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Strategies to Determine Off-Target Effects of Engineered Nucleases

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Genome Editing

Part of the book series: Advances in Experimental Medicine and Biology ((AEMB))

Abstract

Genome editing is greatly facilitated by using engineered nucleases to specifically cleave a pre-selected DNA sequence. Cellular repair of the nuclease-induced DNA breaks by either non-homologous end joining (NHEJ) or homology-directed repair (HDR) allows genome editing in a wide range of organisms and cell lines. However, if a nuclease cleaves at genomic locations other than the intended target, known as “off-target sites”, it can lead to mutations, chromosomal loss or rearrangements, causing gain/loss of function and cytotoxicity. Although zinc finger nucleases (ZFNs), TAL effector nuclease (TALENs), and CRISPR/Cas9 systems have been used successfully to create specific DNA breaks in cells, they lack perfect specificity and may result in off-target cleavage. Methods have been developed to predict and to quantify the off-target cleavage events, which are very important for optimizing nuclease design and determining if the gene editing approaches are highly specific. These methods have the potential to significantly facilitate the design of engineered nucleases for genome editing applications.

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Abbreviations

bp:

Base pairs (of nucleic acid)

Cas9:

CRISPR associated protein 9, an endonuclease from Streptococcus pyogenes

CCR5 :

The chemokine (C-C motif) receptor 5 gene

CRISPR:

Clustered regularly interspaced short palindromic repeats

CRISPR/Cas9:

The combination of guide RNA and associated protein required for DNA cleavage

DSB:

Double strand break

FokI:

An endonuclease derived from Flavobacterium okeanokoites

gRNA:

Guide RNA, used in the CRISPR/Cas9 system

HBB :

The hemoglobin beta gene, also known as beta-globin

HDR:

The homology directed DNA repair pathway

IDLV:

Integrase deficient lentiviral vector

indel:

A short insertion, deletion, or combined insertion and deletion of DNA

NGS:

Next generation sequencing, platforms such as Illumina fall in this category

NHEJ:

The non-homologous end-joining DNA repair pathway

RVD:

Repeat variable di-residue, the two amino acids in TAL repeats that specify the DNA base

SMRT:

Single molecule real-time, a third generation sequencing platform

TALEN:

Transcription activator-like effector nuclease

ZFN:

Zinc finger nuclease

References

  1. Smithies O, Gregg RG, Boggs SS, Koralewski MA, Kucherlapati RS. Insertion of DNA-sequences into the human chromosomal beta-globin locus by homologous recombination. Nature. 1985;317:230–4.

    Article  CAS  PubMed  Google Scholar 

  2. Capecchi MR. Altering the genome by homologous recombination. Science. 1989;244:1288–92.

    Article  CAS  PubMed  Google Scholar 

  3. Marcaida MJ, Muñoz IG, Blanco FJ, Prieto J, Montoya G. Homing endonucleases: from basics to therapeutic applications. Cell Mol Life Sci. 2010;67:727–48.

    Article  CAS  PubMed  Google Scholar 

  4. Silva G, Poirot L, Galetto R, Smith J, Montoya G, Duchateau P, Pâques F. Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy. Curr Gene Ther. 2011;11:11–27.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Porteus MH, Carroll D. Gene targeting using zinc finger nucleases. Nat Biotechnol. 2005;23:967–73.

    Article  CAS  PubMed  Google Scholar 

  6. Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S, Jamieson AC, Porteus MH, Gregory PD, Holmes MC. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature. 2005;435:646–51.

    Article  CAS  PubMed  Google Scholar 

  7. Chandrasegaran S, Smith J. Chimeric restriction enzymes: what is next? Biol Chem. 1999;380:841–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Li T, Huang S, Jiang WZ, Wright D, Spalding MH, Weeks DP, Yang B. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res. 2011;39:359–72.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanove AJ, Voytas DF. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics. 2010;186:757–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U. Breaking the code of DNA binding specificity of TAL-type III effectors. Science. 2009;326:1509–12.

    Article  CAS  PubMed  Google Scholar 

  11. Moscou MJ, Bogdanove AJ. A simple cipher governs DNA recognition by TAL effectors. Science. 2009;326:1501.

    Article  CAS  PubMed  Google Scholar 

  12. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 2013;31:233–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816–21.

    Article  CAS  PubMed  Google Scholar 

  15. Petek LM, Russell DW, Miller DG. Frequent endonuclease cleavage at off-target locations in vivo. Mol Ther. 2010;18:983–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Tadepally HD, Burger G, Aubry M. Evolution of C2H2-zinc finger genes and subfamilies in mammals: species-specific duplication and loss of clusters, genes and effector domains. BMC Evol Biol. 2008;8:176.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Miller J, McLachlan AD, Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985;4:1609–14.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Frankel AD, Berg JM, Pabo CO. Metal-dependent folding of a single zinc finger from transcription factor IIIA. Proc Natl Acad Sci U S A. 1987;84:4841–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wolfe SA, Grant RA, Elrod-Erickson M, Pabo CO. Beyond the "recognition code": structures of two Cys2His2 zinc finger/TATA box complexes. Structure. 2001;9:717–23.

    Article  CAS  PubMed  Google Scholar 

  20. Ramirez CL, Foley JE, Wright DA, Muller-Lerch F, Rahman SH, Cornu TI, Winfrey RJ, Sander JD, Fu F, Townsend JA, et al. Unexpected failure rates for modular assembly of engineered zinc fingers. Nat Methods. 2008;5:374–5.

    Article  CAS  PubMed  Google Scholar 

  21. Li L, Wu LP, Chandrasegaran S. Functional domains in Fok I restriction endonuclease. Proc Natl Acad Sci U S A. 1992;89:4275–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kim YG, Cha J, Chandrasegaran S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci U S A. 1996;93:1156–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bitinaite J, Wah DA, Aggarwal AK, Schildkraut I. FokI dimerization is required for DNA cleavage. Proc Natl Acad Sci U S A. 1998;95:10570–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Porteus MH, Baltimore D. Chimeric nucleases stimulate gene targeting in human cells. Science. 2003;300:763.

    Article  PubMed  Google Scholar 

  25. Gaj T, Guo J, Kato Y, Sirk SJ, Iii CFB. Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nat Methods. 2012;9:805–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Sander JD, Ramirez CL, Linder SJ, Pattanayak V, Shoresh N, Ku M, Foden JA, Reyon D, Bernstein BE, Liu DR, et al. In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites. Nucleic Acids Res. 2013;41, e181.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Pattanayak V, Ramirez CL, Joung JK, Liu DR. Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat Methods. 2011;8:765–70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Gabriel R, Lombardo A, Arens A, Miller JC, Genovese P, Kaeppel C, Nowrouzi A, Bartholomae CC, Wang J, Friedman G, et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat Biotechnol. 2011;29:816–23.

    Article  CAS  PubMed  Google Scholar 

  29. Gupta A, Meng X, Zhu LJ, Lawson ND, Wolfe SA. Zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases. Nucleic Acids Res. 2011;39:381–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Mussolino C, Alzubi J, Fine EJ, Morbitzer R, Cradick TJ, Lahaye T, Bao G, Cathomen T. TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity. Nucleic Acids Res. 2014;42:6762–73.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Elrod-Erickson M, Rould MA, Nekludova L, Pabo CO. Zif268 protein-DNA complex refined at 1.6 A: a model system for understanding zinc finger-DNA interactions. Structure. 1996;4:1171–80.

    Article  CAS  PubMed  Google Scholar 

  32. Choo Y, Klug A. Selection of DNA binding sites for zinc fingers using rationally randomized DNA reveals coded interactions. Proc Natl Acad Sci U S A. 1994;91:11168–72.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Jamieson AC, Kim SH, Wells JA. In vitro selection of zinc fingers with altered DNA-binding specificity. Biochemistry. 1994;33:5689–95.

    Article  CAS  PubMed  Google Scholar 

  34. Rebar EJ, Pabo CO. Zinc finger phage: affinity selection of fingers with new DNA-binding specificities. Science. 1994;263:671–3.

    Article  CAS  PubMed  Google Scholar 

  35. Choo 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 U S A. 1994;91:11163–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Joung JK, Ramm EI, Pabo CO. A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions. Proc Natl Acad Sci U S A. 2000;97:7382–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Joung JK. Identifying and modifying protein-DNA and protein-protein interactions using a bacterial two-hybrid selection system. J Cell Biochem Suppl. 2001;37:53–7.

    Article  PubMed  CAS  Google Scholar 

  38. Durai S, Bosley A, Abulencia AB, Chandrasegaran S, Ostermeier M. A bacterial one-hybrid selection system for interrogating zinc finger-DNA interactions. Comb Chem High Throughput Screen. 2006;9:301–11.

    Article  CAS  PubMed  Google Scholar 

  39. Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, Eichtinger M, Jiang T, Foley JE, Winfrey RJ, Townsend JA, et al. Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell. 2008;31:294–301.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Sander JD, Dahlborg EJ, Goodwin MJ, Cade L, Zhang F, Cifuentes D, Curtin SJ, Blackburn JS, Thibodeau-Beganny S, Qi Y, et al. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). Nat Methods. 2011;8:67–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Moore M, Choo Y, Klug A. Design of polyzinc finger peptides with structured linkers. Proc Natl Acad Sci U S A. 2001;98:1432–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Wolfe SA, Ramm EI, Pabo CO. Combining structure-based design with phage display to create new Cys(2)His(2) zinc finger dimers. Structure. 2000;8:739–50.

    Article  CAS  PubMed  Google Scholar 

  43. Imanishi M, Hori Y, Nagaoka M, Sugiura Y. DNA-bending finger: artificial design of 6-zinc finger peptides with polyglycine linker and induction of DNA bending. Biochemistry. 2000;39:4383–90.

    Article  CAS  PubMed  Google Scholar 

  44. Zhang HS, Liu D, Huang Y, Schmidt S, Hickey R, Guschin D, Su H, Jovin IS, Kunis M, Hinkley S, et al. A designed zinc-finger transcriptional repressor of phospholamban improves function of the failing heart. Mol Ther. 2012;20:1508–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Fine 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, e42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Abarrategui-Pontes C, Creneguy A, Thinard R, Fine EJ, Thepenier V, Fournier LRL, Cradick TJ, Bao G, Tesson L, Podevin G, et al. Codon swapping of zinc finger nucleases confers expression in primary cells and in vivo from a single lentiviral vector. Curr Gene Ther. 2014;14:12.

    Google Scholar 

  47. Choo Y, Klug A. A role in DNA binding for the linker sequences of the first three zinc fingers of TFIIIA. Nucleic Acids Res. 1993;21:3341–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Jantz D, Berg JM. Reduction in DNA-binding affinity of Cys2His2 zinc finger proteins by linker phosphorylation. Proc Natl Acad Sci U S A. 2004;101:7589–93.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Handel EM, Alwin S, Cathomen T. Expanding or restricting the target site repertoire of zinc-finger nucleases: the inter-domain linker as a major determinant of target site selectivity. Mol Ther. 2009;17:104–11.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  50. Schornack S, Meyer A, Römer P, Jordan T, Lahaye T. Gene-for-gene-mediated recognition of nuclear-targeted AvrBs3-like bacterial effector proteins. J Plant Physiol. 2006;163:256–72.

    Article  CAS  PubMed  Google Scholar 

  51. Boch J, Bonas U. Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annu Rev Phytopathol. 2009;48:419–36.

    Article  CAS  Google Scholar 

  52. Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, et al. A TALE nuclease architecture for efficient genome editing. Nat Biotechnol. 2011;29:143–8.

    Article  CAS  PubMed  Google Scholar 

  53. Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 2011;39, e82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Christian ML, Demorest ZL, Starker CG, Osborn MJ, Nyquist MD, Zhang Y, Carlson DF, Bradley P, Bogdanove AJ, Voytas DF. Targeting G with TAL effectors: a comparison of activities of TALENs constructed with NN and NK repeat variable Di-residues. PLoS One. 2012;7, e45383.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Mussolino C, Morbitzer R, Lutge F, Dannemann N, Lahaye T, Cathomen T. A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res. 2011;39:9283–93.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Bedell VM, Wang Y, Campbell JM, Poshusta TL, Starker CG, Krug Ii RG, Tan W, Penheiter SG, Ma AC, Leung AYH, et al. In vivo genome editing using a high-efficiency TALEN system. Nature. 2012;491:114–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Guilinger JP, Pattanayak V, Reyon D, Tsai SQ, Sander JD, Joung JK, Liu DR. Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity. Nat Methods. 2014;11:429–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Schierling B, Dannemann N, Gabsalilow L, Wende W, Cathomen T, Pingoud A. A novel zinc-finger nuclease platform with a sequence-specific cleavage module. Nucleic Acids Res. 2012;40:2623–38.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Beurdeley M, Bietz F, Li J, Thomas S, Stoddard T, Juillerat A, Zhang F, Voytas DF, Duchateau P, Silva GH. Compact designer TALENs for efficient genome engineering. Nat Commun. 2013;4:1762.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  60. Szczepek M, Brondani V, Buchel J, Serrano L, Segal DJ, Cathomen T. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol. 2007;25:786–93.

    Article  CAS  PubMed  Google Scholar 

  61. Miller JC, Holmes MC, Wang J, Guschin DY, Lee Y-L, Rupniewski I, Beausejour CM, Waite AJ, Wang NS, Kim KA, et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol. 2007;25:778–85.

    Article  CAS  PubMed  Google Scholar 

  62. Doyon Y, Vo TD, Mendel MC, Greenberg SG, Wang J, Xia DF, Miller JC, Urnov FD, Gregory PD, Holmes MC. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nat Methods. 2011;8:74–9.

    Article  CAS  PubMed  Google Scholar 

  63. Cade L, Reyon D, Hwang WY, Tsai SQ, Patel S, Khayter C, Joung JK, Sander JD, Peterson RT, Yeh J-RJ. Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs. Nucleic Acids Res. 2012;40:8001–10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Guo J, Gaj T, Barbas 3rd CF. Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases. J Mol Biol. 2010;400:96–107.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Ramirez CL, Certo MT, Mussolino C, Goodwin MJ, Cradick TJ, McCaffrey AP, Cathomen T, Scharenberg AM, Joung JK. Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects. Nucleic Acids Res. 2012;40:5560–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology. 2005;151:2551–61.

    Article  CAS  PubMed  Google Scholar 

  67. Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea. Science. 2010;327:167–70.

    Article  CAS  PubMed  Google Scholar 

  68. Marraffini LA, Sontheimer EJ. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat Rev Genet. 2010;11:181–90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Garneau JE, Dupuis M, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadán AH, Moineau S. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature. 2010;468:67–71.

    Article  CAS  PubMed  Google Scholar 

  70. Hale CR, Zhao P, Olson S, Duff MO, Graveley BR, Wells L, Terns RM, Terns MP. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell. 2009;139:945–56.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Segal DJ, Meckler JF. Genome engineering at the dawn of the golden age. Annu Rev Genomics Hum Genet. 2013;14:135–58.

    Article  CAS  PubMed  Google Scholar 

  72. Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A. 2012;109:E2579–86.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J. RNA-programmed genome editing in human cells. Elife. 2013;2, e00471.

    Article  PubMed  PubMed Central  Google Scholar 

  74. Cornu TI, Cathomen T. Quantification of zinc finger nuclease-associated toxicity. Methods Mol Biol. 2010;649:237–45.

    Article  CAS  PubMed  Google Scholar 

  75. Tesson L, Usal C, Ménoret S, Leung E, Niles BJ, Remy S, Santiago Y, Vincent AI, Meng X, Zhang L, et al. Knockout rats generated by embryo microinjection of TALENs. Nat Biotechnol. 2011;29:695–6.

    Article  CAS  PubMed  Google Scholar 

  76. Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, Cassady JP, Cost GJ, Zhang L, Santiago Y, Miller JC, et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol. 2011;29:731–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Cradick TJ, Fine EJ, Antico CJ, Bao G. CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. 2013;41:9584–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31:827–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Mojica FJ, Díez-Villaseñor C, García-Martínez J, Almendros C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology. 2009;155:733–40.

    Article  CAS  PubMed  Google Scholar 

  80. Shah SA, Erdmann S, Mojica FJ, Garrett RA. Protospacer recognition motifs: mixed identities and functional diversity. RNA Biol. 2013;10:891–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Horvath P, Romero DA, Coûté-Monvoisin AC, Richards M, Deveau H, Moineau S, Boyaval P, Fremaux C, Barrangou R. Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. J Bacteriol. 2008;190:1401–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Fischer S, Maier LK, Stoll B, Brendel J, Fischer E, Pfeiffer F, Dyall-Smith M, Marchfelder A. An archaeal immune system can detect multiple protospacer adjacent motifs (PAMs) to target invader DNA. J Biol Chem. 2012;287:33351–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Cho SW, Kim S, Kim Y, Kweon J, Kim HS, Bae S, Kim J-S. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 2013;24:132–41.

    Article  PubMed  CAS  Google Scholar 

  84. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014;156:935–49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature. 2014;507:62–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Ran FA, Hsu PD, Lin C-Y, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013;154:1380–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L, Church GM. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol. 2013;31:833–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Guilinger JP, Thompson DB, Liu DR. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat Biotechnol. 2014;32:577–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee MJ, Joung JK. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol. 2014;32:569–76.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, Osawa H, Kashiwagi S, Fukami K, Miyata T, Miyoshi H, et al. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell. 2008;132:487–98.

    Article  CAS  PubMed  Google Scholar 

  91. Zembruski NC, Stache V, Haefeli WE, Weiss J. 7-Aminoactinomycin D for apoptosis staining in flow cytometry. Anal Biochem. 2012;429:79–81.

    Article  CAS  PubMed  Google Scholar 

  92. Hockemeyer D, Soldner F, Beard C, Gao Q, Mitalipova M, DeKelver RC, Katibah GE, Amora R, Boydston EA, Zeitler B, et al. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol. 2009;27:851–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Perez EE, Wang J, Miller JC, Jouvenot Y, Kim KA, Liu O, Wang N, Lee G, Bartsevich VV, Lee Y-L, et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol. 2008;26:808–16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Cradick TJ, Ambrosini G, Iseli C, Bucher P, McCaffrey AP. ZFN-site searches genomes for zinc finger nuclease target sites and off-target sites. BMC Bioinformatics. 2011;12:152.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol. 2013;31:839–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Wang X, Wang Y, Wu X, Wang J, Wang Y, Qiu Z, Chang T, Huang H, Lin R-J, Yee J-K. Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors. Nat Biotechnol. 2015;33:175–8.

    Article  CAS  PubMed  Google Scholar 

  97. Wu X, Scott DA, Kriz AJ, Chiu AC, Hsu PD, Dadon DB, Cheng AW, Trevino AE, Konermann S, Chen S, et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat Biotechnol. 2014;32:670–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. O’Geen H, Henry IM, Bhakta MS, Meckler JF, Segal DJ. A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture. Nucleic Acids Res. 2014;43:3389–404.

    Article  CAS  Google Scholar 

  99. Kuscu C, Arslan S, Singh R, Thorpe J, Adli M. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat Biotechnol. 2014;32:677–83.

    Article  CAS  PubMed  Google Scholar 

  100. Kim D, Bae S, Park J, Kim E, Kim S, Yu HR, Hwang J, Kim JI, Kim JS. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods. 2015;12:237–43.

    Article  CAS  PubMed  Google Scholar 

  101. Frock RL, Hu J, Meyers RM, Ho YJ, Kii E, Alt FW. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat Biotechnol. 2015;33:179–86.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Tsai SQ, Zheng Z, Nguyen NT, Liebers M, Topkar VV, Thapar V, Wyvekens N, Khayter C, Iafrate AJ, Le LP, et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 2014;33:187–97.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  103. Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 2013;31:822–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. Reyon D, Tsai SQ, Khayter C, Foden JA, Sander JD, Joung JK. FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol. 2012;30:460–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Abarrategui-Pontes C, Creneguy A, Thinard R, Fine EJ, Thepenier V, Fournier LRL, Cradick TJ, Bao G, Tesson L, Podevin G, et al. Codon swapping of zinc finger nucleases confers expression in primary cells and in vivo from a single lentiviral vector. Curr Gene Ther. 2014;14:365–76.

    Article  PubMed  CAS  Google Scholar 

  106. Grau J, Boch J, Posch S. TALENoffer: genome-wide TALEN off-target prediction. Bioinformatics. 2013;29:2931–2.

    Article  CAS  PubMed  Google Scholar 

  107. Doyle EL, Booher NJ, Standage DS, Voytas DF, Brendel VP, Vandyk JK, Bogdanove AJ. TAL effector-nucleotide targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction. Nucleic Acids Res. 2012;40:W117–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Cradick TJ, Qui 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:e214.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Lin Y, Cradick TJ, Brown MT, Deshmukh H, Ranjan P, Sarode N, Wile BM, Vertino PM, Stewart FJ, Bao G. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res. 2014;42:7473–85.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. Xie 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:e100448.

    Article  PubMed  PubMed Central  Google Scholar 

  111. Babu M, Beloglazova N, Flick R, Graham C, Skarina T, Nocek B, Gagarinova A, Pogoutse O, Brown G, Binkowski A, et al. A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair. Mol Microbiol. 2011;79:484–502.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins A, Iyer V, Huang X, et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Methods. 2014;11:399–402.

    Article  CAS  PubMed  Google Scholar 

  113. Xiao A, Cheng Z, Kong L, Zhu Z, Lin S, Gao G, Zhang B. CasOT: a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics. 2014;21:2014.

    Google Scholar 

  114. Sander JD, Maeder ML, Reyon D, Voytas DF, Joung JK, Dobbs D. ZiFiT (Zinc Finger Targeter): an updated zinc finger engineering tool. Nucleic Acids Res. 2010;38(Suppl):W462–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Ma M, Ye AY, Zheng W, Kong L. A guide RNA sequence design platform for the CRISPR/Cas9 system for model organism genomes. Biomed Res Int. 2013;2013:270805.

    PubMed  PubMed Central  Google Scholar 

  116. Montague 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:W401–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. Guschin DY, Waite AJ, Katibah GE, Miller JC, Holmes MC, Rebar EJ. A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol. 2010;649:247–56.

    Article  CAS  PubMed  Google Scholar 

  118. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protocols. 2013;8:2281–308.

    Article  CAS  PubMed  Google Scholar 

  119. Fine EJ, Cradick TJ, Bao G. Identification of Off-Target Cleavage Sites of Zinc Finger Nucleases and TAL Effector Nucleases Using Predictive Models. In: Storici F, editor, Gene correction. Totowa: Humana Press; 2014. Vol. 1114, pp. 371–83.

    Google Scholar 

  120. Zhu 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, e108424.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  121. Bae 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;30:1473–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by the National Institutes of Health as an NIH Nanomedicine Development Center Award [PN2EY018244 to GB]. E.J.F. was additionally supported by the National Science Foundation Graduate Research Fellowship [DGE-1148903].

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Authors and Affiliations

  1. Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Dr., Atlanta, GA, 30332, USA

    Eli J. Fine, Thomas James Cradick & Gang Bao Ph.D.

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  1. Eli J. Fine
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  2. Thomas James Cradick
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  3. Gang Bao Ph.D.
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Correspondence to Gang Bao Ph.D. .

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Editors and Affiliations

  1. Inst for Cell & Gene Therapy, Universitaetsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany

    Toni Cathomen

  2. University of North Carolina, Chapel Hill, North Carolina, USA

    Matthew Hirsch

  3. Stanford Medical School, Palo Alto, California, USA

    Matthew Porteus

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© 2016 American Society of Gene and Cell Therapy

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Fine, E.J., Cradick, T.J., Bao, G. (2016). Strategies to Determine Off-Target Effects of Engineered Nucleases. In: Cathomen, T., Hirsch, M., Porteus, M. (eds) Genome Editing. Advances in Experimental Medicine and Biology(). Springer, New York, NY. https://doi.org/10.1007/978-1-4939-3509-3_11

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