Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing
Authors: Ciaran M Lee, Thomas J Cradick, Eli J Fine, Gang Bao
Journal: Molecular Therapy (2016)

The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications. The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications. Targeted genome-editing technology continues to create intense excitement with each new technological advance.1Segal DJ Meckler JF Genome engineering at the dawn of the golden age.Annu Rev Genomics Hum Genet. 2013; 14: 135-158Crossref PubMed Scopus (57) Google Scholar,2Cai M Yang Y Targeted genome editing tools for disease modeling and gene therapy.Curr Gene Ther. 2014; 14: 2-9Crossref PubMed Scopus (30) Google Scholar,3Cox DB Platt RJ Zhang F Therapeutic genome editing: prospects and challenges.Nat Med. 2015; 21: 121-131Crossref PubMed Scopus (133) Google Scholar The development of tools to generate DNA breaks, activate,4Wilber A Tschulena U Hargrove PW Kim YS Persons DA Barbas CF et al.A zinc-finger transcriptional activator designed to interact with the gamma-globin gene promoters enhances fetal hemoglobin production in primary human adult erythroblasts.Blood. 2010; 115: 3033-3041Crossref PubMed Scopus (0) Google Scholar repress or label genomic loci,5Cong L Zhou R Kuo YC Cunniff M Zhang F Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains.Nat Commun. 2012; 3: 968Crossref PubMed Scopus (173) Google Scholar,6Ma H Naseri A Reyes-Gutierrez P Wolfe SA Zhang S Pederson T Multicolor CRISPR labeling of chromosomal loci in human cells.Proc Natl Acad Sci USA. 2015; 112: 3002-3007Crossref PubMed Google Scholar and remodel chromatin7Hilton IB D'Ippolito AM Vockley CM Thakore PI Crawford GE Reddy TE et al.Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers.Nat Biotechnol. 2015; 33: 510-517Crossref PubMed Scopus (214) Google Scholar in a controlled, targeted manner will greatly aid the studies of a wide range of biological issues, including gene and genomic functions. The ability to specifically modify the genome also holds great promise for targeted gene therapies. Early work with meganucleases and zinc finger nucleases (ZFNs) showed that targeted site-specific DNA breaks could greatly increase the rate of homology-directed repair (HDR) at the specified locus.8Rouet P Smih F Jasin M Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.Proc Natl Acad Sci USA. 1994; 91: 6064-6068Crossref PubMed Scopus (298) Google Scholar,9Bibikova M Carroll D Segal DJ Trautman JK Smith J Kim YG et al.Stimulation of homologous recombination through targeted cleavage by chimeric nucleases.Mol Cell Biol. 2001; 21: 289-297Crossref PubMed Scopus (301) Google Scholar More recent developments include TAL effector nucleases (TALENs)10Cermak T Doyle EL Christian M Wang L Zhang Y Schmidt C et al.Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting.Nucleic Acids Res. 2011; 39: e82Crossref PubMed Scopus (896) Google Scholar,11Miller JC Tan S Qiao G Barlow KA Wang J Xia DF et al.A TALE nuclease architecture for efficient genome editing.Nat Biotechnol. 2011; 29: 143-148Crossref PubMed Scopus (935) Google Scholar and CRISPR/Cas9 systems (Figure 1).12Jinek M Chylinski K Fonfara I Hauer M Doudna JA Charpentier E A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (2023) Google Scholar,13Ran FA Hsu PD Wright J Agarwala V Scott DA Zhang F Genome engineering using the CRISPR-Cas9 system.Nat Protoc. 2013; 8: 2281-2308Crossref PubMed Scopus (825) Google Scholar,14Cong L Ran FA Cox D Lin S Barretto R Habib N et al.Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (2682) Google Scholar ZFNs consist of zinc finger motifs, which bind to DNA triplets, and the FokI nuclease domain which cleaves DNA upon dimerization.15Kim YG Cha J Chandrasegaran S Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.Proc Natl Acad Sci USA. 1996; 93: 1156-1160Crossref PubMed Scopus (683) Google Scholar,16Smith J Bibikova M Whitby FG Reddy AR Chandrasegaran S Carroll D Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains.Nucleic Acids Res. 2000; 28: 3361-3369Crossref PubMed Scopus (301) Google Scholar TALENs are composed of TAL effectors fused to the FokI nuclease domain and recognize DNA bases via conserved repeats that differ by two residues known as the repeat variable diresidue (RVD), which confers specificity to individual bases.17Boch J Scholze H Schornack S Landgraf A Hahn S Kay S et al.Breaking the code of DNA binding specificity of TAL-type III effectors.Science. 2009; 326: 1509-1512Crossref PubMed Scopus (1026) Google Scholar,18Bogdanove AJ Voytas DF TAL effectors: customizable proteins for DNA targeting.Science. 2011; 333: 1843-1846Crossref PubMed Scopus (459) Google Scholar Unlike ZFNs and TALENs that use protein domains to recognize target DNA sequences, the widely used CRISPR/Cas9 system adapted from Streptococcus pyogenes (Spy) uses both RNA and protein-based DNA recognition. These RNA-guided nucleases (RGENs) use a short guide RNA strand (gRNA), which targets a 20-nucleotide sequence, and the CRISPR associated (Cas) endonuclease Cas9, which binds to the fixed protospacer adjacent motif (PAM) NGG.12Jinek M Chylinski K Fonfara I Hauer M Doudna JA Charpentier E A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (2023) Google Scholar,13Ran FA Hsu PD Wright J Agarwala V Scott DA Zhang F Genome engineering using the CRISPR-Cas9 system.Nat Protoc. 2013; 8: 2281-2308Crossref PubMed Scopus (825) Google Scholar Although there is a strict adherence to PAM recognition, due to the short length of the PAM the specificity of RGENs is largely controlled by gRNA-DNA interaction. With these engineered nucleases, we now have efficient molecular scissors that can cut genomic DNA in cells at preselected locations and introduce mutagenic errors via the nonhomologous end joining (NHEJ) DNA repair pathway for targeted gene knockout or targeted deletion of large chromosomal segments. Alternatively, if an exogenous DNA donor template is introduced in concert with the nuclease, DNA cleavage (DNA double strand breaks or nicks) may trigger endogenous HDR with the supplied DNA donor template, resulting in precise DNA modifications (Figure 1). These abilities have led to the emerging field of genome editing, a new field in engineering and life sciences focusing on precisely modifying genomes using engineered nucleases. With the rapid advancement of genome-editing research, a suite of nuclease design and validation tools has been developed, significantly facilitating nuclease target site selection and experimental validation in terms of on-target and off-target activities. For most of the biological and medical applications of genome editing, high efficiency and high specificity of engineered nucleases are among the most important functional requirements; both are closely related to target site selection. For each endogenous genomic locus, the efficiency of DNA cleavage, both on-target and off-target, depends not only on the intrinsic nuclease activity (such as that of FokI domains and Cas9 protein) but also on target site accessibility and the affinity of DNA binding domain(s) (such as Tal effector domains and gRNA) to the target sequence. The specificity of engineered nucleases is significantly affected by the affinity of nuclease-DNA binding, such as zinc finger—DNA binding (ZFNs), Tal effector—DNA binding (TALENs) and gRNA—DNA hybridization (CRISPR), although the dimerization of FokI domains (ZFNs and TALENs) and the Cas9-PAM interactions may also play important roles. There is a lack of understanding on the behavior and functions of engineered nucleases in living cells, especially the dynamics of their interactions with DNA, and the cell cycle-dependent cleavage activity. Due to the limited biological knowledge and understanding of the structure and dynamics of the cell nucleus, especially chromatin structure, prediction of nuclease target accessibility and cleavage rates in living cells remains difficult. Further, the efficiency of homology directed repair also depends on the design, accessibility, and binding affinity of the donor templates as well. Therefore, experimental validation of target site selection is necessary. Herein, we use "true off-target sites" to indicate the off-target sites that are experimentally confirmed using polymerase chain reaction, sequencing or other methods. In this article, we review some of the web-based tools available for target selection in designing engineered nucleases, and selected experimental methods for quantifying nuclease activity and specificity. Due to space limitations and the rapid development of the genome-editing field, only a subset of available tools will be discussed, rather than having a comprehensive review. Challenges in target selection, especially in predicting off-target effects, and future directions in precision genome editing will also be discussed. A range of bioinformatics and experimental-based nuclease design tools have been developed that aid the target site selection of engineered nucleases. These tools fall into the following three main categories: (i) choice of target sites/design of nucleases, (ii) genomic searches for possible off-target sites, and (iii) determining the level of on- and off-target cleavage rates. A list of the available design tools is given in Table 1, together with a brief description of the functionality for each tool. Most of the tools listed in Table 1 are for the design of CRISPR/Cas9 systems, with a few for ZFNs and TALENs.Table 1Nuclease design toolsDesign toolNuclease designOff-target searchMax # of mismatchActivity analysisFunctionalityCRISPR/Cas9 toolsBenchling; https://benchling.com✓✓4XWeb tools for molecular biology and CRISPR applications, including gRNA design for a gene or genome coordinates. Automatically annotate with the exon and CDS information.Cas-OFFinder; http://www.rgenome.net/cas-offinder/X✓9XWeb site or downloadable program that searches for potential off-target sites for input gRNA and protospacer adjacent motifs (PAMs) in the requested genome or user-defined sequence.57Bae S Park J Kim JS Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases.Bioinformatics. 2014; 30: 1473-1475Crossref PubMed Scopus (115) Google Scholar Search includes DNA or RNA bulges in addition to base mismatches.CCTop; http://crispr.cos.uni-heidelberg.de✓✓5XCRISPR/Cas9 Target online predictor evaluates target sites within an input sequence giving a ranking of the candidates based on the possible off-target sites it identifies in a number of genomes.111Stemmer M Thumberger T Del Sol Keyer M Wittbrodt J Mateo JL CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool.PLoS One. 2015; 10: e0124633Crossref Scopus (37) Google ScholarCHOPCHOP;https://chopchop.rc.fas.harvard.edu✓✓2XA web tool for selecting target sites for CRISPR/Cas9- or TALEN-directed targeting from within an input sequence or chromosomal coordinates in a number of genomes. Locates off-target sites with up to two mismatches.35Montague TG Cruz JM Gagnon JA Church GM Valen E CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing.Nucleic Acids Res. 2014; 42 (Web Server issue): W401-W407Crossref PubMed Scopus (112) Google ScholarCOSMID; https://crispr.bme.gatech.eduX✓3XCRISPR Off-target Sites with Mismatches, Insertions, and Deletions. An exhaustive search tool identifying sites matching user supplied criteria that can include DNA or RNA bulges in addition to base mismatches. Also designs primers for downstream applications, such as next-generation sequencing (NGS).48Cradick TJ Qiu P Lee CM Fine EJ Bao G COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites.Mol Ther Nucleic Acids. 2014; 3: e214Abstract Full Text Full Text PDF PubMed Scopus (37) Google ScholarCRISPR design; http://crispr.mit.edu✓✓4XDesigns gRNA by evaluating possible off-target sites in a number of genomes and outputs with scoring, highlighting gRNA that may have higher specificity.52Hsu PD Scott DA Weinstein JA Ran FA Konermann S Agarwala V et al.DNA targeting specificity of RNA-guided Cas9 nucleases.Nat Biotechnol. 2013; 31: 827-832Crossref PubMed Scopus (871) Google ScholarCRISPR MultiTargeter; http://www.multicrispr.net✓Xn/aXUsers can input sequences to search for unique CRISPR target sites or highly similar or identical target sites in multiple genes or transcripts or within a single gene.112Prykhozhij SV Rajan V Gaston D Berman JN Correction: CRISPR MultiTargeter: a web tool to find common and unique CRISPR single guide RNA targets in a set of similar sequences.PLoS One. 2015; 10: e0138634Google ScholarCRISPR_Scan; http://www.crisprscan.org✓Xn/aPredictionFinds and predicts the cutting efficiency of gRNAs in a given sequence or protein coding gene. Also has UCSC tracks for high and low scoring gRNAs43Moreno-Mateos MA Vejnar CE Beaudoin JD Fernandez JP Mis EK Khokha MK et al.CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo.Nat Methods. 2015; 12: 982-988Crossref PubMed Scopus (0) Google ScholarCRISPR-Plant; http://www.genome.arizona.edu/crispr/✓Xn/aXA CRISPR search tool targeting a range of plant species, identifying spacer sequences located in a selected gene region, gene or larger chromosomal region.RISPRsearch.html CRISPRdirect; http://crispr.dbcls.jp✓X2XSelects target sites from accession numbers, genome locations or input sequences. Outputs the expected number of genomic sites with 8 or 12 bp matching seed sequences.113Naito Y Hino K Bono H Ui-Tei K CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites.Bioinformatics. 2015; 31: 1120-1123Crossref PubMed Scopus (0) Google ScholarCRISPRfinder; http://crispr.u-psud.fr/ Server/XXn/aXLocates CRISPR (repeat) structures in published microbial genomes or in small to very large user- submitted sequences, also can extract the spacer sequences.114Grissa I Vergnaud G Pourcel C CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats.Nucleic Acids Res. 2007; 35 (Web Server issue): W52-W57Crossref PubMed Scopus (544) Google ScholarCRISPRseek; http://mvw.bioconductor.org/packages/release/bioc/html/CRISPRseek.html✓✓4XOpen source software to identify target-specific gRNAs, minimizing off-target cleavage at other sites within any selected genome outputs a list of all possible sgRNAs for a given PAM sequence..115Zhu LJ Holmes BR Aronin N Brodsky MH CRISPRseek: a bioconductor package to identify target-specific guide RNAs for CRISPR-Cas9 genome-editing systems.PLoS One. 2014; 9: e108424Crossref PubMed Scopus (31) Google ScholarCROP-IT; http://cheetah.bioch.virginia.edu/AdliLab/CROP-IT/homepage.html✓✓6, 9XCRISPR/Cas9 Off-target Prediction and Identification Tool assists biologists in designing CRISPR/ CAS9. Predicts and ranks off-target sites in mouse or and human genome.DESKGEN; https://www.deskgen.com/landing/✓✓3XAllows designing gene editing experiments through a full range of genome-editing tools, including providing gRNAs, vectors, donors, and target activity scores and library designs.Dharmacon Configurator; http://dharmacon.gelifesciences.com/gene-editing/crispr-rna-configurator/✓Xn/aXDesigns gRNA targeting sequences based on Entrez Gene ID or Gene symbol, with links to order synthesized RNAs. Also evaluates entered gRNAs.DNA 2.0 Design Tool; https://www.dna20.com/eCommerce/cas9/input✓Xn/aXAllows testing user supplied gRNAs or designs gRNA or pairs of gRNAs targeting a user-specified gene or chromosomal site. gRNAs are evaluated using scoring system.E-CRISP; http://www.e-crisp.org/E-CRISP/✓✓10XDesigns and evaluates CRISPR target sites from a gene symbol, an ENSEMBL ID or a FASTA sequence and designs gRNAs, allowing for a number of different CRISPR uses.116Heigwer F Kerr G Boutros M E-CRISP: fast CRISPR target site identification.Nat Methods. 2014; 11: 122-123Crossref PubMed Scopus (125) Google ScholarflyCRISPR Optimal Target Finder; http://tools.flycrispr.molbio.wisc.edu/targetFinder/✓✓6XIdentifies target sites and evaluates their specificity for use targeting Drosophila melanogaster. Off-target sites evaluated using empirically rooted rules.117Gratz SJ Ukken FP Rubinstein CD Thiede G Donohue LK Cummings AM et al.Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila.Genetics. 2014; 196: 961-971Crossref PubMed Scopus (119) Google ScholarGT-Scan; http://gt-scan.braembl.org.au/gt-scan/✓✓3XGenome Target Scan ranks all potential targets in a er-selected region of a genome in terms of how many putative off-target sites they have, to aid target site selection.118O'Brien A Bailey TL GT-Scan: identifying unique genomic targets.Bioinformatics. 2014; 30: 2673-2675Crossref PubMed Scopus (21) Google ScholarJack Lins gRNA finder; http://spot.colorado.edu/slin/cas9.html✓Xn/aXCRISPR target site locator that searches the sense strand of DNA sequences for PAM sequences and returns the sites target sites of a specified length. Also allows checking for secondary structure.Microhomology-associated Score Calculator; http://www.rgenome.net/mich-calculator/✓Xn/aXEvaluates microhomology in input sequences to evaluate potential for induced frame shifts and knockouts. A scoring system estimates microhomology-associated deletions at nuclease target sites.119Bae S Kweon J Kim HS Kim JS Microhomology-based choice of Cas9 nuclease target sites.Nat Methods. 2014; 11: 705-706Crossref PubMed Scopus (56) Google ScholarsgRNAcas9; http://www.biootools.com✓✓5XA downloadable program software package that designs CRISPR gRNAs, provides cloning sequences with minimized off-target effects and searches for potential off-target cleavage. Designs polymerase chain reaction primers flanking on- or off-target sites.120Xie S Shen B Zhang C Huang X Zhang Y sgRNAcas9: a software package for designing CRISPR sgRNA and evaluating potential off-target cleavage sites.PloS One. 2014; 9: e100448Crossref PubMed Scopus (49) Google ScholarsgRNA Designer; http://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design✓Xn/aPredictionA web-based tool for designing sgRNAs targeting the human or mouse gene. This tool provides guidance as to which sgRNAs are most likely to give high on-target activity, but is not intended for any off-target predictions.41Doench JG Hartenian E Graham DB Tothova Z Hegde M Smith I et al.Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation.Nat Biotechnol. 2014; 32: 1262-1267Crossref PubMed Scopus (807) Google ScholarsgRNA Scorer; https://crispr.med.harvard.edu/sgRNAScorer/✓Xn/aPredictionA sgRNA design tool that was developed using results from an in vivo library-on-library methodology that simultaneously assessed sgRNA activity across ∼1,400 genomic loci.42Chari R Mali P Moosburner M Church GM Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach.Nat Methods. 2015; 12: 823-826Crossref PubMed Scopus (51) Google ScholarWGE; http://www.sanger.ac.uk/htgt/wge/✓✓4XA series of tools using a database of determined single and paired guide strands, including a genome browser environment and pre-scored off-target information.121Hodgkins A Farne A Perera S Grego T Parry-Smith DJ Skarnes WC et al.WGE: a CRISPR database for genome engineering.Bioinformatics. 2015; 31: 3078-3080Crossref PubMed Scopus (10) Google ScholarZFN and TALEN toolsPROGNOS; http://bao.rice.edu/Research/BioinformaticTools/prognos.htmlX✓20XPredicted report of genome-wide nuclease off-target sites exhaustively identifies off-target sites for TALENs or ZFNs, provides a ranked list and designs primers for downstream applications, such as mutation detection or NGS.46Fine EJ Cradick TJ Zhao CL Lin Y Bao G An online bioinformatics tool predicts zinc finger and TALE nuclease off-target cleavage.Nucleic Acids Res. 2014; 42: e42Crossref PubMed Scopus (45) Google ScholarSAPTA; http://bao.rice.edu/Research/BioinformaticTools/TAL_targeter.html✓Xn/aXScoring algorithm for predicting TALE(N) Activity scans an input DNA sequence and designs TAL effectors or TALENs predicted to have the highest activity, particularly when using the di-residues NK that provide more specifically within a supplied gene sequence.39Lin Y Fine EJ Zheng Z Antico CJ Voit RA Porteus MH et al.SAPTA: a new design tool for improving TALE nuclease activity.Nucleic Acid Res. 2014; 42: e47Crossref PubMed Scopus (0) Google ScholarTAL Plasmids Sequence Assembly Tool; http://bao.rice.edu/Research/BioinformaticTools/assembleTALSequences.htmlXXn/aXFacilitates cloning and sequencing of TAL and TALEN plasmids, by generating vector sequence after a user enters target sequences or repeat variable diresidues and assembly method.TALE-N Effector Nuclease Targeter; https://tale-nt.cac.cornell.edu✓✓XTools for designing, evaluating, and assembling custom single and paired TAL effector constructs and TALENs. Includes off-target search and counting.33Doyle EL Booher NJ Standage DS Voytas DF Brendel VP Vandyk JK et al.TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction.Nucleic Acids Res. 2012; 40 (Web Server issue): W117-W122Crossref PubMed Scopus (274) Google ScholarTALgetter/TALENoffer; http://galaxy2.informatik.uni-halle.de:8976✓✓10XPredicts off-target sites for TALEN sites identified in an input sequence. Available as web site, command line program and as a Galaxy server122Grau J Boch J Posch S TALENoffer: genome-wide TALEN off-target prediction.Bioinformatics. 2013; 29: 2931-2932Crossref PubMed Scopus (0) Google ScholarZiFiT; http://zifit.partners.org/ZiFiT/✓XblastXZinc finger targeter originally a tool for identifying of potential zinc finger nuclease (ZFN) sites, but ZiFit has been extended to also contain tools for TALENs and CRISPR nucleases.31Sander JD Zaback P Joung JK Voytas DF Dobbs D Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool.Nucleic Acids Res. 2007; 35 (Web Server issue): W599-W605Crossref PubMed Scopus (115) Google ScholarOther genome-editing resourcesCRISPR-GA; http://54.80.152.219XXn/aQuantificationWeb-based analysis of CRISPR-Cas9 genome editing outcomes to quantify and characterize the indels and mutations through single or paired read NGS.75Güell M Yang L Church GM Genome editing assessment using CRISPR Genome Analyzer (CRISPR-GA).Bioinformatics. 2014; 30: 2968-2970Crossref PubMed Scopus (22) Google ScholarCRISPResso; http://crispresso.rocksXXn/aQuantificationProvides analysis of CRISPR-Cas9 genome editing outcomes (nonhomologous end joining or homology-directed repair) from NGS single or paired reads when compared to uploaded reference sequence.EENdb; http://eendb.zfgenetics.orgXXn/aXDatabases of target sites for reported TALENs, ZFNs and CRISPR/Cas systems in different organisms. Also lists methods and uses for gene editing.123Xiao A Wu Y Yang Z Hu Y Wang W Zhang Y et al.EENdb: a database and knowledge base of ZFNs and TALENs for endonuclease engineering.Nucleic Acids Res. 2013; 41 (Database issue): D415-D422Crossref PubMed Scopus (0) Google ScholarGibson Designer; http://www.sanger.ac.uk/htgt/wge/gibson_designerXXn/aXDesigns human or mouse amplification primers to create targeting vectors by Gibson assembly. Gibson Designer matches vector design with CRISPR sites for the creation of exon deletions.121Hodgkins A Farne A Perera S Grego T Parry-Smith DJ Skarnes WC et al.WGE: a CRISPR database for genome engineering.Bioinformatics. 2015; 31: 3078-3080Crossref PubMed Scopus (10) Google ScholarTIDE; http://tide.nki.nlXXn/aQuantificationTracking of indels by decomposition (TIDE) is a web tool to determine the level and type of mutations introduced by genome editing. Compares Sanger sequencing reactions.64Brinkman EK Chen T Amendola M van Steensel B Easy quantitative assessment of genome editing by sequence trace decomposition.Nucleic Acids Res. 2014; 42: e168Crossref PubMed Google Scholar Open table in a new tab Zinc finger proteins (ZFPs) can be designed to target many novel sequences based on the 3 bp specificity of individual fingers.19Pavletich NP Pabo CO Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A.Science. 1991; 252: 809-817Crossref PubMed Scopus (1642) Google Scholar,20Pabo CO Peisach E Grant RA Design and selection of novel Cys2His2 zinc finger proteins.Annu Rev Biochem. 2001; 70: 313-340Crossref PubMed Scopus (427) Google Scholar Phage display-based selections and rational design techniques have been used by certain companies and research labs to generate high-affinity ZFPs and ZFNs.21Rebar EJ Pabo CO Zinc finger phage: affinity selection of fingers with new DNA-binding specificities.Science. 1994; 263: 671-673Crossref PubMed Google Scholar,22Jamieson AC Wang H Kim SH A zinc finger directory for high-affinity DNA recognition.Proc Natl Acad Sci USA. 1996; 93: 12834-12839Crossref PubMed Scopus (87) Google Scholar,23Segal DJ Dreier B Beerli RR Barbas 3rd CF Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences.Proc Natl Acad Sci USA. 1999; 96: 2758-2763Crossref PubMed Scopus (302) Google Scholar,24Choo Y Klug A Toward a code for the interactions of zinc fingers with DNA: selection of randomized fingers displayed on phage.Proc Natl Acad Sci USA. 1994; 91: 11163-11167Crossref PubMed Scopus (291) Google Scholar,25Porteus MH Baltimore D Chimeric nucleases stimulate gene targeting in human cells.Science. 2003; 300: 763Crossref PubMed Scopus (430) Google Scholar,26Urnov FD Miller JC Lee YL Beausejour CM Rock JM Augustus S et al.Highly efficient endogenous human gene correction using designed zinc-finger nucleases.Nature. 2005; 435: 646-651Crossref PubMed Scopus (937) Google Scholar However, zinc finger (ZF) design remains difficult due to positional effects and a lack of straightforward ZFP design principles—a number of amino acid sequences in a given finger can specify a given triplet, but the activity of any given zinc finger is strongly dependent on its position in the ZFP and the nature of the neighboring zinc fingers.27Isalan M Choo Y Klug A Synergy between adjacent zinc fingers in sequence-specific DNA recognition.Proc Natl Acad Sci USA. 1997; 94: 5617-5621Crossref PubMed Scopus (120) Google Scholar,28Ramirez CL Foley JE Wright DA Müller-Lerch F Rahman SH Cornu TI et al.Unexpected failure rates for modular assembly of engineered zinc fingers.Nat Methods. 2008; 5: 374-375Crossref PubMed Scopus (224) Google Scholar,29Sander JD Dahlborg EJ Goodwin MJ Cade L Zhang F Cifuentes D et al.Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA).Nat Methods. 2011; 8: 67-69Crossref PubMed Scopus (274) Google Scholar Tools such as ZiFit were developed to address this issue by taking the context dependence into account. However, designing a highly active and specific ZFN pair remains challenging.30Mandell JG Barbas 3rd CF Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleases.Nucleic Acids Res. 2006; 34 (Web Server issue): W516-W523Crossref PubMed Scopus (182) Google Scholar,31Sander JD Zaback P Joung JK Voytas DF Dobbs D Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool.Nucleic Acids Res. 2007; 35 (Web Server issue): W599-W605Crossref PubMed Scopus (115) Google Scholar Alternatively, a bacteria two-hybrid screening platform is also available for custom ZFP production.32Maeder ML Thibodeau-Beganny S Osiak A Wright DA Anthony RM Eichtinger M et al.Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification.Mol Cell. 2008; 31: 294-301Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar However, the substantial amount of work required has limited its use outside of a small number of dedicated labs. For designing TALENs, the DNA-targeting specificity of TAL effector RVDs is more straightforward than that of ZFs, allowing easier design of TALENs. There are four main RVDs, one for each DNA base.17Boch J Scholze H Schornack S Landgraf A Hahn S Kay S et al.Breaking the cod