Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The emerging and uncultivated potential of CRISPR technology in plant science

Abstract

The application of clustered regularly interspaced short palindromic repeats (CRISPR) for genetic manipulation has revolutionized life science over the past few years. CRISPR was first discovered as an adaptive immune system in bacteria and archaea, and then engineered to generate targeted DNA breaks in living cells and organisms. During the cellular DNA repair process, various DNA changes can be introduced. The diverse and expanding CRISPR toolbox allows programmable genome editing, epigenome editing and transcriptome regulation in plants. However, challenges in plant genome editing need to be fully appreciated and solutions explored. This Review intends to provide an informative summary of the latest developments and breakthroughs of CRISPR technology, with a focus on achievements and potential utility in plant biology. Ultimately, CRISPR will not only facilitate basic research, but also accelerate plant breeding and germplasm development. The application of CRISPR to improve germplasm is particularly important in the context of global climate change as well as in the face of current agricultural, environmental and ecological challenges.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Applications of CRISPR technology in plant cells.
Fig. 2: Repurposing CRISPR as a recruiting platform.
Fig. 3: Diverse CRISPR expression and multiplex systems.
Fig. 4: Revolutionizing plant breeding by combining CRISPR with other cutting-edge technologies.
Fig. 5: Genetic screens with CRISPR libraries in whole plants and plant cells.

References

  1. 1.

    Bibikova, M., Golic, M., Golic, K. G. & Carroll, D. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161, 1169–1175 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Christian, M. et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186, 757–761 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    CAS  Article  Google Scholar 

  4. 4.

    Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  Google Scholar 

  5. 5.

    Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Li, J.-F. et al. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat. Biotechnol. 31, 688–691 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Nekrasov, V., Staskawicz, B., Weigel, D., Jones, J. D. G. & Kamoun, S. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 691–693 (2013).

    Article  CAS  PubMed  Google Scholar 

  8. 8.

    Shan, Q. et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol. 31, 686–688 (2013).

    Article  CAS  PubMed  Google Scholar 

  9. 9.

    Chen, K., Wang, Y., Zhang, R., Zhang, H. & Gao, C. CRISPR/Cas genome editing and precision plant breeding in agriculture. Annu. Rev. Plant Biol. 70, 667–697 (2019).

    Article  CAS  PubMed  Google Scholar 

  10. 10.

    Zhang, Y., Massel, K., Godwin, I. D. & Gao, C. Applications and potential of genome editing in crop improvement. Genome Biol. 19, 210 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Ma, X., Zhu, Q., Chen, Y. & Liu, Y.-G. CRISPR/Cas9 platforms for genome editing in plants: developments and applications. Mol. Plant 9, 961–974 (2016).

    Article  CAS  PubMed  Google Scholar 

  12. 12.

    Schindele, P., Wolter, F. & Puchta, H. Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13. FEBS Lett. 592, 1954–1967 (2018).

    Article  CAS  PubMed  Google Scholar 

  13. 13.

    Yin, K., Gao, C. & Qiu, J.-L. Progress and prospects in plant genome editing. Nat. Plants 3, 17107 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Kim, J.-S. Precision genome engineering through adenine and cytosine base editing. Nat. Plants 4, 148–151 (2018).

    Article  CAS  PubMed  Google Scholar 

  15. 15.

    Huang, T. K. & Puchta, H. CRISPR/Cas-mediated gene targeting in plants: finally a turn for the better for homologous recombination. Plant Cell Rep. 38, 443–453 (2019).

    Article  CAS  PubMed  Google Scholar 

  16. 16.

    Mahas, A., Neal Stewart, C. & Mahfouz, M. M. Harnessing CRISPR/Cas systems for programmable transcriptional and post-transcriptional regulation. Biotechnol. Adv. 36, 295–310 (2018).

    Article  CAS  PubMed  Google Scholar 

  17. 17.

    Makarova, K. S. et al. An updated evolutionary classification of CRISPR–Cas systems. Nat. Rev. Microbiol. 13, 722–736 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Burstein, D. et al. New CRISPR–Cas systems from uncultivated microbes. Nature 542, 237–241 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Harrington, L. B. et al. Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science 362, 839–842 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Liu, J.-J. et al. CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature 566, 218 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Shmakov, S. et al. Discovery and functional characterization of diverse Class 2 CRISPR-Cas systems. Mol. Cell 60, 385–397 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Shmakov, S. et al. Diversity and evolution of class 2 CRISPR–Cas systems. Nat. Rev. Microbiol. 15, 169–182 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Yan, W. X. et al. Functionally diverse type V CRISPR-Cas systems. Science 363, 88–91 (2019).

    Article  CAS  Google Scholar 

  24. 24.

    Ma, X. et al. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol. Plant 8, 1274–1284 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Fauser, F., Schiml, S. & Puchta, H. Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J. 79, 348–359 (2014).

    Article  CAS  Google Scholar 

  26. 26.

    Wang, Z.-P. et al. Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol. 16, 144 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Lee, K. et al. Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize. Plant Biotechnol. J. 17, 839–842 (2019).

    Article  CAS  Google Scholar 

  28. 28.

    Michno, J.-M. et al. CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme. GM Crops Food 6, 243–252 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Ran, F. A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Kleinstiver, B. P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Anders, C., Bargsten, K. & Jinek, M. Structural plasticity of PAM recognition by engineered variants of the RNA-guided endonuclease Cas9. Mol. Cell 61, 895–902 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Nishimasu, H. et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 361, 1259–1262 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Hu, X. et al. Expanding the range of CRISPR/Cas9 genome editing in rice. Mol. Plant 9, 943–945 (2016).

    Article  CAS  PubMed  Google Scholar 

  37. 37.

    Hu, X., Meng, X., Liu, Q., Li, J. & Wang, K. Increasing the efficiency of CRISPR-Cas9-VQR precise genome editing in rice. Plant Biotechnol. J. 16, 292–297 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Wang, J. et al. xCas9 expands the scope of genome editing with reduced efficiency in rice. Plant Biotechnol. J. 17, 709–711 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Hua, K., Tao, X., Han, P., Wang, R. & Zhu, J.-K. Genome engineering in rice using Cas9 variants that recognize NG PAM sequences. Mol. Plant https://doi.org/10.1016/j.molp.2019.03.009 (2019).

  40. 40.

    Zhong, Z. et al. Improving plant genome editing with high-fidelity xCas9 and non-canonical PAM-targeting Cas9-NG. Mol. Plant https://doi.org/10.1016/j.molp.2019.03.011 (2019).

  41. 41.

    Li, J. et al. Plant genome editing using xCas9 with expanded PAM compatibility. J. Genet. Genomics https://doi.org/10.1016/j.jgg.2019.03.004 (2019).

  42. 42.

    Endo, M. et al. Genome editing in plants by engineered CRISPR–Cas9 recognizing NG PAM. Nat. Plants 5, 14–17 (2018).

    Google Scholar 

  43. 43.

    Ren, B. et al. Cas9-NG greatly expands the targeting scope of genome-editing toolkit by recognizing NG and other atypical PAMs in rice. Mol. Plant https://doi.org/10.1016/j.molp.2019.03.010 (2019).

  44. 44.

    Negishi, K. et al. An adenine base editor with expanded targeting scope using SpCas9-NGv1 in rice. Plant Biotechnol. J. https://doi.org/10.1111/pbi.13120 (2019).

  45. 45.

    Wang, M. et al. Optimizing baseeditors for improved efficiency and expanded editing scope in rice. Plant Biotechnol. J. https://doi.org/10.1111/pbi.13124 (2019).

  46. 46.

    Ge, Z. et al. Engineered xCas9 and SpCas9-NG variants broaden PAM recognition sites to generate mutations in Arabidopsis plants. Plant Biotechnol. J. https://doi.org/10.1111/pbi.13148 (2019).

  47. 47.

    Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Friedland, A. E. et al. Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications. Genome Biol. 16, 257 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Kleinstiver, B. P. et al. Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition. Nat. Biotechnol. 33, 1293–1298 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Kaya, H., Mikami, M., Endo, A., Endo, M. & Toki, S. Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9. Sci. Rep. 6, 26871 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Steinert, J., Schiml, S., Fauser, F. & Puchta, H. Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus. Plant J. 84, 1295–1305 (2015).

    Article  CAS  PubMed  Google Scholar 

  52. 52.

    Jia, H., Xu, J., Orbović, V., Zhang, Y. & Wang, N. Editing Citrus genome via SaCas9/sgRNA system. Front. Plant Sci. 8, 2135 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Müller, M. et al. Streptococcus thermophilus CRISPR-Cas9 systems enable specific editing of the human genome. Mol. Ther. 24, 636–644 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Lee, C. M., Cradick, T. J. & Bao, G. The Neisseria meningitidis CRISPR-Cas9 system enables specific genome editing in mammalian cells. Mol. Ther. 24, 645–654 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Hirano, H. et al. Structure and engineering of Francisella novicida Cas9. Cell 164, 950–961 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Esvelt, K. M. et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat. Methods 10, 1116–21 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Kim, E. et al. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat. Commun. 8, 14500 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Chatterjee, P., Jakimo, N. & Jacobson, J. M. Minimal PAM specificity of a highly similar SpCas9 ortholog. Sci. Adv. 4, eaau0766 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Karvelis, T. et al. Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements. Genome Biol. 16, 253 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Rousseau, B. A., Hou, Z., Gramelspacher, M. J. & Zhang, Y. Programmable RNA cleavage and recognition by a natural CRISPR-Cas9 system from Neisseria meningitidis. Mol. Cell 69, 906–914 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. 61.

    Jakimo, N., Chatterjee, P., Nip, L. & Jacobson, J. M. A Cas9 with complete PAM recognition for adenine dinucleotides. Preprint at https://www.biorxiv.org/node/129129.abstract (2018).

  62. 62.

    Zetsche, B. et al. Cpf1 Is a single RNA-guided endonuclease of a Class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Yamano, T. et al. Crystal structure of Cpf1 in complex with guide RNA and target DNA. Cell 165, 949–962 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. 64.

    Kim, D. et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat. Biotechnol. 34, 863–868 (2016).

    Article  CAS  Google Scholar 

  65. 65.

    Kleinstiver, B. P. et al. Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat. Biotechnol. 34, 869–874 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. 66.

    Tang, X. et al. A CRISPR–Cpf1 system for efficient genome editing and transcriptional repression in plants. Nat. Plants 3, 17103 (2017).

    Article  PubMed  Google Scholar 

  67. 67.

    Zhong, Z. et al. Plant genome editing using FnCpf1 and LbCpf1 nucleases at redefined and altered PAM sites. Mol. Plant 11, 999–1002 (2018).

    Article  CAS  PubMed  Google Scholar 

  68. 68.

    Endo, A., Masafumi, M., Kaya, H. & Toki, S. Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida. Sci. Rep. 6, 38169 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. 69.

    Hu, X., Wang, C., Liu, Q., Fu, Y. & Wang, K. Targeted mutagenesis in rice using CRISPR-Cpf1 system. J. Genet. Genom. 44, 71–73 (2017).

    Article  Google Scholar 

  70. 70.

    Wang, M., Mao, Y., Lu, Y., Tao, X. & Zhu, J. Multiplex gene editing in rice using the CRISPR-Cpf1 system. Mol. Plant 10, 1011–1013 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. 71.

    Yin, X. et al. CRISPR-Cas9 and CRISPR-Cpf1 mediated targeting of a stomatal developmental gene EPFL9 in rice. Plant Cell Rep. 36, 745–757 (2017).

    Article  CAS  PubMed  Google Scholar 

  72. 72.

    Begemann, M. B. et al. Precise insertion and guided editing of higher plant genomes using Cpf1 CRISPR nucleases. Sci. Rep. 7, 11606 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. 73.

    Bernabé‐Orts, J. M. et al. Assessment of Cas12a-mediated gene editing efficiency in plants. Plant Biotechnol. J. https://doi.org/10.1111/pbi.13113 (2019).

  74. 74.

    Kim, H. et al. CRISPR/Cpf1-mediated DNA-free plant genome editing. Nat. Commun. 8, 14406 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. 75.

    Li, B. et al. Robust CRISPR/Cpf1(Cas12a) mediated genome editing in allotetraploid cotton (G. hirsutum). Plant Biotechnol. J. https://doi.org/10.1111/pbi.13147 (2019).

  76. 76.

    Jia, H., Orbović, V. & Wang, N. CRISPR-LbCas12a-mediated modification of citrus. Plant Biotechnol. J. https://doi.org/10.1111/pbi.13109 (2019).

  77. 77.

    Zhang, X. et al. Multiplex gene regulation by CRISPR-ddCpf1. Cell Discov. 3, 17018 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. 78.

    Gao, L. et al. Engineered Cpf1 variants with altered PAM specificities. Nat. Biotechnol. 35, 789 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. 79.

    Tóth, E. et al. Mb- and FnCpf1 nucleases are active in mammalian cells: activities and PAM preferences of four wild-type Cpf1 nucleases and of their altered PAM specificity variants. Nucleic Acids Res. 46, 10272–10285 (2018).

    PubMed  PubMed Central  Google Scholar 

  80. 80.

    Li, S. et al. Expanding the scope of CRISPR/Cpf1-mediated genome editing in rice. Mol. Plant 11, 995–998 (2018).

    Article  CAS  PubMed  Google Scholar 

  81. 81.

    Kleinstiver, B. P. et al. Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat. Biotechnol. 37, 276 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. 82.

    Zetsche, B. et al. A survey of genome editing activity for 16 Cpf1 orthologs. Preprint at https://www.biorxiv.org/content/10.1101/134015v1.article-info (2017).

  83. 83.

    Teng, F. et al. Enhanced mammalian genome editing by new Cas12a orthologs with optimized crRNA scaffolds. Genome Biol. 20, 15 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  84. 84.

    Liu, L. et al. C2c1-sgRNA complex structure reveals RNA-guided DNA cleavage mechanism. Mol. Cell 65, 310–322 (2017).

    Article  CAS  PubMed  Google Scholar 

  85. 85.

    Yang, H., Gao, P., Rajashankar, K. R. & Patel, D. J. PAM-dependent target DNA recognition and cleavage by C2c1 CRISPR-Cas endonuclease. Cell 167, 1814–1828 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. 86.

    Teng, F. et al. Repurposing CRISPR-Cas12b for mammalian genome engineering. Cell Discov. 4, 63 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. 87.

    Strecker, J. et al. Engineering of CRISPR-Cas12b for human genome editing. Nat. Commun. 10, 212 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. 88.

    Dolan, A. E. et al. Introducing a spectrum of long-range genomic deletions in human embryonic stem cells using Type I CRISPR-Cas. Mol. Cell 74, 1–15 (2019).

    Article  CAS  Google Scholar 

  89. 89.

    Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. & Vale, R. D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. 90.

    Leonetti, M. D., Sekine, S., Kamiyama, D., Weissman, J. S. & Huang, B. A scalable strategy for high-throughput GFP tagging of endogenous human proteins. Proc. Natl Acad. Sci. USA 113, E3501–E3508 (2016).

    Article  CAS  PubMed  Google Scholar 

  91. 91.

    Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583–588 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. 92.

    Lowder, L. G. et al. Robust transcriptional activation in plants using multiplexed CRISPR-Act2.0 and mTALE-Act systems. Mol. Plant 11, 245–256 (2018).

    Article  CAS  PubMed  Google Scholar 

  93. 93.

    Shao, S. et al. Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system. Nucleic Acids Res. 44, e86 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. 94.

    Shechner, D. M., Hacisuleyman, E., Younger, S. T. & Rinn, J. L. Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display. Nat. Methods 12, 664–670 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. 95.

    Zalatan, J. G. et al. Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell 160, 339–350 (2015).

    Article  CAS  Google Scholar 

  96. 96.

    Ma, H. et al. Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat. Biotechnol. 34, 528–530 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. 97.

    Carlson-Stevermer, J. et al. Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing. Nat. Commun. 8, 1711 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. 98.

    Qin, P. et al. Live cell imaging of low- and non-repetitive chromosome loci using CRISPR-Cas9. Nat. Commun. 8, 14725 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. 99.

    Ma, H. et al. CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging. Nat. Methods 15, 928 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. 100.

    Kunii, A. et al. Three-component repurposed technology for enhanced expression: highly accumulable transcriptional activators via branched tag arrays. CRISPR J. 1, 337–347 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. 101.

    Abudayyeh, O. O. et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. 102.

    Abudayyeh, O. O. et al. RNA targeting with CRISPR–Cas13. Nature 550, 280–284 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. 103.

    Cox, D. B. T. et al. RNA editing with CRISPR-Cas13. Science 358, 1019–1027 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. 104.

    Gootenberg, J. S. et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356, 438–442 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. 105.

    Aman, R. et al. RNA virus interference via CRISPR/Cas13a system in plants. Genome Biol. 19, 1 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. 106.

    Aman, R. et al. Engineering RNA virus interference via the CRISPR/Cas13 machinery in Arabidopsis. Viruses 10, 732 (2018).

    Article  CAS  PubMed Central  Google Scholar 

  107. 107.

    Lowder, L., Malzahn, A. & Qi, Y. Rapid evolution of manifold CRISPR systems for plant genome editing. Front. Plant Sci. 7, 1683 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  108. 108.

    Gao, Y. & Zhao, Y. Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J. Integr. Plant Biol. 56, 343–349 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. 109.

    Gao, Y. et al. Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development. Proc. Natl Acad. Sci. USA 112, 2275–2280 (2015).

    Article  CAS  PubMed  Google Scholar 

  110. 110.

    Tang, X. et al. A single transcript CRISPR-Cas9 system for efficient genome editing in plants. Mol. Plant 9, 1088–1091 (2016).

    Article  CAS  PubMed  Google Scholar 

  111. 111.

    Tang, X. et al. Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing. Plant Biotechnol. J. https://doi.org/10.1111/pbi.13068 (2018).

  112. 112.

    Wang, M. et al. Multiplex gene editing in rice with simplified CRISPR-Cpf1 and CRISPR-Cas9 systems: simplified single transcriptional unit CRISPR systems. J. Integr. Plant Biol. 60, 626–631 (2018).

    Article  CAS  PubMed  Google Scholar 

  113. 113.

    Yoshioka, S., Fujii, W., Ogawa, T., Sugiura, K. & Naito, K. Development of a mono-promoter-driven CRISPR/Cas9 system in mammalian cells. Sci. Rep. 5, 18341 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. 114.

    Ding, D., Chen, K., Chen, Y., Li, H. & Xie, K. Engineering introns to express RNA guides for Cas9- and Cpf1-mediated multiplex genome editing. Mol. Plant 11, 542–552 (2018).

    Article  CAS  PubMed  Google Scholar 

  115. 115.

    Lowder, L. G. et al. A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol. 169, 971–985 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. 116.

    Vazquez-Vilar, M. et al. A modular toolbox for gRNA–Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods 12, 10 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. 117.

    Xing, H.-L. et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 14, 327 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. 118.

    Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  Google Scholar 

  119. 119.

    Mikami, M., Toki, S. & Endo, M. In Planta processing of the SpCas9–gRNA complex. Plant Cell Physiol. 58, 1857–1867 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  120. 120.

    Li, S. et al. Synthesis-dependent repair of Cpf1-induced double strand DNA breaks enables targeted gene replacement in rice. J. Exp. Bot. 69, 4715–4721 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  121. 121.

    Xie, K., Minkenberg, B. & Yang, Y. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc. Natl Acad. Sci. USA 112, 3570–3575 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. 122.

    Wang, W., Akhunova, A., Chao, S. & Akhunov, E. Optimizing multiplex CRISPR/Cas9-based genome editing for wheat. Preprint at https://www.biorxiv.org/content/10.1101/051342v1 (2016).

  123. 123.

    Qi, W. et al. High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize. BMC Biotechnol. 16, 58 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. 124.

    Tsai, S. Q. et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat. Biotechnol. 32, 569–576 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  125. 125.

    Yan, Q. et al. Multiplex CRISPR/Cas9-based genome engineering enhanced by Drosha-mediated sgRNA-shRNA structure. Sci. Rep. 6, 38970 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  126. 126.

    Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  127. 127.

    Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016).

    Article  CAS  Google Scholar 

  128. 128.

    Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  129. 129.

    Conticello, S. G. The AID/APOBEC family of nucleic acid mutators. Genome Biol. 9, 229 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  130. 130.

    Kunz, C., Saito, Y. & Schär, P. DNA Repair in mammalian cells: Mismatched repair: variations on a theme. Cell. Mol. Life Sci. CMLS 66, 1021–1038 (2009).

    Article  CAS  PubMed  Google Scholar 

  131. 131.

    Mol, C. D. et al. Crystal structure of human uracil-DNA glycosylase in complex with a protein inhibitor: Protein mimicry of DNA. Cell 82, 701–708 (1995).

    Article  CAS  PubMed  Google Scholar 

  132. 132.

    Komor, A. C. et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T: Abase editors with higher efficiency and product purity. Sci. Adv. 3, eaao4774 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  133. 133.

    Wang, L. et al. Enhanced base editing by co-expression of free uracil DNA glycosylase inhibitor. Cell Res. 27, 1289–1292 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  134. 134.

    Zafra, M. P. et al. Optimized base editors enable efficient editing in cells, organoids and mice. Nat. Biotechnol. 36, 888–893 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  135. 135.

    Kuscu, C. & Adli, M. CRISPR-Cas9-AID base editor is a powerful gain-of-function screening tool. Nat. Methods 13, 983–984 (2016).

    Article  CAS  PubMed  Google Scholar 

  136. 136.

    Gehrke, J. M. et al. An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities. Nat. Biotechnol. 36, 977–982 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  137. 137.

    Wang, X. et al. Efficient base editing in methylated regions with a human APOBEC3A-Cas9 fusion. Nat. Biotechnol. 36, 946–949 (2018).

    Article  CAS  PubMed  Google Scholar 

  138. 138.

    Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  139. 139.

    Molla, K. A. & Yang, Y. CRISPR/Cas-mediated base editing: technical considerations and practical applications. Trends Biotechnol. https://doi.org/10.1016/j.tibtech.2019.03.008 (2019).

  140. 140.

    Hua, K., Tao, X. & Zhu, J.-K. Expanding the base editing scope in rice by using Cas9 variants. Plant Biotechnol. J. 17, 499–504 (2019).

    Article  PubMed  Google Scholar 

  141. 141.

    Li, J., Sun, Y., Du, J., Zhao, Y. & Xia, L. Generation of targeted point mutations in rice by a modified CRISPR/Cas9 system. Mol. Plant 10, 526–529 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  142. 142.

    Lu, Y. & Zhu, J.-K. Precise editing of a target base in the rice genome using a modified CRISPR/Cas9 system. Mol. Plant 10, 523–525 (2017).

    Article  CAS  PubMed  Google Scholar 

  143. 143.

    Ren, B. et al. A CRISPR/Cas9 toolkit for efficient targeted base editing to induce genetic variations in rice. Sci. China Life Sci. 60, 516–519 (2017).

    Article  PubMed  Google Scholar 

  144. 144.

    Zong, Y. et al. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nat. Biotechnol. 35, 438–440 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  145. 145.

    Zong, Y. et al. Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A. Nat. Biotechnol. 36, 950–953 (2018).

    Article  CAS  Google Scholar 

  146. 146.

    Ren, B. et al. Improved base editor for efficiently inducing genetic variations in rice with CRISPR/Cas9-guided hyperactive hAID mutant. Mol. Plant 11, 623–626 (2018).

    Article  CAS  PubMed  Google Scholar 

  147. 147.

    Shimatani, Z. et al. Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nat. Biotechnol. 35, 441–443 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  148. 148.

    Yan, F. et al. Highly efficient A·T to G·C base editing by Cas9n-guided tRNA adenosine deaminase in rice. Mol. Plant 11, 631–634 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  149. 149.

    Kang, B.-C. et al. Precision genome engineering through adenine base editing in plants. Nat. Plants 4, 427–431 (2018).

    Article  CAS  Google Scholar 

  150. 150.

    Matthews, M. M. et al. Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity. Nat. Struct. Mol. Biol. 23, 426–433 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  151. 151.

    Montiel-Gonzalez, M. F., Vallecillo-Viejo, I., Yudowski, G. A. & Rosenthal, J. J. C. Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing. Proc. Natl Acad. Sci. USA 110, 18285–18290 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  152. 152.

    Jin, S. et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364, 292–295 (2019).

    CAS  Google Scholar 

  153. 153.

    Gajula, K. S. Designing an elusive C•G→G•C CRISPR base editor. Trends Biochem. Sci. 44, 91–94 (2019).

    Article  CAS  PubMed  Google Scholar 

  154. 154.

    Čermák, T., Baltes, N. J., Čegan, R., Zhang, Y. & Voytas, D. F. High-frequency, precise modification of the tomato genome. Genome Biol. 16, 232 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  155. 155.

    Sun, Y. et al. Engineering herbicide-resistant rice plants through CRISPR/Cas9-mediated homologous recombination of Acetolactate Synthase. Mol. Plant 9, 628–631 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  156. 156.

    Malzahn, A., Lowder, L. & Qi, Y. Plant genome editing with TALEN and CRISPR. Cell Biosci. 7, 21 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  157. 157.

    Li, H. et al. Design and specificity of long ssDNA donors for CRISPR-based knock-in. Preprint at https://www.biorxiv.org/content/10.1101/178905v1 (2017).

  158. 158.

    Richardson, C. D., Ray, G. J., DeWitt, M. A., Curie, G. L. & Corn, J. E. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat. Biotechnol. 34, 339–344 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  159. 159.

    Baker, O. et al. The contribution of homology arms to nuclease-assisted genome engineering. Nucleic Acids Res. 45, 8105–8115 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  160. 160.

    Fauser, F. et al. In planta gene targeting. Proc. Natl Acad. Sci. USA 109, 7535–7540 (2012).

    Article  PubMed  Google Scholar 

  161. 161.

    Schiml, S., Fauser, F. & Puchta, H. The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny. Plant J. 80, 1139–1150 (2014).

    Article  CAS  PubMed  Google Scholar 

  162. 162.

    Zhang, J.-P. et al. Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. Genome Biol. 18, 35 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  163. 163.

    Li, J. et al. Efficient allelic replacement in rice by gene editing: A case study of the NRT1.1B gene. J. Integr. Plant Biol. 60, 536–540 (2018).

    Article  CAS  PubMed  Google Scholar 

  164. 164.

    Baltes, N. J., Gil-Humanes, J., Cermak, T., Atkins, P. A. & Voytas, D. F. DNA replicons for plant genome engineering. Plant Cell 26, 151–163 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  165. 165.

    Dahan‐Meir, T. et al. Efficient in planta gene targeting in tomato using geminiviral replicons and the CRISPR/Cas9 system. Plant J. 95, 5–16 (2018).

    Article  CAS  PubMed  Google Scholar 

  166. 166.

    Wang, M. et al. Gene targeting by homology-directed repair in rice using a geminivirus-based CRISPR/Cas9 system. Mol. Plant 10, 1007–1010 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  167. 167.

    Cunningham, F. J., Goh, N. S., Demirer, G. S., Matos, J. L. & Landry, M. P. Nanoparticle-mediated delivery towards advancing plant genetic engineering. Trends Biotechnol. 36, 882–897 (2018).

    Article  CAS  PubMed  Google Scholar 

  168. 168.

    Demirer, G. S. et al. High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants. Nat. Nanotechnol. 14, 456–464 (2019).

    Article  CAS  PubMed  Google Scholar 

  169. 169.

    Ghanta, K. S. et al. 5′ Modifications improve potency and efficacy of DNA donors for precision genome editing. Preprint at https://www.biorxiv.org/content/10.1101/354480v1.full (2018).

  170. 170.

    Ma, M. et al. Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-Avidin/Biotin-donor DNA system. Cell Res. 27, 578–581 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  171. 171.

    Aird, E. J., Lovendahl, K. N., Martin, A. S., Harris, R. S. & Gordon, W. R. Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template. Commun. Biol. 1, 54 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  172. 172.

    Keskin, H. et al. Transcript-RNA-templated DNA recombination and repair. Nature 515, 436–439 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  173. 173.

    Li, S. et al. Precise gene replacement in rice by RNA transcript-templated homologous recombination. Nat. Biotechnol. 37, 445–450 (2019).

    Article  CAS  PubMed  Google Scholar 

  174. 174.

    Jayathilaka, K. et al. A chemical compound that stimulates the human homologous recombination protein RAD51. Proc. Natl Acad. Sci. USA 105, 15848–15853 (2008).

    Article  PubMed  Google Scholar 

  175. 175.

    Song, J. et al. RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency. Nat. Commun. 7, 10548 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  176. 176.

    Schuermann, D., Molinier, J., Fritsch, O. & Hohn, B. The dual nature of homologous recombination in plants. Trends Genet. 21, 172–181 (2005).

    Article  CAS  PubMed  Google Scholar 

  177. 177.

    Shaked, H., Melamed-Bessudo, C. & Levy, A. A. High-frequency gene targeting in Arabidopsis plants expressing the yeast RAD54 gene. Proc. Natl Acad. Sci. USA 102, 12265–12269 (2005).

    Article  CAS  PubMed  Google Scholar 

  178. 178.

    Chen, X. et al. In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting. Nat. Commun. 8, 657 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  179. 179.

    Chu, V. T. et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 33, 543–548 (2015).

    Article  CAS  Google Scholar 

  180. 180.

    Maruyama, T. et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat. Biotechnol. 33, 538–542 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  181. 181.

    Robert, F., Barbeau, M., Éthier, S., Dostie, J. & Pelletier, J. Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing. Genome Med. 7, 93 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  182. 182.

    Qi, Y. et al. Increasing frequencies of site-specific mutagenesis and gene targeting in Arabidopsis by manipulating DNA repair pathways. Genome Res. 23, 547–554 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  183. 183.

    Endo, M., Mikami, M. & Toki, S. Biallelic gene targeting in rice. Plant Physiol. 170, 667–677 (2016).

    Article  CAS  PubMed  Google Scholar 

  184. 184.

    Rodríguez-Leal, D., Lemmon, Z. H., Man, J., Bartlett, M. E. & Lippman, Z. B. Engineering quantitative trait variation for crop improvement by genome editing. Cell 171, 470–480 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  185. 185.

    Zhang, H. et al. Genome editing of upstream open reading frames enables translational control in plants. Nat. Biotechnol. 36, 894–898 (2018).

    Article  CAS  PubMed  Google Scholar 

  186. 186.

    Li, Z., Xiong, X., Wang, F., Liang, J. & Li, J.-F. Gene disruption through base editing-induced messenger RNA missplicing in plants. New Phytol. 222, 1139–1148 (2019).

    Article  CAS  PubMed  Google Scholar 

  187. 187.

    Xue, C., Zhang, H., Lin, Q., Fan, R. & Gao, C. Manipulating mRNA splicing by base editing in plants. Sci. China Life Sci. 61, 1293–1300 (2018).

    Article  CAS  PubMed  Google Scholar 

  188. 188.

    Piatek, A. et al. RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors. Plant Biotechnol. J. 13, 578–589 (2015).

    Article  CAS  PubMed  Google Scholar 

  189. 189.

    Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  190. 190.

    Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  191. 191.

    Zhang, X. et al. Gene activation in human cells using CRISPR/Cpf1-p300 and CRISPR/Cpf1-SunTag systems. Protein Cell 9, 380–383 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  192. 192.

    Li, Z. et al. A potent Cas9-derived gene activator for plant and mammalian cells. Nat. Plants 3, 930–936 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  193. 193.

    Papikian, A., Liu, W., Gallego-Bartolomé, J. & Jacobsen, S. E. Site-specific manipulation of Arabidopsis loci using CRISPR-Cas9 SunTag systems. Nat. Commun. 10, 729 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  194. 194.

    Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  195. 195.

    Cheng, A. W. et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 23, 1163–1171 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  196. 196.

    Farzadfard, F., Perli, S. D. & Lu, T. K. Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. ACS Synth. Biol. 2, 604–613 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  197. 197.

    Deaner, M., Mejia, J. & Alper, H. S. Enabling graded and large-scale multiplex of desired genes using a dual-mode dCas9 activator in Saccharomyces cerevisiae. ACS Synth. Biol. 6, 1931–1943 (2017).

    Article  CAS  PubMed  Google Scholar 

  198. 198.

    Kiani, S. et al. Cas9 gRNA engineering for genome editing, activation and repression. Nat. Methods 12, 1051–1054 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  199. 199.

    Breinig, M. et al. Multiplexed orthogonal genome editing and transcriptional activation by Cas12a. Nat. Methods 16, 51–54 (2019).

    Article  CAS  PubMed  Google Scholar 

  200. 200.

    Braun, S. M. G. et al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat. Commun. 8, 560 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  201. 201.

    Huang, Y.-H. et al. DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A. Genome Biol. 18, 176 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  202. 202.

    Liao, H.-K. et al. In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation. Cell 171, 1495–1507 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  203. 203.

    Morita, S. et al. Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat. Biotechnol. 34, 1060–1065 (2016).

    Article  CAS  Google Scholar 

  204. 204.

    Gallego-Bartolomé, J. et al. Targeted DNA demethylation of the Arabidopsis genome using the human TET1 catalytic domain. Proc. Natl Acad. Sci. USA 115, E2125–E2134 (2018).

    Article  CAS  PubMed  Google Scholar 

  205. 205.

    Galonska, C. et al. Genome-wide tracking of dCas9-methyltransferase footprints. Nat. Commun. 9, 597 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  206. 206.

    O’Connell, M. R. et al. Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature 516, 263–266 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  207. 207.

    Batra, R. et al. Elimination of toxic microsatellite repeat expansion RNA by RNA-targeting Cas9. Cell 170, 899–912 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  208. 208.

    Xiang, G., Zhang, X., An, C., Cheng, C. & Wang, H. Temperature effect on CRISPR-Cas9 mediated genome editing. J. Genet. Genom. 44, 199–205 (2017).

    Article  Google Scholar 

  209. 209.

    Moreno-Mateos, M. A. et al. CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing. Nat. Commun. 8, 2024 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  210. 210.

    LeBlanc Chantal. et al. Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress. Plant J. 93, 377–386 (2018).

    Article  CAS  PubMed  Google Scholar 

  211. 211.

    Malzahn, A. A. et al. Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis. BMC Biol. 17, 9 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  212. 212.

    LeBlanc, C. et al. Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress. Plant J. 93, 377–386 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  213. 213.

    Gao, X., Chen, J., Dai, X., Zhang, D. & Zhao, Y. An effective strategy for reliably isolating heritable and Cas9-free Arabidopsis mutants generated by CRISPR/Cas9-mediated genome editing. Plant Physiol. 171, 1794–1800 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  214. 214.

    Lu, H.-P. et al. CRISPR-S: an active interference element for a rapid and inexpensive selection of genome-edited, transgene-free rice plants. Plant Biotechnol. J. 15, 1371–1373 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  215. 215.

    Iaffaldano, B., Zhang, Y. & Cornish, K. CRISPR/Cas9 genome editing of rubber producing dandelion Taraxacum kok-saghyz using Agrobacterium rhizogenes without selection. Ind. Crops Prod. 89, 356–362 (2016).

    Article  CAS  Google Scholar 

  216. 216.

    Zhang, Y. et al. Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nat. Commun. 7, 12617 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  217. 217.

    Woo, J. W. et al. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat. Biotechnol. 33, 1162–1164 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  218. 218.

    Malnoy, M. et al. DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 ribonucleoproteins. Front. Plant Sci. 7, 1904 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  219. 219.

    Subburaj, S. et al. Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins. Plant Cell Rep. 35, 1535–1544 (2016).

    Article  CAS  PubMed  Google Scholar 

  220. 220.

    Li, J. et al. Whole genome sequencing reveals rare off-target mutations and considerable inherent genetic or/and somaclonal variations in CRISPR/Cas9-edited cotton plants. Plant Biotechnol. J. 17, 858–868 (2019).

    Article  CAS  PubMed  Google Scholar 

  221. 221.

    Liang, Z. et al. Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nat. Commun. 8, 14261 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  222. 222.

    Svitashev, S., Schwartz, C., Lenderts, B., Young, J. K. & Cigan, A. M. Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes. Nat. Commun. 7, 13274 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  223. 223.

    Toda, E. et al. An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice. Nat. Plants 5, 363–368 (2019).

    Article  CAS  PubMed  Google Scholar 

  224. 224.

    He, Y. et al. Programmed self-elimination of the CRISPR/Cas9 construct greatly accelerates the isolation of edited and transgene-free rice plants. Mol. Plant 11, 1210–1213 (2018).

    Article  CAS  PubMed  Google Scholar 

  225. 225.

    Ryder, P., McHale, M., Fort, A. & Spillane, C. Generation of stable nulliplex autopolyploid lines of Arabidopsis thaliana using CRISPR/Cas9 genome editing. Plant Cell Rep. 36, 1005–1008 (2017).

    Article  CAS  PubMed  Google Scholar 

  226. 226.

    Shan, S. et al. Application of CRISPR/Cas9 to Tragopogon (Asteraceae), an evolutionary model for the study of polyploidy. Mol. Ecol. Resour. 18, 1427–1443 (2018).

    Article  CAS  PubMed  Google Scholar 

  227. 227.

    Andersson, M. et al. Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts. Plant Cell Rep. 36, 117–128 (2017).

    Article  CAS  PubMed  Google Scholar 

  228. 228.

    Braatz, J. et al. CRISPR-Cas9 targeted mutagenesis leads to simultaneous modification of different homoeologous gene copies in polyploid oilseed Rape (Brassica napus). Plant Physiol. 174, 935–942 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  229. 229.

    Gao, W. et al. Genome editing in cotton with the CRISPR/Cas9 system. Front. Plant Sci. 8, 1364 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  230. 230.

    Jiang, W. Z. et al. Significant enhancement of fatty acid composition in seeds of the allohexaploid, Camelina sativa, using CRISPR/Cas9 gene editing. Plant Biotechnol. J. 15, 648–657 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  231. 231.

    Liu, Y. et al. Targeted mutagenesis in tetraploid switchgrass (Panicum virgatum L.) using CRISPR/Cas9. Plant Biotechnol. J. 16, 381–393 (2018).

    Article  CAS  PubMed  Google Scholar 

  232. 232.

    Morineau, C. et al. Selective gene dosage by CRISPR-Cas9 genome editing in hexaploid Camelina sativa. Plant Biotechnol. J. 15, 729–739 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  233. 233.

    Wang, Y. et al. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat. Biotechnol. 32, 947–951 (2014).

    Article  CAS  PubMed  Google Scholar 

  234. 234.

    Zhang, Y. et al. Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nat. Commun. 7, 12617 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  235. 235.

    Wang, W. et al. Transgenerational CRISPR-Cas9 activity facilitates multiplex gene editing in allopolyploid wheat. CRISPR J. 1, 65–74 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  236. 236.

    Zhang, Z. et al. Development of an Agrobacterium-delivered CRISPR/Cas9 system for wheat genome editing. Plant Biotechnol. J. https://doi.org/10.1111/pbi.13088 (2019).

  237. 237.

    Jia, H. et al. Genome editing of the disease susceptibility gene CsLOB1 in citrus confers resistance to citrus canker. Plant Biotechnol. J. 15, 817–823 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  238. 238.

    Fauser, F., Schiml, S. & Puchta, H. Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J. 79, 348–359 (2014).

    Article  CAS  Google Scholar 

  239. 239.

    Feng, Z. et al. Efficient genome editing in plants using a CRISPR/Cas system. Cell Res. 23, 1229–1232 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  240. 240.

    Shan, Q. et al. ZFN, TALEN and CRISPR-Cas9 mediated homology directed gene insertion in Arabidopsis: a disconnect between somatic and germinal cells. J. Genet. Genom. 45, 681–684 (2018).

    Article  Google Scholar 

  241. 241.

    Mao, Y. et al. Development of germ-line-specific CRISPR-Cas9 systems to improve the production of heritable gene modifications in Arabidopsis. Plant Biotechnol. J. 14, 519–532 (2016).

    Article  CAS  PubMed  Google Scholar 

  242. 242.

    Eid, A., Ali, Z. & Mahfouz, M. M. High efficiency of targeted mutagenesis in Arabidopsis via meiotic promoter-driven expression of Cas9 endonuclease. Plant Cell Rep. 35, 1555–1558 (2016).

    Article  CAS  PubMed  Google Scholar 

  243. 243.

    Feng, Z. et al. A highly efficient cell division-specific CRISPR/Cas9 system generates homozygous mutants for multiple genes in Arabidopsis. Int. J. Mol. Sci. 19, 3925 (2018).

    Article  CAS  PubMed Central  Google Scholar 

  244. 244.

    Li, H.-J., Liu, N.-Y., Shi, D.-Q., Liu, J. & Yang, W.-C. YAO is a nucleolar WD40-repeat protein critical for embryogenesis and gametogenesis in Arabidopsis. BMC Plant Biol. 10, 169 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  245. 245.

    Yan, L. et al. High-efficiency genome editing in Arabidopsis using YAO promoter-driven CRISPR/Cas9 system. Mol. Plant 8, 1820–1823 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  246. 246.

    Tsutsui, H. & Higashiyama, T. pKAMA-ITACHI vectors for highly efficient CRISPR/Cas9-mediated gene knockout in Arabidopsis thaliana. Plant Cell Physiol. 58, 46–56 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  247. 247.

    Miki, D., Zhang, W., Zeng, W., Feng, Z. & Zhu, J.-K. CRISPR/Cas9-mediated gene targeting in Arabidopsis using sequential transformation. Nat. Commun. 9, 1967 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  248. 248.

    Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat. Biotechnol. 36, 765–771 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  249. 249.

    Feng, Z. et al. Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc. Natl Acad. Sci. USA 111, 4632–4637 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  250. 250.

    Tang, X. et al. A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 (Cas12a) nucleases in rice. Genome Biol. 19, 84 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  251. 251.

    Rees, H. A., Wilson, C., Doman, J. L. & Liu, D. R. Analysis and minimization of cellular RNA editing by DNA adenine base editors. Sci. Adv. 5, eaax5717 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  252. 252.

    Grünewald, J. et al. Transcriptome-wide off-target RNA editing induced byCRISPR-guided DNA base editors. Nature 569, 433 (2019).

    Article  CAS  PubMed  Google Scholar 

  253. 253.

    Kleinstiver, B. P. et al. High-fidelity CRISPR-Cas9 variants with undetectable genome-wide off-targets. Nature 529, 490–495 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  254. 254.

    Slaymaker, I. M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  255. 255.

    Chen, J. S. et al. Enhanced proofreading governs CRISPR–Cas9 targeting accuracy. Nature 550, 407–410 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  256. 256.

    Casini, A. et al. A highly specific SpCas9 variant is identified by in vivo screening in yeast. Nat. Biotechnol. 36, 265–271 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  257. 257.

    Lee, J. K. et al. Directed evolution of CRISPR-Cas9 to increase its specificity. Nat. Commun. 9, 3048 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  258. 258.

    Vakulskas, C. A. et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat. Med. 24, 1216 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  259. 259.

    Zhang, D. et al. Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases. Genome Biol. 18, 191 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  260. 260.

    Liang, Z., Chen, K., Yan, Y., Zhang, Y. & Gao, C. Genotyping genome-edited mutations in plants using CRISPR ribonucleoprotein complexes. Plant Biotechnol. J. 16, 2053–2062 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  261. 261.

    Zhang, Q. et al. Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention. Plant Mol. Biol. 96, 445–456 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  262. 262.

    Lowe, K. et al. Morphogenic regulators Baby boom and Wuschel improve monocot transformation. Plant Cell 28, 1998–2015 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  263. 263.

    Mookkan, M., Nelson-Vasilchik, K., Hague, J., Zhang, Z. J. & Kausch, A. P. Selectable marker independent transformation of recalcitrant maize inbred B73 and sorghum P898012 mediated by morphogenic regulators BABY BOOM and WUSCHEL2. Plant Cell Rep. 36, 1477–1491 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  264. 264.

    Khanday, I., Skinner, D., Yang, B., Mercier, R. & Sundaresan, V. A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds. Nature 565, 91 (2019).

    Article  CAS  PubMed  Google Scholar 

  265. 265.

    Wang, C. et al. Clonal seeds from hybrid rice by simultaneous genome engineering of meiosis and fertilization genes. Nat. Biotechnol. 37, 283–286 (2019).

    Article  CAS  PubMed  Google Scholar 

  266. 266.

    d’Erfurth, I. et al. Turning meiosis into mitosis. PLoS Biol. 7, e1000124 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  267. 267.

    Mieulet, D. et al. Turning rice meiosis into mitosis. Cell Res. 26, 1242–1254 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  268. 268.

    Kelliher, T. et al. One-step genome editing of elite crop germplasm during haploid induction. Nat. Biotechnol. 37, 287–292 (2019).

    Article  CAS  PubMed  Google Scholar 

  269. 269.

    Wang, B. et al. Development of a haploid-inducer mediated genome editing (IMGE) system for accelerating maize breeding. Mol. Plant 12, 597–602 (2019).

    Article  CAS  PubMed  Google Scholar 

  270. 270.

    Lemmon, Z. H. et al. Rapid improvement of domestication traits in an orphan crop by genome editing. Nat. Plants 4, 766 (2018).

    Article  CAS  Google Scholar 

  271. 271.

    Li, T. et al. Domestication of wild tomato is accelerated by genome editing. Nat. Biotechnol. 36, 1160–1163 (2018).

    Article  CAS  Google Scholar 

  272. 272.

    Zsögön, A. et al. De novo domestication of wild tomato using genome editing. Nat. Biotechnol. 36, 1211–1216 (2018).

    Article  CAS  Google Scholar 

  273. 273.

    Sarno, R. et al. Programming sites of meiotic crossovers using Spo11 fusion proteins. Nucleic Acids Res. 45, e164 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  274. 274.

    Jacobs, T. B., Zhang, N., Patel, D. & Martin, G. B. Generation of a collection of mutant tomato lines using pooled CRISPR libraries. Plant Physiol. 174, 2023–2037 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  275. 275.

    Lu, Y. et al. Genome-wide targeted mutagenesis in rice using the CRISPR/Cas9 system. Mol. Plant 10, 1242–1245 (2017).

    Article  CAS  PubMed  Google Scholar 

  276. 276.

    Meng, X. et al. Construction of a genome-wide mutant library in rice using CRISPR/Cas9. Mol. Plant 10, 1238–1241 (2017).

    Article  CAS  PubMed  Google Scholar 

  277. 277.

    Butt, H. et al. CRISPR directed evolution of the spliceosome for resistance to splicing inhibitors. Genome Biol. 20, 73 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  278. 278.

    Boesch, P. et al. DNA repair in organelles: Pathways, organization, regulation, relevance in disease and aging. Biochim. Biophys. Acta BBA - Mol. Cell Res. 1813, 186–200 (2011).

    Article  CAS  Google Scholar 

  279. 279.

    Srivastava, S. & Moraes, C. T. Manipulating mitochondrial DNA heteroplasmy by a mitochondrially targeted restriction endonuclease. Hum. Mol. Genet. 10, 3093–3099 (2001).

    Article  CAS  PubMed  Google Scholar 

  280. 280.

    Tanaka, M. et al. Gene therapy for mitochondrial disease by delivering restriction endonuclease SmaI into mitochondria. J. Biomed. Sci. 9, 534–541 (2002).

    CAS  PubMed  Google Scholar 

  281. 281.

    Bacman, S. R., Williams, S. L., Pinto, M., Peralta, S. & Moraes, C. T. Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs. Nat. Med. 19, 1111–1113 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  282. 282.

    Gammage, P. A., Rorbach, J., Vincent, A. I., Rebar, E. J. & Minczuk, M. Mitochondrially targeted ZFNs for selective degradation of pathogenic mitochondrial genomes bearing large‐scale deletions or point mutations. EMBO Mol. Med. 6, 458–466 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  283. 283.

    Gammage, P. A., Moraes, C. T. & Minczuk, M. Mitochondrial genome engineering: the revolution may not be CRISPR-Ized. Trends Genet. 34, 101–110 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  284. 284.

    Jo, A. et al. Efficient mitochondrial genome editing by CRISPR/Cas9. BioMed. Res. Int. 2015, 305716 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  285. 285.

    Piatek, A. A., Lenaghan, S. C. & Neal Stewart, C. Advanced editing of the nuclear and plastid genomes in plants. Plant Sci. 273, 42–49 (2018).

    Article  CAS  PubMed  Google Scholar 

  286. 286.

    Ruf, S. et al. High-efficiency generation of fertile transplastomic Arabidopsis plants. Nat. Plants 5, 282 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  287. 287.

    Bae, S., Kweon, J., Kim, H. S. & Kim, J.-S. Microhomology-based choice of Cas9 nuclease target sites. Nat. Methods 11, 705–706 (2014).

    Article  CAS  Google Scholar 

  288. 288.

    van Overbeek, M. et al. DNA repair profiling reveals nonrandom outcomes at Cas9-mediated breaks. Mol. Cell 63, 633–646 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  289. 289.

    Bothmer, A. et al. Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus. Nat. Commun. 8, 13905 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  290. 290.

    Chang, H. H. Y. et al. Different DNA end configurations dictate which NHEJ components are most important for joining efficiency. J. Biol. Chem. 291, 24377–24389 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  291. 291.

    Allen, F. et al. Predicting the mutations generated by repair of Cas9-induced double-strand breaks. Nat. Biotechnol. 37, 64–72 (2018).

    Article  CAS  Google Scholar 

  292. 292.

    Chakrabarti, A. M. et al. Target-specific precision of CRISPR-mediated genome editing. Mol. Cell 73, 699–713 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  293. 293.

    Dreissig, S. et al. Live-cell CRISPR imaging in plants reveals dynamic telomere movements. Plant J. Cell Mol. Biol. 91, 565–573 (2017).

    Article  CAS  Google Scholar 

  294. 294.

    Ma, H. et al. Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat. Biotechnol. 34, 528–530 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  295. 295.

    Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. & Vale, R. D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  296. 296.

    Fujita, T. & Fujii, H. Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochem. Biophys. Res. Commun. 439, 132–136 (2013).

    Article  CAS  PubMed  Google Scholar 

  297. 297.

    Nishimasu, H. et al. Crystal Structure of Staphylococcus aureus Cas9. Cell 162, 1113–1126 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  298. 298.

    Kaya, H., Ishibashi, K. & Toki, S. A split Staphylococcus aureus Cas9 as a compact genome-editing tool in plants. Plant Cell Physiol. 58, 643–649 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Due to limited space, we could not cite all the related literature. We apologize to the authors whose work was not cited in this Review. Our plant genome engineering research is supported by the National Science Foundation Plant Genome Research Program (grant no. IOS-1758745), Biotechnology Risk Assessment Grant Program (grant no. 2018-33522-28789) from the US Department of Agriculture, the Foundation for Food and Agriculture Research (grant no. 593603) and Syngenta Biotechnology.

Author information

Affiliations

Authors

Contributions

Y.Z., A.A.M., S.S. and Y.Q. wrote the manuscript. Y.Z. prepared the figures. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Yiping Qi.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information: Nature Plants thanks S. Toki, L. Xia and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhang, Y., Malzahn, A.A., Sretenovic, S. et al. The emerging and uncultivated potential of CRISPR technology in plant science. Nat. Plants 5, 778–794 (2019). https://doi.org/10.1038/s41477-019-0461-5

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing