Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing

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.

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

[2]  David A. Scott,et al.  Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity , 2013, Cell.

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

[4]  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.

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

[6]  David A. Scott,et al.  In vivo genome editing using Staphylococcus aureus Cas9 , 2015, Nature.

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

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

[9]  Xiaoling Wang,et al.  Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors , 2015, Nature Biotechnology.

[10]  Xingxu Huang,et al.  sgRNAcas9: A Software Package for Designing CRISPR sgRNA and Evaluating Potential Off-Target Cleavage Sites , 2014, PloS one.

[11]  Jeffrey C. Miller,et al.  A rapid and general assay for monitoring endogenous gene modification. , 2010, Methods in molecular biology.

[12]  Euan A Ashley,et al.  Clinical interpretation and implications of whole-genome sequencing. , 2014, JAMA.

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

[14]  Peng Qiu,et al.  COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites , 2014, Molecular therapy. Nucleic acids.

[15]  Christopher M. Vockley,et al.  Epigenome editing by a CRISPR/Cas9-based acetyltransferase activates genes from promoters and enhancers , 2015, Nature Biotechnology.

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

[17]  Eric P. Bennett,et al.  High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs , 2014, Nucleic acids research.

[18]  Jong-il Kim,et al.  Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells , 2015, Nature Methods.

[19]  Yoon-Sang Kim,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. , 2010, Blood.

[20]  Meagan E. Sullender,et al.  Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation , 2014, Nature Biotechnology.

[21]  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.

[22]  Mazhar Adli,et al.  Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease , 2014, Nature Biotechnology.

[23]  Mazhar Adli,et al.  Cas9-chromatin binding information enables more accurate CRISPR off-target prediction , 2015, Nucleic acids research.

[24]  Jin-Soo Kim,et al.  Surrogate reporters for enrichment of cells with nuclease-induced mutations , 2011, Nature Methods.

[25]  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.

[26]  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.

[27]  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.

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

[29]  Jun Liu,et al.  Zinc-finger nickase-mediated insertion of the lysostaphin gene into the beta-casein locus in cloned cows , 2013, Nature Communications.

[30]  S H Kim,et al.  A zinc finger directory for high-affinity DNA recognition. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

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

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

[33]  A Klug,et al.  Synergy between adjacent zinc fingers in sequence-specific DNA recognition. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

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

[35]  Hidemasa Bono,et al.  CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites , 2014, Bioinform..

[36]  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..

[37]  Volker Brendel,et al.  TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction , 2012, Nucleic Acids Res..

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

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

[40]  Randall J. Platt,et al.  Therapeutic genome editing: prospects and challenges , 2015, Nature Medicine.

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

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

[43]  Kira S. Makarova,et al.  Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems , 2013, Nucleic acids research.

[44]  P. Rouet,et al.  Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

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

[46]  Daniel F. Voytas,et al.  Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool , 2007, Nucleic Acids Res..

[47]  Jonathon T. Hill,et al.  Poly peak parser: Method and software for identification of unknown indels using sanger sequencing of polymerase chain reaction products , 2014, Developmental dynamics : an official publication of the American Association of Anatomists.

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

[49]  Yan Zhang,et al.  TALE nickase-mediated SP110 knockin endows cattle with increased resistance to tuberculosis , 2015, Proceedings of the National Academy of Sciences.

[50]  David A. Scott,et al.  Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells , 2014, Nature Biotechnology.

[51]  M. Rowicka,et al.  Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing , 2013, Nature Methods.

[52]  Sangsu Bae,et al.  Microhomology-based choice of Cas9 nuclease target sites , 2014, Nature Methods.

[53]  H. Brunner.  Annual Review of Genomics and Human Genetics , 2001, European Journal of Human Genetics.

[54]  Prashant Mali,et al.  Orthogonal Cas9 Proteins for RNA-Guided Gene Regulation and Editing , 2013, Nature Methods.

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

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

[57]  William C. Skarnes,et al.  WGE: a CRISPR database for genome engineering , 2015, Bioinform..

[58]  Gang Bao,et al.  Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing. , 2014, Cell reports.

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

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

[61]  Jin-Soo Kim,et al.  Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases , 2014, Bioinform..

[62]  Charles E. Vejnar,et al.  CRISPRscan: designing highly efficient sgRNAs for CRISPR/Cas9 targeting in vivo , 2015, Nature Methods.

[63]  Ciaran M. Lee,et al.  Correction of the ΔF508 Mutation in the Cystic Fibrosis Transmembrane Conductance Regulator Gene by Zinc-Finger Nuclease Homology-Directed Repair , 2012, BioResearch open access.

[64]  M. Boutros,et al.  E-CRISP: fast CRISPR target site identification , 2014, Nature Methods.

[65]  C. Rubinstein,et al.  Highly Specific and Efficient CRISPR/Cas9-Catalyzed Homology-Directed Repair in Drosophila , 2014, Genetics.

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

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

[68]  David A. Scott,et al.  Genome engineering using the CRISPR-Cas9 system , 2013, Nature Protocols.

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

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

[71]  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.

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

[73]  Gang Bao,et al.  The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells , 2016, Molecular therapy : the journal of the American Society of Gene Therapy.

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

[75]  Jin-Soo Kim,et al.  Genotyping with CRISPR-Cas-derived RNA-guided endonucleases , 2014, Nature Communications.

[76]  Feng Zhang,et al.  Genome engineering using CRISPR-Cas9 system. , 2015, Methods in molecular biology.

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

[78]  Jeffrey G. Mandell,et al.  Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleases , 2006, Nucleic Acids Res..

[79]  George M. Church,et al.  Genome editing assessment using CRISPR Genome Analyzer (CRISPR-GA) , 2014, Bioinform..

[80]  Timothy L. Bailey,et al.  GT-Scan: identifying unique genomic targets , 2014, Bioinform..

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

[82]  Richard L. Frock,et al.  Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases , 2014, Nature Biotechnology.

[83]  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.

[84]  Yi Yang,et al.  Targeted genome editing tools for disease modeling and gene therapy. , 2014, Current gene therapy.

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

[86]  Gang Bao,et al.  CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences , 2014, Nucleic acids research.

[87]  Joshua A. Arribere,et al.  Efficient Marker-Free Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans , 2014, Genetics.

[88]  Martin J. Aryee,et al.  GUIDE-Seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases , 2014, Nature Biotechnology.

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

[90]  C. Pabo,et al.  Zinc finger phage: affinity selection of fingers with new DNA-binding specificities. , 1994, Science.

[91]  Desheng Liang,et al.  TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus. , 2014, Biochemical and biophysical research communications.

[92]  Claudio Mussolino,et al.  TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity , 2014, Nucleic acids research.

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

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

[95]  M. Lomax,et al.  Biological consequences of radiation-induced DNA damage: relevance to radiotherapy. , 2013, Clinical oncology (Royal College of Radiologists (Great Britain)).

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

[97]  George M. Church,et al.  CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing , 2014, Nucleic Acids Res..

[98]  Jun Nakamura,et al.  Endogenous versus exogenous DNA adducts: their role in carcinogenesis, epidemiology, and risk assessment. , 2011, Toxicological sciences : an official journal of the Society of Toxicology.

[99]  L. Zhu,et al.  CRISPRseek: A Bioconductor Package to Identify Target-Specific Guide RNAs for CRISPR-Cas9 Genome-Editing Systems , 2014, PloS one.

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

[101]  Bradley E. Bernstein,et al.  In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites , 2013, Nucleic acids research.

[102]  Daniel Gaston,et al.  CRISPR MultiTargeter: A Web Tool to Find Common and Unique CRISPR Single Guide RNA Targets in a Set of Similar Sequences , 2015, PloS one.

[103]  B. van Steensel,et al.  Easy quantitative assessment of genome editing by sequence trace decomposition , 2014, Nucleic acids research.

[104]  Yanni Lin,et al.  Designing and testing the activities of TAL effector nucleases. , 2014, Methods in molecular biology.

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

[106]  Gang Bao,et al.  SAPTA: a new design tool for improving TALE nuclease activity , 2014, Nucleic acids research.

[107]  David R. Liu,et al.  Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification , 2014, Nature Biotechnology.

[108]  Stefan Posch,et al.  TALENoffer: genome-wide TALEN off-target prediction , 2013, Bioinform..

[109]  G. Church,et al.  Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach , 2015, Nature Methods.

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

[111]  Henriette O'Geen,et al.  A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture , 2014, bioRxiv.

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

[113]  Oliver Pelz,et al.  E-TALEN: a web tool to design TALENs for genome engineering , 2013, Nucleic acids research.

[114]  Gang Bao,et al.  Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering , 2015, PloS one.

[115]  Ibtissem Grissa,et al.  CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats , 2007, Nucleic Acids Res..

[116]  Martin J. Aryee,et al.  Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing , 2014, Nature Biotechnology.

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

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

[119]  Shaojie Zhang,et al.  Multicolor CRISPR labeling of chromosomal loci in human cells , 2015, Proceedings of the National Academy of Sciences.

[120]  Joana A. Vidigal,et al.  In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system , 2014, Nature.

[121]  Gang Bao,et al.  An online bioinformatics tool predicts zinc finger and TALE nuclease off-target cleavage , 2013, Nucleic acids research.

[122]  Maximilian Müller,et al.  Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome. , 2016, Molecular therapy : the journal of the American Society of Gene Therapy.

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