R. W. Davis,et al.  Replacement of chromosome segments with altered DNA sequences constructed in vitro. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[63]  Chang Tong,et al.  Rapid and cost-effective gene targeting in rat embryonic stem cells by TALENs. , 2012, Journal of genetics and genomics = Yi chuan xue bao.

[64]  Feng Zhang,et al.  Selection-Free Zinc-Finger Nuclease Engineering by Context-Dependent Assembly (CoDA) , 2010, Nature Methods.

[65]  B. Dujon,et al.  Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae , 1995, Molecular and cellular biology.

[66]  Bing Yang,et al.  Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice , 2013, Nucleic acids research.

[67]  Philip Bradley,et al.  The Crystal Structure of TAL Effector PthXo1 Bound to Its DNA Target , 2012, Science.

[68]  Pilar Blancafort,et al.  Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA-binding proteins. , 2003, Biochemistry.

[69]  Eli J. Fine,et al.  DNA targeting specificity of RNA-guided Cas9 nucleases , 2013, Nature Biotechnology.

[70]  T. Serikawa,et al.  Efficient gene targeting by TAL effector nucleases coinjected with exonucleases in zygotes , 2013, Scientific Reports.

[71]  P. Gregory,et al.  Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery , 2007, Nature Biotechnology.

[72]  Adam James Waite,et al.  An improved zinc-finger nuclease architecture for highly specific genome editing , 2007, Nature Biotechnology.

[73]  Daniel F. Voytas,et al.  Compact designer TALENs for efficient genome engineering , 2013, Nature Communications.

[74]  Luke A. Gilbert,et al.  CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes , 2013, Cell.

[75]  Melissa M. Harrison,et al.  Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease , 2013, Genetics.

[76]  Jeffry D Sander,et al.  FLAsH assembly of TALeNs for high-throughput genome editing , 2022 .

[77]  J. Keith Joung,et al.  FLASH Assembly of TALENs Enables High-Throughput Genome Editing , 2012, Nature Biotechnology.

[78]  D J Segal,et al.  Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[79]  Qi Zhou,et al.  Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems , 2013, Nature Biotechnology.

[80]  Yongxiang Zhao,et al.  Heritable gene targeting in the mouse and rat using a CRISPR-Cas system , 2013, Nature Biotechnology.

[81]  J. Gall,et al.  Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases , 2008, Proceedings of the National Academy of Sciences.

[82]  E. Rebar,et al.  Genome editing with engineered zinc finger nucleases , 2010, Nature Reviews Genetics.

[83]  J. Keith Joung,et al.  TALENs: a widely applicable technology for targeted genome editing , 2012, Nature Reviews Molecular Cell Biology.

[84]  R. Guyomard,et al.  An Immune-Related Gene Evolved into the Master Sex-Determining Gene in Rainbow Trout, Oncorhynchus mykiss , 2012, Current Biology.

[85]  Dana Carroll,et al.  Design, construction and in vitro testing of zinc finger nucleases , 2006, Nature Protocols.

[86]  M. McVey,et al.  Synthesis-dependent microhomology-mediated end joining accounts for multiple types of repair junctions , 2010, Nucleic acids research.

[87]  Neville E. Sanjana,et al.  Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells , 2014, Science.

[88]  Mario R. Capecchi,et al.  Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes , 1988, Nature.

[89]  Rudolf Jaenisch,et al.  Toward eliminating HLA class I expression to generate universal cells from allogeneic donors. , 2013, Blood.

[90]  D. F. Carlson,et al.  Efficient nonmeiotic allele introgression in livestock using custom endonucleases , 2013, Proceedings of the National Academy of Sciences.

[91]  Feng Zhang,et al.  High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases , 2010, Proceedings of the National Academy of Sciences.

[92]  Prashant Mali,et al.  Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. , 2009, Cell stem cell.

[93]  A Klug,et al.  Selection of DNA binding sites for zinc fingers using rationally randomized DNA reveals coded interactions. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[94]  I. Thorey,et al.  Efficient Immunoglobulin Gene Disruption and Targeted Replacement in Rabbit Using Zinc Finger Nucleases , 2011, PloS one.

[95]  Jingyun Li,et al.  Heritable Targeted Inactivation of Myostatin Gene in Yellow Catfish (Pelteobagrus fulvidraco) Using Engineered Zinc Finger Nucleases , 2011, PloS one.

[96]  T. Lahaye,et al.  Assembly of custom TALE-type DNA binding domains by modular cloning , 2011, Nucleic acids research.

[97]  Feng Zhang,et al.  Targeted Mutagenesis of Duplicated Genes in Soybean with Zinc-Finger Nucleases1[W][OA] , 2011, Plant Physiology.

[98]  Jeffrey C. Miller,et al.  Highly efficient endogenous human gene correction using designed zinc-finger nucleases , 2005, Nature.

[99]  Sylvestre Marillonnet,et al.  Assembly of Designer TAL Effectors by Golden Gate Cloning , 2011, PloS one.

[100]  Jennifer A. Doudna,et al.  Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation , 2014, Science.

[101]  Ryan M. O’Connell,et al.  Targeting Human MicroRNA Genes Using Engineered Tal-Effector Nucleases (TALENs) , 2013, PloS one.

[102]  Xiaojun Zhu,et al.  Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos , 2013, Cell Research.

[103]  Jens Boch,et al.  TAL effector RVD specificities and efficiencies , 2012, Nature Biotechnology.

[104]  George M. Church,et al.  Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers , 2012, Nucleic acids research.

[105]  Mario R. Capecchi,et al.  High frequency targeting of genes to specific sites in the mammalian genome , 1986, Cell.

[106]  Li Wang,et al.  Erratum: Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting (Nucleic Acids Research (2011) 39 (e82) DOI: 10.1093/nar/gkr218) , 2011 .

[107]  Jeffry D. Sander,et al.  Heritable and Precise Zebrafish Genome Editing Using a CRISPR-Cas System , 2013, PloS one.

[108]  A. McCaffrey,et al.  Zinc-finger nucleases as a novel therapeutic strategy for targeting hepatitis B virus DNAs. , 2010, Molecular therapy : the journal of the American Society of Gene Therapy.

[109]  Eunji Kim,et al.  Targeted chromosomal deletions in human cells using zinc finger nucleases. , 2010, Genome research.

[110]  Thomas Gaj,et al.  Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases. , 2010, Journal of molecular biology.

[111]  A Klug,et al.  Repetitive zinc‐binding domains in the protein transcription factor IIIA from Xenopus oocytes. , 1985, The EMBO journal.

[112]  J. Berg,et al.  Proposed structure for the zinc-binding domains from transcription factor IIIA and related proteins. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[113]  Elo Leung,et al.  Targeted Genome Editing Across Species Using ZFNs and TALENs , 2011, Science.

[114]  Xia Li,et al.  High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases , 2013, Nucleic acids research.

[115]  R. Kucherlapati,et al.  Insertion of DNA sequences into the human chromosomal β-globin locus by homologous recombination , 1985, Nature.

[116]  Elo Leung,et al.  A TALE nuclease architecture for efficient genome editing , 2011, Nature Biotechnology.

[117]  Seung Woo Cho,et al.  Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease , 2013, Nature Biotechnology.

[118]  Hojun Li,et al.  In vivo genome editing restores hemostasis in a mouse model of hemophilia , 2011, Nature.

[119]  J. Haber.  Mating-Type Genes and MAT Switching in Saccharomyces cerevisiae , 2012, Genetics.

[120]  D. F. Carlson,et al.  Precision editing of large animal genomes. , 2012, Advances in genetics.

[121]  David R. Liu,et al.  Revealing Off-Target Cleavage Specificities of Zinc Finger Nucleases by In Vitro Selection , 2011, Nature Methods.

[122]  Matthew J. Moscou,et al.  A Simple Cipher Governs DNA Recognition by TAL Effectors , 2009, Science.

[123]  D J Segal,et al.  Development of Zinc Finger Domains for Recognition of the 5′-ANN-3′ Family of DNA Sequences and Their Use in the Construction of Artificial Transcription Factors* , 2001, The Journal of Biological Chemistry.

[124]  T. Hocking,et al.  Heritable Targeted Gene Disruption in Zebrafish Using Designed Zinc Finger Nucleases , 2008, Nature Biotechnology.

[125]  E. Marois,et al.  Targeted Mutagenesis in the Malaria Mosquito Using TALE Nucleases , 2013, PloS one.

[126]  Y. Doyon,et al.  Precise genome modification in the crop species Zea mays using zinc-finger nucleases , 2009, Nature.

[127]  R. Tuli,et al.  RNA-Guided Genome Editing for Target Gene Mutations in Wheat , 2013, G3: Genes, Genomes, Genetics.

[128]  D. Carroll,et al.  Donor DNA Utilization During Gene Targeting with Zinc-Finger Nucleases , 2013, G3: Genes, Genomes, Genetics.

[129]  John A. Calarco,et al.  Heritable Custom Genomic Modifications in Caenorhabditis elegans via a CRISPR–Cas9 System , 2013, Genetics.

[130]  E. Amaya,et al.  Highly efficient bi-allelic mutation rates using TALENs in Xenopus tropicalis , 2012, Biology Open.

[131]  Fyodor Urnov,et al.  Chromosomal translocations induced at specified loci in human stem cells , 2009, Proceedings of the National Academy of Sciences.

[132]  Ruhong Zhou,et al.  Comprehensive Interrogation of Natural TALE DNA Binding Modules and Transcriptional Repressor Domains , 2012, Nature Communications.

[133]  Tal Pupko,et al.  Native homing endonucleases can target conserved genes in humans and in animal models , 2011, Nucleic acids research.

[134]  Gary D. Stormo,et al.  An optimized two-finger archive for ZFN-mediated gene targeting , 2012, Nature Methods.

[135]  George M. Church,et al.  Heritable genome editing in C. elegans via a CRISPR-Cas9 system , 2013, Nature Methods.

[136]  Toni Cathomen,et al.  Expanding or restricting the target site repertoire of zinc-finger nucleases: the inter-domain linker as a major determinant of target site selectivity. , 2009, Molecular therapy : the journal of the American Society of Gene Therapy.

[137]  Jeffrey C. Miller,et al.  An unbiased genome-wide analysis of zinc-finger nuclease specificity , 2011, Nature Biotechnology.

[138]  Detlef Weigel,et al.  Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease , 2013, Nature Biotechnology.

[139]  R. Rothstein.  One-step gene disruption in yeast. , 1983, Methods in enzymology.

[140]  Laurent Poirot,et al.  Characterization of three loci for homologous gene targeting and transgene expression , 2013, Biotechnology and bioengineering.

[141]  Elo Leung,et al.  Knockout rats generated by embryo microinjection of TALENs , 2011, Nature Biotechnology.

[142]  Peng Li,et al.  Generation of RAG 1- and 2-deficient rabbits by embryo microinjection of TALENs , 2013, Cell Research.

[143]  S Chandrasegaran,et al.  Functional domains in Fok I restriction endonuclease. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[144]  H. Sezutsu,et al.  Targeted mutagenesis in the silkworm Bombyx mori using zinc finger nuclease mRNA injection. , 2010, Insect biochemistry and molecular biology.

[145]  George Church,et al.  Optimization of scarless human stem cell genome editing , 2013, Nucleic acids research.

[146]  S Chandrasegaran,et al.  Chimeric restriction endonuclease. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[147]  Biao Lu,et al.  A do-it-yourself protocol for simple transcription activator-like effector assembly , 2013, Biological Procedures Online.

[148]  Fayza Daboussi,et al.  Overcoming Transcription Activator-like Effector (TALE) DNA Binding Domain Sensitivity to Cytosine Methylation*♦ , 2012, The Journal of Biological Chemistry.

[149]  C. Case,et al.  Validated Zinc Finger Protein Designs for All 16 GNN DNA Triplet Targets* , 2002, The Journal of Biological Chemistry.

[150]  Jin-Soo Kim,et al.  Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases , 2014, Genome research.

[151]  H. Ariga,et al.  Efficient Targeted Mutagenesis in Medaka Using Custom-Designed Transcription Activator-Like Effector Nucleases , 2013, Genetics.

[152]  M. Bibikova,et al.  Efficient Gene Targeting in Drosophila With Zinc-Finger Nucleases , 2006, Genetics.

[153]  S Chandrasegaran,et al.  Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains. , 2000, Nucleic acids research.

[154]  Eunji Kim,et al.  Precision genome engineering with programmable DNA-nicking enzymes , 2012, Genome research.

[155]  Kevin Kim,et al.  A TALEN genome-editing system for generating human stem cell-based disease models. , 2013, Cell stem cell.

[156]  Dana Carroll,et al.  Heritable Gene Knockout in Caenorhabditis elegans by Direct Injection of Cas9–sgRNA Ribonucleoproteins , 2013, Genetics.

[157]  Nieng Yan,et al.  Structural Basis for Sequence-Specific Recognition of DNA by TAL Effectors , 2012, Science.

[158]  A Klug,et al.  Improved DNA binding specificity from polyzinc finger peptides by using strings of two-finger units. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[159]  M. Llinás,et al.  Site-specific genome editing in Plasmodium falciparum using engineered zinc-finger nucleases , 2012, Nature Methods.

[160]  A. Bradley,et al.  Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells , 2011, Nature.

[161]  B. Kleinstiver,et al.  Monomeric site-specific nucleases for genome editing , 2012, Proceedings of the National Academy of Sciences.

[162]  C. Barbas,et al.  Targeted gene knockout by direct delivery of ZFN proteins , 2012, Nature Methods.

[163]  Ryo Takeuchi,et al.  Tapping natural reservoirs of homing endonucleases for targeted gene modification , 2011, Proceedings of the National Academy of Sciences.

[164]  Neville E Sanjana,et al.  A transcription activator-like effector toolbox for genome engineering , 2012, Nature Protocols.

[165]  Jeffry D. Sander,et al.  Engineering Designer Transcription Activator‐‐Like Effector Nucleases (TALENs) by REAL or REAL‐Fast Assembly , 2012, Current protocols in molecular biology.

[166]  Chad A. Cowan,et al.  Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. , 2013, Cell stem cell.

[167]  A. James,et al.  orco mutant mosquitoes lose strong preference for humans and are not repelled by volatile DEET , 2013, Nature.

[168]  Moon-Soo Kim,et al.  Quantitative analysis of TALE–DNA interactions suggests polarity effects , 2013, Nucleic acids research.

[169]  R. Jaenisch,et al.  Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases , 2009, Nature Biotechnology.

[170]  Ignacio Anegon,et al.  Knockout Rats via Embryo Microinjection of Zinc-Finger Nucleases , 2009, Science.

[171]  E. Lander,et al.  Genetic Screens in Human Cells Using the CRISPR-Cas9 System , 2013, Science.

[172]  L. Liaw,et al.  Targeted Genome Modification in Mice Using Zinc-Finger Nucleases , 2010, Genetics.

[173]  Jeffry D. Sander,et al.  Efficient In Vivo Genome Editing Using RNA-Guided Nucleases , 2013, Nature Biotechnology.

[174]  Takashi Yamamoto,et al.  Targeted mutagenesis in sea urchin embryos using TALENs , 2014, Development, growth & differentiation.

[175]  D. Lu,et al.  Zinc-finger-nucleases mediate specific and efficient excision of HIV-1 proviral DNA from infected and latently infected human T cells , 2013, Nucleic acids research.

[176]  A. Fire,et al.  Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans , 1998, Nature.

[177]  Carlos F. Barbas,et al.  Chimeric TALE recombinases with programmable DNA sequence specificity , 2012, Nucleic acids research.

[178]  H. Sezutsu,et al.  Efficient disruption of endogenous Bombyx gene by TAL effector nucleases. , 2013, Insect biochemistry and molecular biology.

[179]  Sheng Huang,et al.  TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain , 2010, Nucleic Acids Res..

[180]  Igor Antoshechkin,et al.  A large-scale in vivo analysis reveals that TALENs are significantly more mutagenic than ZFNs generated using context-dependent assembly , 2013, Nucleic acids research.

[181]  TAL effector nucleases induce mutations at a pre-selected location in the genome of primary barley transformants , 2013, Plant Molecular Biology.

[182]  C. Gersbach,et al.  Highly active zinc-finger nucleases by extended modular assembly , 2013, Genome research.

[183]  J. Orange,et al.  Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases , 2008, Nature Biotechnology.

[184]  G. Church,et al.  CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering , 2013, Nature Biotechnology.

[185]  D. Carroll.  Genome Engineering With Zinc-Finger Nucleases , 2011, Genetics.

[186]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[187]  J. Bitinaite,et al.  FokI dimerization is required for DNA cleavage. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[188]  Margherita Neri,et al.  Site-specific integration and tailoring of cassette design for sustainable gene transfer , 2011, Nature Methods.

[189]  Bo Zhang,et al.  Highly Efficient Genome Modifications Mediated by CRISPR/Cas9 in Drosophila , 2013, Genetics.

[190]  David R. Liu,et al.  High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity , 2013, Nature Biotechnology.

[191]  Feng Zhang,et al.  CRISPR-assisted editing of bacterial genomes , 2013, Nature Biotechnology.

[192]  Tetsushi Sakuma,et al.  Efficient TALEN construction and evaluation methods for human cell and animal applications , 2013, Genes to cells : devoted to molecular & cellular mechanisms.

[193]  An Xiao,et al.  Heritable gene targeting in zebrafish using customized TALENs , 2011, Nature Biotechnology.

[194]  N. Pavletich,et al.  Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A , 1991, Science.

[195]  Stephen C. Ekker,et al.  Mojo Hand, a TALEN design tool for genome editing applications , 2013, BMC Bioinformatics.

[196]  A. Klug,et al.  Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases , 2008, Proceedings of the National Academy of Sciences.

[197]  J. Keith Joung,et al.  Improved Somatic Mutagenesis in Zebrafish Using Transcription Activator-Like Effector Nucleases (TALENs) , 2012, PloS one.

[198]  X. Cui,et al.  Targeted integration in rat and mouse embryos with zinc-finger nucleases , 2011, Nature Biotechnology.

[199]  Dana Carroll,et al.  Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. , 2002, Genetics.

[200]  Yongping Huang,et al.  The CRISPR/Cas System mediates efficient genome engineering in Bombyx mori , 2013, Cell Research.

[201]  Colby G Starker,et al.  In vivo Genome Editing Using High Efficiency TALENs , 2012, Nature.

[202]  J R Desjarlais,et al.  Toward rules relating zinc finger protein sequences and DNA binding site preferences. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[203]  B. González,et al.  Modular system for the construction of zinc-finger libraries and proteins , 2010, Nature Protocols.

[204]  A. Bogdanove,et al.  TAL Effectors: Customizable Proteins for DNA Targeting , 2011, Science.

[205]  Kenichi Nakajima,et al.  Colored Fluorescent Silk Made by Transgenic Silkworms , 2013 .

[206]  Jiyeon Kweon,et al.  TALENs and ZFNs are associated with different mutation signatures , 2013, Nature Methods.

[207]  Gabriela Plesa,et al.  Efficient clinical scale gene modification via zinc finger nuclease-targeted disruption of the HIV co-receptor CCR5. , 2013, Human gene therapy.

[208]  Michelle A. E. Anderson,et al.  TALEN-Based Gene Disruption in the Dengue Vector Aedes aegypti , 2013, PloS one.

[209]  J. Doudna,et al.  A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity , 2012, Science.

[210]  Jac A. Nickoloff,et al.  Gene Conversion Tracts from Double-Strand Break Repair in Mammalian Cells , 1998, Molecular and Cellular Biology.

[211]  David J. Rawlings,et al.  Tracking genome engineering outcome at individual DNA breakpoints , 2011, Nature Methods.

[212]  Daniel F. Voytas,et al.  Simple Methods for Generating and Detecting Locus-Specific Mutations Induced with TALENs in the Zebrafish Genome , 2012, PLoS genetics.

[213]  B. Stoddard,et al.  Homing endonucleases: from microbial genetic invaders to reagents for targeted DNA modification. , 2011, Structure.

[214]  Dana Carroll,et al.  Enhancing Gene Targeting with Designed Zinc Finger Nucleases , 2003, Science.

[215]  An integrated chip for the high-throughput synthesis of transcription activator-like effectors. , 2012, Angewandte Chemie.

[216]  Lihua Julie Zhu,et al.  Zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases , 2010, Nucleic Acids Res..

[217]  P. Rouet,et al.  Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. , 1994, Molecular and cellular biology.

[218]  J. Durocher,et al.  Mutation detection using Surveyor nuclease. , 2004, BioTechniques.

[219]  Ronnie J Winfrey,et al.  Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification. , 2008, Molecular cell.

[220]  Erin L. Doyle,et al.  Targeting DNA Double-Strand Breaks with TAL Effector Nucleases , 2010, Genetics.

[221]  B. Dujon,et al.  Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus. , 1992, Genetics.

[222]  M. Tomishima,et al.  Cancer translocations in human cells induced by zinc finger and TALE nucleases , 2013, Genome research.

[223]  K. Golic,et al.  Ends-out, or replacement, gene targeting in Drosophila , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[224]  Dana Carroll,et al.  A CRISPR approach to gene targeting. , 2012, Molecular therapy : the journal of the American Society of Gene Therapy.

[225]  Ronnie J Winfrey,et al.  High frequency modification of plant genes using engineered zinc finger nucleases , 2009, Nature.

[226]  B. Torbett,et al.  Zinc-finger nuclease editing of human cxcr4 promotes HIV-1 CD4(+) T cell resistance and enrichment. , 2012, Molecular therapy : the journal of the American Society of Gene Therapy.

[227]  Jun Li,et al.  Targeted genome modification of crop plants using a CRISPR-Cas system , 2013, Nature Biotechnology.

[228]  Y. Doyon,et al.  Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme , 2012, Genome research.

[229]  Takashi Yamamoto,et al.  Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc‐finger nucleases , 2012, Development, growth & differentiation.

[230]  James E. DiCarlo,et al.  RNA-Guided Human Genome Engineering via Cas9 , 2013, Science.

[231]  Nicholas E. Propson,et al.  Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis , 2013, Proceedings of the National Academy of Sciences.

[232]  Jun Zhang,et al.  Generation of gene-modified mice via Cas9/RNA-mediated gene targeting , 2013, Cell Research.

[233]  Rotem Sorek,et al.  CRISPR-mediated adaptive immune systems in bacteria and archaea. , 2013, Annual review of biochemistry.

[234]  T. Cathomen,et al.  Inactivation of Hepatitis B Virus Replication in Cultured Cells and In Vivo with Engineered Transcription Activator-Like Effector Nucleases , 2013, Molecular therapy : the journal of the American Society of Gene Therapy.

[235]  Toni Cathomen,et al.  Unexpected failure rates for modular assembly of engineered zinc fingers , 2008, Nature Methods.

[236]  Shondra M. Pruett-Miller,et al.  Gene correction by homologous recombination with zinc finger nucleases in primary cells from a mouse model of a generic recessive genetic disease. , 2010, Molecular therapy : the journal of the American Society of Gene Therapy.

[237]  Tetsushi Sakuma,et al.  Tissue-specific and ubiquitous gene knockouts by TALEN electroporation provide new approaches to investigating gene function in Ciona , 2014, Development.

[238]  Mike Boxem,et al.  CRISPR/Cas9-Targeted Mutagenesis in Caenorhabditis elegans , 2013, Genetics.

[239]  Jeffrey C. Miller,et al.  Generation of a triple‐gene knockout mammalian cell line using engineered zinc‐finger nucleases , 2010, Biotechnology and bioengineering.

[240]  Erin L. Doyle,et al.  Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting , 2011, Nucleic acids research.

[241]  D. Voytas,et al.  Comparing ZFNs and TALENs for Gene Targeting in Drosophila , 2013 .

[242]  Eunji Kim,et al.  Targeted chromosomal duplications and inversions in the human genome using zinc finger nucleases. , 2012, Genome research.

[243]  Morgan L. Maeder,et al.  In Situ Genetic Correction of the Sickle Cell Anemia Mutation in Human Induced Pluripotent Stem Cells Using Engineered Zinc Finger Nucleases , 2011, Stem cells.

[244]  A Klug,et al.  Design of polyzinc finger peptides with structured linkers. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[245]  Thuy D Vo,et al.  Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures , 2011, Nature Methods.

[246]  Yoshio Koyanagi,et al.  Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus , 2013, Scientific Reports.

[247]  Lei Zhang,et al.  Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome. , 2010, Genome research.

[248]  Claudio Mussolino,et al.  A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity , 2011, Nucleic acids research.

[249]  I. Dawid,et al.  Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs) , 2012, Proceedings of the National Academy of Sciences.

[250]  Claudio Mussolino,et al.  Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects , 2012, Nucleic acids research.

[251]  Xiaohui Xie,et al.  Biallelic genome modification in F0 Xenopus tropicalis embryos using the CRISPR/Cas system , 2013, Genesis.

[252]  S. Latt.  Sister chromatid exchange formation. , 1981, Annual review of genetics.

[253]  B. Zeitler,et al.  Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases , 2011, Proceedings of the National Academy of Sciences.

[254]  J R Desjarlais,et al.  Use of a zinc-finger consensus sequence framework and specificity rules to design specific DNA binding proteins. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[255]  Tobias Schmidt,et al.  A ligation-independent cloning technique for high-throughput assembly of transcription activator–like effector genes , 2012, Nature Biotechnology.

[256]  Jens Boch,et al.  Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors , 2009, Science.

[257]  Marilyn Fisher,et al.  Simple and efficient CRISPR/Cas9‐mediated targeted mutagenesis in Xenopus tropicalis , 2013, Genesis.

[258]  Shondra M Pruett-Miller,et al.  High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases , 2011, Nature Methods.

[259]  R. Paro,et al.  An efficient strategy for TALEN-mediated genome engineering in Drosophila , 2013, Nucleic acids research.

[260]  Thuy D. Vo,et al.  Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells , 2011, Nature Cell Biology.

[261]  Wolfgang Wurst,et al.  Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides , 2013, Proceedings of the National Academy of Sciences.

[262]  S Chandrasegaran,et al.  Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[263]  Seung Woo Cho,et al.  Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly. , 2009, Genome research.

[264]  S. Diederichs,et al.  Noncoding RNA gene silencing through genomic integration of RNA destabilizing elements using zinc finger nucleases. , 2011, Genome research.

[265]  Jennifer Doudna,et al.  RNA-programmed genome editing in human cells , 2013, eLife.

[266]  C. Barbas,et al.  ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. , 2013, Trends in biotechnology.

[267]  Y. Kamei,et al.  Targeted disruption of exogenous EGFP gene in medaka using zinc‐finger nucleases , 2012, Development, growth & differentiation.

[268]  M. Spalding,et al.  High-efficiency TALEN-based gene editing produces disease-resistant rice , 2012, Nature Biotechnology.

[269]  E. Shklarman,et al.  Nontransgenic Genome Modification in Plant Cells1[W][OA] , 2010, Plant Physiology.

[270]  Honglin Liu,et al.  Targeted editing of goat genome with modular-assembly zinc finger nucleases based on activity prediction by computational molecular modeling , 2013, Molecular Biology Reports.

[271]  S. Wolfe,et al.  Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases , 2013, Genome research.

[272]  K. Sekiguchi,et al.  Equarin is involved as an FGF signaling modulator in chick lens differentiation. , 2012, Developmental biology.

[273]  D. Voytas,et al.  Rapid and efficient gene modification in rice and Brachypodium using TALENs. , 2013, Molecular plant.

[274]  Rudolf Jaenisch,et al.  One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering , 2013, Cell.

[275]  P. Hegemann,et al.  Nuclear gene targeting in Chlamydomonas using engineered zinc-finger nucleases. , 2013, The Plant journal : for cell and molecular biology.

[276]  Takahito Watanabe,et al.  Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases , 2012, Nature Communications.

[277]  L. Schouls,et al.  Identification of genes that are associated with DNA repeats in prokaryotes , 2002, Molecular microbiology.

[278]  Hui Zhao,et al.  Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis , 2014, Development.

[279]  Dana Carroll,et al.  Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells , 2006, Proceedings of the National Academy of Sciences.

[280]  Steven Lin,et al.  Precise and Heritable Genome Editing in Evolutionarily Diverse Nematodes Using TALENs and CRISPR/Cas9 to Engineer Insertions and Deletions , 2013, Genetics.

[281]  Cameron S. Osborne,et al.  LMO2-Associated Clonal T Cell Proliferation in Two Patients after Gene Therapy for SCID-X1 , 2003, Science.

[282]  Alfred Pingoud,et al.  A novel zinc-finger nuclease platform with a sequence-specific cleavage module , 2011, Nucleic acids research.

[283]  George H. Silva,et al.  High Frequency Targeted Mutagenesis Using Engineered Endonucleases and DNA-End Processing Enzymes , 2013, PloS one.