Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9

  • Koo, Taeyoung (Center for Genome Engineering, Institute for Basic Science) ;
  • Lee, Jungjoon (The Institute of Molecular Biology and Genetics) ;
  • Kim, Jin-Soo (Center for Genome Engineering, Institute for Basic Science)
  • Received : 2015.04.17
  • Accepted : 2015.04.30
  • Published : 2015.06.30


Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid off-target mutations.


Supported by : IBS


  1. Veres, A., Gosis, B.S., Ding, Q., Collins, R., Ragavendran, A., Brand, H., Erdin, S., Cowan, C.A., Talkowski, M.E., and Musunuru, K. (2014). Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing. Cell Stem Cell 15, 27-30.
  2. Vouillot, L., Thelie, A., and Pollet, N. (2015). Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3 (Bethesda) 5, 407-415.
  3. Wu, X., Scott, D.A., Kriz, A.J., Chiu, A.C., Hsu, P.D., Dadon, D.B., Cheng, A.W., Trevino, A.E., Konermann, S., Chen, S., et al. (2014). Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol. 32, 670-676.
  4. Yusa, K., Rashid, S.T., Strick-Marchand, H., Varela, I., Liu, P.Q., Paschon, D.E., Miranda, E., Ordonez, A., Hannan, N.R., Rouhani, F.J., et al. (2011). Targeted gene correction of alpha1-antitrypsin deficiency in induced pluripotent stem cells. Nature 478, 391-394.
  5. Zhu, X., Xu, Y., Yu, S., Lu, L., Ding, M., Cheng, J., Song, G., Gao, X., Yao, L., Fan, D., et al. (2014). An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system. Sci. Rep. 4, 6420.
  6. Zuris, J.A., Thompson, D.B., Shu, Y., Guilinger, J.P., Bessen, J.L., Hu, J.H., Maeder, M.L., Joung, J.K., Chen, Z.Y. and Liu, D.R. (2015). Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73-80.
  7. Mussolino, C., Morbitzer, R., Lutge, F., Dannemann, N., Lahaye, T. and Cathomen, T. (2011). A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res. 39, 9283-9293.
  8. Parant, J.M., George, S.A., Pryor, R., Wittwer, C.T., and Yost, H.J. (2009). A rapid and efficient method of genotyping zebrafish mutants. Dev. Dyn. 238, 3168-3174.
  9. Park, C.Y., Kim, J., Kweon, J., Son, J.S., Lee, J.S., Yoo, J.E., Cho, S.R., Kim, J.H., Kim, J.S., and Kim, D.W. (2014). Targeted inversion and reversion of the blood coagulation factor 8 gene in human iPS cells using TALENs. Proc. Natl. Acad. Sci. USA 111, 9253-9258.
  10. Pattanayak, V., Ramirez, C.L., Joung, J.K., and Liu, D.R. (2011). Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat. Methods 8, 765-770.
  11. Pattanayak, V., Lin, S., Guilinger, J.P., Ma, E., Doudna, J.A., and Liu, D.R. (2013). High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839-843.
  12. Ramakrishna, S., Kwaku Dad, A.B., Beloor, J., Gopalappa, R., Lee, S.K., and Kim, H. (2014). Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res. 24, 1020-1027.
  13. Ran, F.A., Hsu, P.D., Lin, C.Y., Gootenberg, J.S., Konermann, S., Trevino, A.E., Scott, D.A., Inoue, A., Matoba, S., Zhang, Y., et al. (2013). Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380-1389.
  14. Ran, F.A., Cong, L., Yan, W.X., Scott, D.A., Gootenberg, J.S., Kriz, A.J., Zetsche, B., Shalem, O., Wu, X., Makarova, K.S., et al. (2015). In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186-191.
  15. Smith, C., Gore, A., Yan, W., Abalde-Atristain, L., Li, Z., He, C., Wang, Y., Brodsky, R.A., Zhang, K., Cheng, L., et al. (2014). Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs. Cell Stem Cell 15, 12-13.
  16. Tebas, P., Stein, D., Tang, W.W., Frank, I., Wang, S.Q., Lee, G., Spratt, S.K., Surosky, R.T., Giedlin, M.A., Nichol, G., et al. (2014). Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl. J. Med. 370, 901-910.
  17. Tsai, S.Q., Zheng, Z., Nguyen, N.T., Liebers, M., Topkar, V.V., Thapar, V., Wyvekens, N., Khayter, C., Iafrate, A.J., Le, L.P., et al. (2015). GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187-197.
  18. Urnov, F.D., Miller, J.C., Lee, Y.L., Beausejour, C.M., Rock, J.M., Augustus, S., Jamieson, A.C., Porteus, M.H., Gregory, P.D., and Holmes, M.C. (2005). Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646-651.
  19. Kim, E., Kim, S., Kim, D.H., Choi, B.S., Choi, I.Y., and Kim, J.S. (2012). Precision genome engineering with programmable DNA-nicking enzymes. Genome Res. 22, 1327-1333.
  20. Kim, Y., Kweon, J., Kim, A., Chon, J.K., Yoo, J.Y., Kim, H.J., Kim, S., Lee, C., Jeong, E., Chung, E., et al. (2013a). A library of TAL effector nucleases spanning the human genome. Nat. Biotechnol. 31, 251-258.
  21. Kim, Y., Kweon, J., and Kim, J.S. (2013b). TALENs and ZFNs are associated with different mutation signatures. Nat. Methods 10, 185.
  22. Kim, Y.K., Wee, G., Park, J., Kim, J., Baek, D., Kim, J.S., and Kim, V.N. (2013c). TALEN-based knockout library for human microRNAs. Nat. Struct. Mol. Biol. 20, 1458-1464.
  23. Kim, J.M., Kim, D., Kim, S., and Kim, J.S. (2014a). Genotyping with CRISPR-Cas-derived RNA-guided endonucleases. Nat. Commun. 5, 3157.
  24. Kim, S., Kim, D., Cho, S.W., Kim, J., and Kim, J.S. (2014b). Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012- 1019.
  25. Kim, D., Bae, S., Park, J., Kim, E., Kim, S., Yu, H.R., Hwang, J., Kim, J.I. and Kim, J.S. (2015). Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat. Methods 12, 237-243.
  26. Kuscu, C., Arslan, S., Singh, R., Thorpe, J., and Adli, M. (2014). Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat. Biotechnol. 32, 677-683.
  27. Lee, H.J., Kim, E., and Kim, J.S. (2010). Targeted chromosomal deletions in human cells using zinc finger nucleases. Genome Res. 20, 81-89.
  28. Lee, H.J., Kweon, J., Kim, E., Kim, S. and Kim, J.S. (2012) .Targeted chromosomal duplications and inversions in the human genome using zinc finger nucleases. Genome Res. 22, 539-548.
  29. Lin, Y., Cradick, T.J., Brown, M.T., Deshmukh, H., Ranjan, P., Sarode, N., Wile, B.M., Vertino, P.M., Stewart, F.J. and Bao, G. (2014). CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res. 42, 7473-7485.
  30. Mali, P., Aach, J., Stranges, P.B., Esvelt, K.M., Moosburner, M., Kosuri, S., Yang, L. and Church, G.M. (2013a). CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833-838.
  31. Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E., Norville, J.E., and Church, G.M. (2013b). RNA-guided human genome engineering via Cas9. Science 339, 823-826.
  32. Miller, J.C., Tan, S., Qiao, G., Barlow, K.A., Wang, J., Xia, D.F., Meng, X., Paschon, D.E., Leung, E., Hinkley, S.J., et al. (2011). A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29, 143-148.
  33. Moscou, M.J., and Bogdanove, A.J. (2009). A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501.
  34. Bae, S., Park, J., and Kim, J.S. (2014). Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473-1475.
  35. Bibikova, M., Beumer, K., Trautman, J.K., and Carroll, D. (2003). Enhancing gene targeting with designed zinc finger nucleases. Science 300, 764.
  36. Bitinaite, J., Wah, D.A., Aggarwal, A.K., and Schildkraut, I. (1998).FokI dimerization is required for DNA cleavage. Proc. Natl. Acad. Sci. USA 95, 10570-10575.
  37. Boch, J., Scholze, H., Schornack, S., Landgraf, A., Hahn, S., Kay, S., Lahaye, T., Nickstadt, A., and Bonas, U. (2009). Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509-1512.
  38. Brunet, E., Simsek, D., Tomishima, M., DeKelver, R., Choi, V.M., Gregory, P., Urnov, F., Weinstock, D.M., and Jasin, M. (2009). Chromosomal translocations induced at specified loci in human stem cells. Proc. Natl. Acad. Sci. USA 106, 10620-10625.
  39. Cho, S.W., Kim, S., Kim, J.M., and Kim, J.S. (2013a). Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230-232.
  40. Cho, S.W., Lee, J., Carroll, D., Kim, J.S., and Lee, J. (2013b). Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics 195, 1177-1180.
  41. Cho, S.W., Kim, S., Kim, Y., Kweon, J., Kim, H.S., Bae, S., and Kim, J.S. (2014). Analysis of off-target effects of CRISPR/Casderived RNA-guided endonucleases and nickases. Genome Res. 24, 132-141.
  42. Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823.
  43. Cradick, T.J., Fine, E.J., Antico, C.J., and Bao, G. (2013) CRISPR/as9 systems targeting beta-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. 41, 9584-9592.
  44. Crosetto, N., Mitra, A., Silva, M.J., Bienko, M., Dojer, N., Wang, Q., Karaca, E., Chiarle, R., Skrzypczak, M., Ginalski, K., et al. (2013). Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat. Methods 10, 361-365.
  45. Frock, R.L., Hu, J., Meyers, R.M., Ho, Y.J., Kii, E., and Alt, F.W. (2015). Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat. Biotechnol. 33, 179-186.
  46. Fu, Y., Foden, J.A., Khayter, C., Maeder, M.L., Reyon, D., Joung, J.K., and Sander, J.D. (2013). High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822-826.
  47. Fu, Y., Sander, J.D., Reyon, D., Cascio, V.M., and Joung, J.K. (2014). Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279-284.
  48. Gabriel, R., Lombardo, A., Arens, A., Miller, J.C., Genovese, P., Kaeppel, C., Nowrouzi, A., Bartholomae, C.C., Wang, J., Friedman, G., et al. (2011). An unbiased genome-wide analysis of zincfinger nuclease specificity. Nat. Biotechnol. 29, 816-823.
  49. Hendel, A., Kildebeck, E.J., Fine, E.J., Clark, J.T., Punjya, N., Sebastiano, V., Bao, G., and Porteus, M.H. (2014). Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing. Cell Rep. 7, 293-305.
  50. Hsu, P.D., Scott, D.A., Weinstein, J.A., Ran, F.A., Konermann, S., Agarwala, V., Li, Y., Fine, E.J., Wu, X., Shalem, O., et al. (2013). DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827-832.
  51. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., and Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821.
  52. Jinek, M., East, A., Cheng, A., Lin, S., Ma, E., and Doudna, J. (2013). RNA-programmed genome editing in human cells. Elife 2, e00471.
  53. Kim, H., and Kim, J.S. (2014). A guide to genome engineering with programmable nucleases. Nat. Rev. Genet. 15, 321-334.
  54. Kim, Y.G., Cha, J., and Chandrasegaran, S. (1996). Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. USA 93, 1156-1160.
  55. Kim, H.J., Lee, H.J., Kim, H., Cho, S.W., and Kim, J.S. (2009). Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly. Genome Res. 19, 1279-1288.
  56. Kim, J.S., Lee, H.J., and Carroll, D. (2010). Genome editing with modularly assembled zinc-finger nucleases. Nat. Methods 7, 91; author reply 91-92.
  57. Kim, H., Um, E., Cho, S.R., Jung, C., and Kim, J.S. (2011). Surrogate reporters for enrichment of cells with nucleaseinduced mutations. Nat. Methods 8, 941-943.

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