Table of Contents Author Guidelines Submit a Manuscript
International Journal of Genomics
Volume 2018, Article ID 1652567, 12 pages
https://doi.org/10.1155/2018/1652567
Review Article

Protein Engineering Strategies to Expand CRISPR-Cas9 Applications

1Department of Biology, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, University of São Paulo, São Paulo, SP, Brazil
2Department of Biochemistry and Immunology, Faculdade de Medicina de Ribeirão Preto, University of São Paulo, São Paulo, SP, Brazil
3Department of Chemistry, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, University of São Paulo, São Paulo, SP, Brazil

Correspondence should be addressed to Lucas F. Ribeiro; rb.psu@oriebirfsacul

Received 21 March 2018; Accepted 6 June 2018; Published 2 August 2018

Academic Editor: Raul A. Platero

Copyright © 2018 Lucas F. Ribeiro et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Linked References

  1. P. Mali, K. M. Esvelt, and G. M. Church, “Cas9 as a versatile tool for engineering biology,” Nature Methods, vol. 10, no. 10, pp. 957–963, 2013. View at Publisher · View at Google Scholar · View at Scopus
  2. P. D. Hsu, E. S. Lander, and F. Zhang, “Development and applications of CRISPR-Cas9 for genome engineering,” Cell, vol. 157, no. 6, pp. 1262–1278, 2014. View at Publisher · View at Google Scholar · View at Scopus
  3. M. F. Copeland, M. C. Politz, and B. F. Pfleger, “Application of TALEs, CRISPR/Cas and sRNAs as trans-acting regulators in prokaryotes,” Current Opinion in Biotechnology, vol. 29, pp. 46–54, 2014. View at Publisher · View at Google Scholar · View at Scopus
  4. R. Barrangou and J. A. Doudna, “Applications of CRISPR technologies in research and beyond,” Nature Biotechnology, vol. 34, no. 9, pp. 933–941, 2016. View at Publisher · View at Google Scholar · View at Scopus
  5. R. Barrangou and P. Horvath, “A decade of discovery: CRISPR functions and applications,” Nature Microbiology, vol. 2, article 17092, 2017. View at Publisher · View at Google Scholar · View at Scopus
  6. S. L. Morgan, N. C. Mariano, A. Bermudez et al., “Manipulation of nuclear architecture through CRISPR-mediated chromosomal looping,” Nature Communications, vol. 8, 2017. View at Publisher · View at Google Scholar · View at Scopus
  7. N. Hao, K. E. Shearwin, and I. B. Dodd, “Programmable DNA looping using engineered bivalent dCas9 complexes,” Nature Communications, vol. 8, no. 1, p. 1628, 2017. View at Publisher · View at Google Scholar · View at Scopus
  8. M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, and E. Charpentier, “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, vol. 337, no. 6096, pp. 816–821, 2012. View at Publisher · View at Google Scholar · View at Scopus
  9. F. J. M. Mojica, C. Díez-Villaseñor, J. García-Martínez, and C. Almendros, “Short motif sequences determine the targets of the prokaryotic CRISPR defence system,” Microbiology, vol. 155, no. 3, pp. 733–740, 2009. View at Publisher · View at Google Scholar · View at Scopus
  10. D. Rath, L. Amlinger, A. Rath, and M. Lundgren, “The CRISPR-Cas immune system: biology, mechanisms and applications,” Biochimie, vol. 117, pp. 119–128, 2015. View at Publisher · View at Google Scholar · View at Scopus
  11. I. Fonfara, A. le Rhun, K. Chylinski et al., “Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems,” Nucleic Acids Research, vol. 42, no. 4, pp. 2577–2590, 2014. View at Publisher · View at Google Scholar · View at Scopus
  12. K. M. Esvelt, P. Mali, J. L. Braff, M. Moosburner, S. J. Yaung, and G. M. Church, “Orthogonal Cas9 proteins for RNA-guided gene regulation and editing,” Nature Methods, vol. 10, no. 11, pp. 1116–1121, 2013. View at Publisher · View at Google Scholar · View at Scopus
  13. F. J. M. Mojica and F. Rodriguez-Valera, “The discovery of CRISPR in archaea and bacteria,” The FEBS Journal, vol. 283, no. 17, pp. 3162–3169, 2016. View at Publisher · View at Google Scholar · View at Scopus
  14. F. J. M. Mojica and L. Montoliu, “On the origin of CRISPR-Cas technology: from prokaryotes to mammals,” Trends in Microbiology, vol. 24, no. 10, pp. 811–820, 2016. View at Publisher · View at Google Scholar · View at Scopus
  15. D. Burstein, L. B. Harrington, S. C. Strutt et al., “New CRISPR-Cas systems from uncultivated microbes,” Nature, vol. 542, no. 7640, pp. 237–241, 2017. View at Publisher · View at Google Scholar · View at Scopus
  16. F. Jiang and J. A. Doudna, “CRISPR–Cas9 structures and mechanisms,” Annual Review of Biophysics, vol. 46, no. 1, pp. 505–529, 2017. View at Publisher · View at Google Scholar · View at Scopus
  17. H. Chen, J. Choi, and S. Bailey, “Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease,” The Journal of Biological Chemistry, vol. 289, no. 19, pp. 13284–13294, 2014. View at Publisher · View at Google Scholar · View at Scopus
  18. G. Gasiunas, R. Barrangou, P. Horvath, and V. Siksnys, “Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria,” Proceedings of the National Academy of Sciences, vol. 109, no. 39, pp. E2579–E2586, 2012. View at Publisher · View at Google Scholar · View at Scopus
  19. C. Anders, O. Niewoehner, A. Duerst, and M. Jinek, “Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease,” Nature, vol. 513, no. 7519, pp. 569–573, 2014. View at Publisher · View at Google Scholar · View at Scopus
  20. M. Jinek, F. Jiang, D. W. Taylor et al., “Structures of Cas9 endonucleases reveal RNA-mediated conformational activation,” Science, vol. 343, no. 6176, article 1247997, 2014. View at Publisher · View at Google Scholar · View at Scopus
  21. S. H. Sternberg, S. Redding, M. Jinek, E. C. Greene, and J. A. Doudna, “DNA interrogation by the CRISPR RNA-guided endonuclease Cas9,” Nature, vol. 507, no. 7490, pp. 62–67, 2014. View at Publisher · View at Google Scholar · View at Scopus
  22. M. D. Szczelkun, M. S. Tikhomirova, T. Sinkunas et al., “Direct observation of R-loop formation by single RNA-guided Cas9 and cascade effector complexes,” Proceedings of the National Academy of Sciences, vol. 111, no. 27, pp. 9798–9803, 2014. View at Publisher · View at Google Scholar · View at Scopus
  23. L. Cong, F. A. Ran, D. Cox et al., “Multiplex genome engineering using CRISPR/Cas systems,” Science, vol. 339, no. 6121, pp. 819–823, 2013. View at Publisher · View at Google Scholar · View at Scopus
  24. W. Jiang, D. Bikard, D. Cox, F. Zhang, and L. A. Marraffini, “RNA-guided editing of bacterial genomes using CRISPR-Cas systems,” Nature Biotechnology, vol. 31, no. 3, pp. 233–239, 2013. View at Publisher · View at Google Scholar · View at Scopus
  25. V. Pattanayak, S. Lin, J. P. Guilinger, E. Ma, J. A. Doudna, and D. R. Liu, “High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity,” Nature Biotechnology, vol. 31, no. 9, pp. 839–843, 2013. View at Publisher · View at Google Scholar · View at Scopus
  26. G. J. Gibson and M. Yang, “What rheumatologists need to know about CRISPR/Cas9,” Nature Reviews Rheumatology, vol. 13, no. 4, pp. 205–216, 2017. View at Publisher · View at Google Scholar · View at Scopus
  27. J. Luo, “CRISPR/Cas9: from genome engineering to cancer drug discovery,” Trends in Cancer, vol. 2, no. 6, pp. 313–324, 2016. View at Publisher · View at Google Scholar · View at Scopus
  28. S. Q. Tsai and J. K. Joung, “Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases,” Nature Reviews Genetics, vol. 17, no. 5, pp. 300–312, 2016. View at Publisher · View at Google Scholar · View at Scopus
  29. J. A. Doudna and C. A. Gersbach, “Genome editing: the end of the beginning,” Genome Biology, vol. 16, no. 1, p. 292, 2015. View at Publisher · View at Google Scholar · View at Scopus
  30. D. B. T. Cox, R. J. Platt, and F. Zhang, “Therapeutic genome editing: prospects and challenges,” Nature Medicine, vol. 21, no. 2, pp. 121–131, 2015. View at Publisher · View at Google Scholar · View at Scopus
  31. R. Aroul-Selvam, T. Hubbard, and R. Sasidharan, “Domain insertions in protein structures,” Journal of Molecular Biology, vol. 338, no. 4, pp. 633–641, 2004. View at Publisher · View at Google Scholar · View at Scopus
  32. M. Wang and G. Caetano-Anollés, “The evolutionary mechanics of domain organization in proteomes and the rise of modularity in the protein world,” Structure, vol. 17, no. 1, pp. 66–78, 2009. View at Publisher · View at Google Scholar · View at Scopus
  33. L. F. Ribeiro, T. D. Warren, and M. Ostermeier, “Construction of protein switches by domain insertion and directed evolution,” Methods in Molecular Biology, vol. 1596, pp. 43–55, 2017. View at Publisher · View at Google Scholar · View at Scopus
  34. J. Tullman, N. Nicholes, M. R. Dumont, L. F. Ribeiro, and M. Ostermeier, “Enzymatic protein switches built from paralogous input domains,” Biotechnology and Bioengineering, vol. 113, no. 4, pp. 852–858, 2016. View at Publisher · View at Google Scholar · View at Scopus
  35. L. F. Ribeiro, N. Nicholes, J. Tullman et al., “Insertion of a xylanase in xylose binding protein results in a xylose-stimulated xylanase,” Biotechnology for Biofuels, vol. 8, no. 1, p. 118, 2015. View at Publisher · View at Google Scholar · View at Scopus
  36. L. F. Ribeiro, J. Tullman, N. Nicholes et al., “A xylose-stimulated xylanase-xylose binding protein chimera created by random nonhomologous recombination,” Biotechnology for Biofuels, vol. 9, no. 1, p. 119, 2016. View at Publisher · View at Google Scholar · View at Scopus
  37. M. Ostermeier, “Engineering allosteric protein switches by domain insertion,” Protein Engineering, Design and Selection, vol. 18, no. 8, pp. 359–364, 2005. View at Publisher · View at Google Scholar · View at Scopus
  38. V. Stein and K. Alexandrov, “Synthetic protein switches: design principles and applications,” Trends in Biotechnology, vol. 33, no. 2, pp. 101–110, 2015. View at Publisher · View at Google Scholar · View at Scopus
  39. B. Chen, L. A. Gilbert, B. A. Cimini et al., “Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system,” Cell, vol. 155, no. 7, pp. 1479–1491, 2013. View at Publisher · View at Google Scholar · View at Scopus
  40. L. R. Polstein and C. A. Gersbach, “A light-inducible CRISPR-Cas9 system for control of endogenous gene activation,” Nature Chemical Biology, vol. 11, no. 3, pp. 198–200, 2015. View at Publisher · View at Google Scholar · View at Scopus
  41. M. F. Bolukbasi, A. Gupta, S. Oikemus et al., “DNA-binding-domain fusions enhance the targeting range and precision of Cas9,” Nature Methods, vol. 12, no. 12, pp. 1150–1156, 2015. View at Publisher · View at Google Scholar · View at Scopus
  42. B. Maji, C. L. Moore, B. Zetsche et al., “Multidimensional chemical control of CRISPR-Cas9,” Nature Chemical Biology, vol. 13, no. 1, pp. 9–11, 2017. View at Publisher · View at Google Scholar · View at Scopus
  43. M. Berrondo, M. Ostermeier, and J. J. Gray, “Structure prediction of domain insertion proteins from structures of individual domains,” Structure, vol. 16, no. 4, pp. 513–527, 2008. View at Publisher · View at Google Scholar · View at Scopus
  44. L. F. Ribeiro, G. P. Furtado, M. R. Lourenzoni et al., “Engineering bifunctional laccase-xylanase chimeras for improved catalytic performance,” The Journal of Biological Chemistry, vol. 286, no. 50, pp. 43026–43038, 2011. View at Publisher · View at Google Scholar · View at Scopus
  45. B. Pierre, J. W. Labonte, T. Xiong et al., “Molecular determinants for protein stabilization by insertional fusion to a thermophilic host protein,” Chembiochem, vol. 16, no. 16, pp. 2392–2402, 2015. View at Publisher · View at Google Scholar · View at Scopus
  46. C. S. Kim, B. Pierre, M. Ostermeier, L. L. Looger, and J. R. Kim, “Enzyme stabilization by domain insertion into a thermophilic protein,” Protein Engineering Design & Selection, vol. 22, no. 10, pp. 615–623, 2009. View at Publisher · View at Google Scholar · View at Scopus
  47. X. H. Zhang, L. Y. Tee, X. G. Wang, Q. S. Huang, and S. H. Yang, “Off-target effects in CRISPR/Cas9-mediated genome engineering,” Molecular Therapy - Nucleic Acids, vol. 4, article e264, 2015. View at Publisher · View at Google Scholar · View at Scopus
  48. S. Q. Tsai, N. Wyvekens, C. Khayter et al., “Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing,” Nature Biotechnology, vol. 32, no. 6, pp. 569–576, 2014. View at Publisher · View at Google Scholar · View at Scopus
  49. J. P. Guilinger, D. B. Thompson, and D. R. Liu, “Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification,” Nature Biotechnology, vol. 32, no. 6, pp. 577–582, 2014. View at Publisher · View at Google Scholar · View at Scopus
  50. A. K. Singh Gautam, S. Balakrishnan, and P. Venkatraman, “Direct ubiquitin independent recognition and degradation of a folded protein by the eukaryotic proteasomes-origin of intrinsic degradation signals,” PLoS One, vol. 7, no. 4, p. e34864, 2012. View at Publisher · View at Google Scholar · View at Scopus
  51. A. C. Komor, Y. B. Kim, M. S. Packer, J. A. Zuris, and D. R. Liu, “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage,” Nature, vol. 533, no. 7603, pp. 420–424, 2016. View at Publisher · View at Google Scholar · View at Scopus
  52. N. M. Gaudelli, A. C. Komor, H. A. Rees et al., “Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage,” Nature, vol. 551, no. 7681, pp. 464–471, 2017. View at Publisher · View at Google Scholar · View at Scopus
  53. H. A. Rees, A. C. Komor, W. H. Yeh et al., “Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery,” Nature Communications, vol. 8, 2017. View at Publisher · View at Google Scholar · View at Scopus
  54. Y. B. Kim, A. C. Komor, J. M. Levy, M. S. Packer, K. T. Zhao, and D. R. Liu, “Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions,” Nature Biotechnology, vol. 35, no. 4, pp. 371–376, 2017. View at Publisher · View at Google Scholar · View at Scopus
  55. J. M. Gerhke, O. R. Cervantes, M. Kendell Clement, L. Pinello, and J. Keith Joung, “High-precision CRISPR-Cas9 base editors with minimized bystander and off-target mutations,” bioRxiv, article 273938, 2018. View at Publisher · View at Google Scholar
  56. Y. Nihongaki, S. Yamamoto, F. Kawano, H. Suzuki, and M. Sato, “CRISPR-Cas9-based photoactivatable transcription system,” Chemistry & Biology, vol. 22, no. 2, pp. 169–174, 2015. View at Publisher · View at Google Scholar · View at Scopus
  57. Y. Gao, X. Xiong, S. Wong, E. J. Charles, W. A. Lim, and L. S. Qi, “Complex transcriptional modulation with orthogonal and inducible dCas9 regulators,” Nature Methods, vol. 13, no. 12, pp. 1043–1049, 2016. View at Publisher · View at Google Scholar · View at Scopus
  58. K. I. Liu, M. N. B. Ramli, C. W. A. Woo et al., “A chemical-inducible CRISPR-Cas9 system for rapid control of genome editing,” Nature Chemical Biology, vol. 12, no. 11, pp. 980–987, 2016. View at Publisher · View at Google Scholar · View at Scopus
  59. A. Amabile, A. Migliara, P. Capasso et al., “Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing,” Cell, vol. 167, no. 1, pp. 219–232.e14, 2016. View at Publisher · View at Google Scholar · View at Scopus
  60. P. Stepper, G. Kungulovski, R. Z. Jurkowska et al., “Efficient targeted DNA methylation with chimeric dCas9-Dnmt3a-Dnmt3L methyltransferase,” Nucleic Acids Research, vol. 45, no. 4, pp. 1703–1713, 2017. View at Publisher · View at Google Scholar · View at Scopus
  61. A. Vojta, P. Dobrinić, V. Tadić et al., “Repurposing the CRISPR-Cas9 system for targeted DNA methylation,” Nucleic Acids Research, vol. 44, no. 12, pp. 5615–5628, 2016. View at Publisher · View at Google Scholar · View at Scopus
  62. J. I. McDonald, H. Celik, L. E. Rois et al., “Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation,” Biology Open, vol. 5, no. 6, pp. 866–874, 2016. View at Publisher · View at Google Scholar · View at Scopus
  63. T. Xiong, G. E. Meister, R. E. Workman et al., “Targeted DNA methylation in human cells using engineered dCas9-methyltransferases,” Scientific Reports, vol. 7, no. 1, p. 6732, 2017. View at Publisher · View at Google Scholar · View at Scopus
  64. N. A. Kearns, H. Pham, B. Tabak et al., “Functional annotation of native enhancers with a Cas9-histone demethylase fusion,” Nature Methods, vol. 12, no. 5, pp. 401–403, 2015. View at Publisher · View at Google Scholar · View at Scopus
  65. I. B. Hilton, A. M. D'Ippolito, C. M. Vockley et al., “Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers,” Nature Biotechnology, vol. 33, no. 5, pp. 510–517, 2015. View at Publisher · View at Google Scholar · View at Scopus
  66. T. Anton, S. Bultmann, H. Leonhardt, and Y. Markaki, “Visualization of specific DNA sequences in living mouse embryonic stem cells with a programmable fluorescent CRISPR/Cas system,” Nucleus, vol. 5, no. 2, pp. 163–172, 2014. View at Publisher · View at Google Scholar · View at Scopus
  67. H. Ma, A. Naseri, P. Reyes-Gutierrez, S. A. Wolfe, S. Zhang, and T. Pederson, “Multicolor CRISPR labeling of chromosomal loci in human cells,” Proceedings of the National Academy of Sciences, vol. 112, no. 10, pp. 3002–3007, 2015. View at Publisher · View at Google Scholar · View at Scopus
  68. N. H. Shah and T. W. Muir, “Inteins: nature’s gift to protein chemists,” Chemical Science, vol. 5, no. 2, pp. 446–461, 2014. View at Publisher · View at Google Scholar · View at Scopus
  69. S. H. Peck, I. Chen, and D. R. Liu, “Directed evolution of a small-molecule-triggered intein with improved splicing properties in mammalian cells,” Chemistry & Biology, vol. 18, no. 5, pp. 619–630, 2011. View at Publisher · View at Google Scholar · View at Scopus
  70. K. M. Davis, V. Pattanayak, D. B. Thompson, J. A. Zuris, and D. R. Liu, “Small molecule-triggered Cas9 protein with improved genome-editing specificity,” Nature Chemical Biology, vol. 11, no. 5, pp. 316–318, 2015. View at Publisher · View at Google Scholar · View at Scopus
  71. X. X. Zhou, X. Zou, H. K. Chung et al., “A single-chain photoswitchable CRISPR-Cas9 architecture for light-inducible gene editing and transcription,” ACS Chemical Biology, vol. 13, no. 2, pp. 443–448, 2017. View at Publisher · View at Google Scholar · View at Scopus
  72. X. X. Zhou, L. Z. Fan, P. Li, K. Shen, and M. Z. Lin, “Optical control of cell signaling by single-chain photoswitchable kinases,” Science, vol. 355, no. 6327, pp. 836–842, 2017. View at Publisher · View at Google Scholar · View at Scopus
  73. G. T. Hess, L. Frésard, K. Han et al., “Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells,” Nature Methods, vol. 13, no. 12, pp. 1036–1042, 2016. View at Publisher · View at Google Scholar · View at Scopus
  74. F. Richter, I. Fonfara, B. Bouazza et al., “Engineering of temperature- and light-switchable Cas9 variants,” Nucleic Acids Research, vol. 44, no. 20, pp. 10003–10014, 2016. View at Publisher · View at Google Scholar · View at Scopus
  75. B. L. Oakes, D. C. Nadler, A. Flamholz et al., “Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch,” Nature Biotechnology, vol. 34, no. 6, pp. 646–651, 2016. View at Publisher · View at Google Scholar · View at Scopus
  76. H. Nishimasu, F. A. Ran, P. D. Hsu et al., “Crystal structure of Cas9 in complex with guide RNA and target DNA,” Cell, vol. 156, no. 5, pp. 935–949, 2014. View at Publisher · View at Google Scholar · View at Scopus
  77. I. M. Slaymaker, L. Gao, B. Zetsche, D. A. Scott, W. X. Yan, and F. Zhang, “Rationally engineered Cas9 nucleases with improved specificity,” Science, vol. 351, no. 6268, pp. 84–88, 2016. View at Publisher · View at Google Scholar · View at Scopus
  78. B. P. Kleinstiver, V. Pattanayak, M. S. Prew et al., “High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects,” Nature, vol. 529, no. 7587, pp. 490–495, 2016. View at Publisher · View at Google Scholar · View at Scopus
  79. V. Siksnys and G. Gasiunas, “Rewiring Cas9 to target new PAM sequences,” Molecular Cell, vol. 61, no. 6, pp. 793-794, 2016. View at Publisher · View at Google Scholar · View at Scopus
  80. E. Zinn and L. H. Vandenberghe, “Adeno-associated virus: fit to serve,” Current Opinion in Virology, vol. 8, pp. 90–97, 2014. View at Publisher · View at Google Scholar · View at Scopus
  81. K. Chamberlain, J. M. Riyad, and T. Weber, “Expressing transgenes that exceed the packaging capacity of adeno-associated virus capsids,” Human Gene Therapy Methods, vol. 27, no. 1, pp. 1–12, 2016. View at Publisher · View at Google Scholar · View at Scopus
  82. A. V. Wright, S. H. Sternberg, D. W. Taylor et al., “Rational design of a split-Cas9 enzyme complex,” Proceedings of the National Academy of Sciences, vol. 112, no. 10, pp. 2984–2989, 2015. View at Publisher · View at Google Scholar · View at Scopus
  83. D. J. J. Truong, K. Kühner, R. Kühn et al., “Development of an intein-mediated split-Cas9 system for gene therapy,” Nucleic Acids Research, vol. 43, no. 13, pp. 6450–6458, 2015. View at Publisher · View at Google Scholar · View at Scopus
  84. Y. Nihongaki, F. Kawano, T. Nakajima, and M. Sato, “Photoactivatable CRISPR-Cas9 for optogenetic genome editing,” Nature Biotechnology, vol. 33, no. 7, pp. 755–760, 2015. View at Publisher · View at Google Scholar · View at Scopus
  85. B. Zetsche, S. E. Volz, and F. Zhang, “A split-Cas9 architecture for inducible genome editing and transcription modulation,” Nature Biotechnology, vol. 33, no. 2, pp. 139–142, 2015. View at Publisher · View at Google Scholar · View at Scopus
  86. B. P. Kleinstiver, M. S. Prew, S. Q. Tsai et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities,” Nature, vol. 523, no. 7561, pp. 481–485, 2015. View at Publisher · View at Google Scholar · View at Scopus
  87. B. P. Kleinstiver, M. S. Prew, S. Q. Tsai et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition,” Nature Biotechnology, vol. 33, no. 12, pp. 1293–1298, 2015. View at Publisher · View at Google Scholar · View at Scopus
  88. R. Heler, A. V. Wright, M. Vucelja, D. Bikard, J. A. Doudna, and L. A. Marraffini, “Mutations in Cas9 enhance the rate of acquisition of viral spacer sequences during the CRISPR-Cas immune response,” Molecular Cell, vol. 65, no. 1, pp. 168–175, 2017. View at Publisher · View at Google Scholar · View at Scopus
  89. J. H. Hu, S. M. Miller, M. H. Geurts et al., “Evolved Cas9 variants with broad PAM compatibility and high DNA specificity,” Nature, vol. 556, no. 7699, pp. 57–63, 2018. View at Publisher · View at Google Scholar
  90. F. Atroshi, A. Rizzo, T. Westermarck, and T. Ali-Vehmas, “Effects of tamoxifen, melatonin, coenzyme Q10, and L-carnitine supplementation on bacterial growth in the presence of mycotoxins,” Pharmacological Research, vol. 38, no. 4, pp. 289–295, 1998. View at Publisher · View at Google Scholar · View at Scopus
  91. X. Liu, E. Pisha, D. A. Tonetti et al., “Antiestrogenic and DNA damaging effects induced by tamoxifen and toremifene metabolites,” Chemical Research in Toxicology, vol. 16, no. 7, pp. 832–837, 2003. View at Publisher · View at Google Scholar · View at Scopus
  92. P. W. Fan, F. Zhang, and J. L. Bolton, “4-Hydroxylated metabolites of the antiestrogens tamoxifen and toremifene are metabolized to unusually stable quinone methides,” Chemical Research in Toxicology, vol. 13, no. 1, pp. 45–52, 2000. View at Publisher · View at Google Scholar · View at Scopus
  93. C. M. Nowak, S. Lawson, M. Zerez, and L. Bleris, “Guide RNA engineering for versatile Cas9 functionality,” Nucleic Acids Research, vol. 44, pp. 9555–9564, 2016. View at Publisher · View at Google Scholar · View at Scopus