Applying CRISPR/Cas for genome engineering in plants: the best is yet to come

Current Opinion in Plant Biology

Volumn 36, Issue , 2017, Pages 1-8

  Puchta, Holger ^a  

^a KARLSRUHE INSTITUTE OF TECHNOLOGY   (Germany)

Author keywords

[No Author keywords available]

Indexed keywords

ANIMAL; CRISPR CAS SYSTEM; DNA END JOINING REPAIR; GENE INACTIVATION; GENETIC ENGINEERING; PLANT GENOME; TRENDS;

ANIMALS; CRISPR-CAS SYSTEMS; DNA END-JOINING REPAIR; GENE KNOCKOUT TECHNIQUES; GENETIC ENGINEERING; GENOME, PLANT;

EID: 84999711685     PISSN: 13695266     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.pbi.2016.11.011     Document Type: Review

References (79)

1. 1 Puchta, H., Dujon, B., Hohn, B., Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination. Proc Natl Acad Sci U S A 93 (1996), 5055–5060.
2. 2 Salomon, S., Puchta, H., Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells. EMBO J 17 (1998), 6086–6095, 10.1093/emboj/17.20.6086.
3. 3 Puchta, H., The repair of double-strand breaks in plants: mechanisms and consequences for genome evolution. J Exp Bot 56 (2005), 1–14, 10.1093/jxb/eri025.
4. 4 Kirik, A., Salomon, S., Puchta, H., Species-specific double-strand break repair and genome evolution in plants. EMBO J 19 (2000), 5562–5566, 10.1093/emboj/19.20.5562.
5. 5 Siebert, R., Puchta, H., Efficient repair of genomic double-strand breaks by homologous recombination between directly repeated sequences in the plant genome. Plant Cell 14 (2002), 1121–1131.
6. 6 Chandrasegaran, S., Carroll, D., Origins of programmable nucleases for genome engineering. J Mol Biol 428:Pt B (2016), 963–989, 10.1016/j.jmb.2015.10.014.
7. 7 Voytas, D.F., Plant genome engineering with sequence-specific nucleases. Annu Rev Plant Biol 64 (2013), 327–350, 10.1146/annurev-arplant-042811-105552.
8. 8 Puchta, H., Fauser, F., Synthetic nucleases for genome engineering in plants: prospects for a bright future. Plant J Cell Mol Biol 78 (2014), 727–741, 10.1111/tpj.12338.
9. 9•• Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., Charpentier, E., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science (New York, NY) 337 (2012), 816–821, 10.1126/science.1225829 Till now the most important article of molecular biology in the 21st century.
10. 10 Shan, Q., Wang, Y., Li, J., Zhang, Y., Chen, K., Liang, Z., Zhang, K., Liu, J., Xi, J.J., Qiu, J.-L., et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol 31 (2013), 686–688, 10.1038/nbt.2650.
11. 11 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 (2013), 691–693, 10.1038/nbt.2655.
12. 12 Li, J.-F., Norville, J.E., Aach, J., McCormack, M., Zhang, D., Bush, J., Church, G.M., Sheen, J., Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol 31 (2013), 688–691, 10.1038/nbt.2654.
13. 13•• Wang, Y., Cheng, X., Shan, Q., Zhang, Y., Liu, J., Gao, C., Qiu, J.-L., Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol 32 (2014), 947–951, 10.1038/nbt.2969 The first impressive example of the power of genome engineering for breeding: the production of wheat that is resistant to powdery mildew.
14. 14 Zhang, D., Li, Z., Li, J.-F., Targeted gene manipulation in plants using the CRISPR/Cas technology. J Genet Genomics (Trans.) Yi chuan xue bao, 43, 2016, 251–262, 10.1016/j.jgg.2016.03.001.
15. 15 Luo, M., Gilbert, B., Ayliffe, M., Applications of CRISPR/Cas9 technology for targeted mutagenesis, gene replacement and stacking of genes in higher plants. Plant Cell Rep 35 (2016), 1439–1450, 10.1007/s00299-016-1989-8.
16. 16 Ma, X., Zhu, Q., Chen, Y., Liu, Y.-G., CRISPR/Cas9 platforms for genome editing in plants: developments and applications. Mol Plant 9 (2016), 961–974, 10.1016/j.molp.2016.04.009.
17. 17 Schiml, S., Puchta, H., Revolutionizing plant biology: multiple ways of genome engineering by CRISPR/Cas. Plant Methods, 12, 2016, 8, 10.1186/s13007-016-0103-0.
18. 18 Liu, D., Hu, R., Palla, K.J., Tuskan, G.A., Yang, X., Advances and perspectives on the use of CRISPR/Cas9 systems in plant genomics research. Curr Opin Plant Biol 30 (2016), 70–77, 10.1016/j.pbi.2016.01.007.
19. 19 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 Cell Mol Biol 79 (2014), 348–359, 10.1111/tpj.12554.
20. 20 Sprink, T., Eriksson, D., Schiemann, J., Hartung, F., Regulatory hurdles for genome editing: process- vs. product-based approaches in different regulatory contexts. Plant Cell Rep 35 (2016), 1493–1506, 10.1007/s00299-016-1990-2.
21. 21• Marton, I., Zuker, A., Shklarman, E., Zeevi, V., Tovkach, A., Roffe, S., Ovadis, M., Tzfira, T., Vainstein, A., Nontransgenic genome modification in plant cells. Plant Physiol 154 (2010), 1079–1087, 10.1104/pp.110.164806 First demonstration that viral synthetic nucleases can be delivered by viral RNA to produce plant mutants without recombinant DNA.
22. 22 Ali, Z., Abul-faraj, A., Li, L., Ghosh, N., Piatek, M., Mahjoub, A., Aouida, M., Piatek, A., Baltes, N.J., Voytas, D.F., et al. Efficient virus-mediated genome editing in plants using the CRISPR/Cas9 system. Mol Plant 8 (2015), 1288–1291, 10.1016/j.molp.2015.02.011.
23. 23 Luo, S., Li, J., Stoddard, T.J., Baltes, N.J., Demorest, Z.L., Clasen, B.M., Coffman, A., Retterath, A., Mathis, L., Voytas, D.F., et al. Non-transgenic plant genome editing using purified sequence-specific nucleases. Mol Plant 8 (2015), 1425–1427, 10.1016/j.molp.2016.04.009.
24. 24 Stoddard, T.J., Clasen, B.M., Baltes, N.J., Demorest, Z.L., Voytas, D.F., Zhang, F., Luo, S., Targeted mutagenesis in plant cells through transformation of sequence-specific nuclease mRNA. PLOS ONE, 11, 2016, e0154634, 10.1371/journal.pone.0154634.
25. 25• Woo, J.W., Kim, J., Kwon, S.I., Corvalan, C., Cho, S.W., Kim, H., Kim, S.-G., Kim, S.-T., Choe, S., Kim, J.-S., DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol 33 (2015), 1162–1164, 10.1038/nbt.3389 First demonstration that Cas9 can be applied as protein DNA complex for mutation induction in plants.
26. 26 Endo, M., Mikami, M., Toki, S., Multigene knockout utilizing off-target mutations of the CRISPR/Cas9 system in rice. Plant Cell Physiol 56 (2015), 41–47, 10.1093/pcp/pcu154.
27. 27 Feng, Z., Mao, Y., Xu, N., Zhang, B., Wei, P., Yang, D.-L., Wang, Z., Zhang, Z., Zheng, R., Yang, L., et al. Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci U S A 111 (2014), 4632–4637, 10.1073/pnas.1400822111.
28. 28 Mikami, M., Toki, S., Endo, M., Precision targeted mutagenesis via Cas9 paired nickases in rice. Plant Cell Physiol 57 (2016), 1058–1068, 10.1093/pcp/pcw049.
29. 29 Kim, D., Kim, S., Kim, S., Park, J., Kim, J.-S., Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq. Genome Res 26 (2016), 406–415, 10.1101/gr.199588.115.
30. 30 Kleinstiver, B.P., Pattanayak, V., Prew, M.S., Tsai, S.Q., Nguyen, N.T., Zheng, Z., Joung, J.K., High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529 (2016), 490–495, 10.1038/nature16526.
31. 31 Ran, F.A., Hsu, P.D., Lin, C.-Y., Gootenberg, J.S., Konermann, S., Trevino, A.E., Scott, D.A., Inoue, A., Matoba, S., Zhang, Y., et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154 (2013), 1380–1389, 10.1016/j.cell.2013.08.021.
32. 32 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 Cell Mol Biol 80 (2014), 1139–1150, 10.1111/tpj.12704.
33. 33• Schiml, S., Fauser, F., Puchta, H., Repair of adjacent single-strand breaks is often accompanied by the formation of tandem sequence duplications in plant genomes. Proc Natl Acad Sci U S A 113 (2016), 7266–7271, 10.1073/pnas.1603823113 Shows that by the paired nickase approach mutations can be induced efficiently in genetic, intergenic and heterochromatic region of the plant genome.
34. 34 Swinnen, G., Goossens, A., Pauwels, L., Lessons from domestication: targeting Cis-regulatory elements for crop improvement. Trends Plant Sci 21 (2016), 506–515, 10.1016/j.tplants.2016.01.014.
35. 35 Yan, W., Chen, D., Kaufmann, K., Efficient multiplex mutagenesis by RNA-guided Cas9 and its use in the characterization of regulatory elements in the AGAMOUS gene. Plant Methods, 12, 2016, 23, 10.1186/s13007-016-0125-7.
36. 36 Rajagopal, N., Srinivasan, S., Kooshesh, K., Guo, Y., Edwards, M.D., Banerjee, B., Syed, T., Emons, B.J.M., Gifford, D.K., Sherwood, R.I., High-throughput mapping of regulatory DNA. Nat Biotechnol 34 (2016), 167–174, 10.1038/nbt.3468.
37. 37 Korkmaz, G., Lopes, R., Ugalde, A.P., Nevedomskaya, E., Han, R., Myacheva, K., Zwart, W., Elkon, R., Agami, R., Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9. Nat Biotechnol 34 (2016), 192–198, 10.1038/nbt.3450.
38. 38 Zhou, H., Liu, B., Weeks, D.P., Spalding, M.H., Yang, B., Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Res 42 (2014), 10903–10914, 10.1093/nar/gku806.
39. 39 Ordon, J., Gantner, J., Kemna, J., Schwalgun, L., Reschke, M., Streubel, J., Boch, J., Stuttmann, J., Generation of chromosomal deletions in dicotyledonous plants employing a user-friendly genome editing toolkit. Plant J Cell Mol Biol, 2016, 10.1111/tpj.13319.
40. 40 Park, C.-Y., Sung, J.J., Kim, D.-W., Genome editing of structural variations: modeling and gene correction. Trends Biotechnol 34 (2016), 548–561, 10.1016/j.tibtech.2016.02.011.
41. 41 Qi, Y., Li, X., Zhang, Y., Starker, C.G., Baltes, N.J., Zhang, F., Sander, J.D., Reyon, D., Joung, J.K., Voytas, D.F., Targeted deletion and inversion of tandemly arrayed genes in Arabidopsis thaliana using zinc finger nucleases. G3 (Bethesda, Md) 3 (2013), 1707–1715, 10.1534/g3.113.006270.
42. 42 Pacher, M., Schmidt-Puchta, W., Puchta, H., Two unlinked double-strand breaks can induce reciprocal exchanges in plant genomes via homologous recombination and nonhomologous end joining. Genetics 175 (2007), 21–29, 10.1534/genetics.106.065185.
43. 43 Choi, K., Henderson, I.R., Meiotic recombination hotspots – a comparative view. Plant J Cell Mol Biol 83 (2015), 52–61, 10.1111/tpj.12870.
44. 44•• Pecina, A., Smith, K.N., Mezard, C., Murakami, H., Ohta, K., Nicolas, A., Targeted stimulation of meiotic recombination. Cell 111 (2002), 173–184 Demonstrated that at least in yeast, mitotic recombination can be controlled by fusion of a DNA binding domain with DSB inducing proteins.
45. 45 Sadhu, M.J., Bloom, J.S., Day, L., Kruglyak, L., CRISPR-directed mitotic recombination enables genetic mapping without crosses. Science (New York, NY) 352 (2016), 1113–1116, 10.1126/science.aaf5124.
46. 46 Steinert, J., Schiml, S., Puchta, H., Homology-based double-strand break-induced genome engineering in plants. Plant Cell Rep, 2016, 10.1007/s00299-016-1981-3.
47. 47 Knoll, A., Fauser, F., Puchta, H., DNA recombination in somatic plant cells: mechanisms and evolutionary consequences. Chromosome Res 22 (2014), 191–201, 10.1007/s10577-014-9415-y.
48. 48• Fauser, F., Roth, N., Pacher, M., Ilg, G., Sánchez-Fernández, R., Biesgen, C., Puchta, H., In planta gene targeting. Proc Natl Acad Sci U S A 109 (2012), 7535–7540, 10.1073/pnas.1202191109 Shows that DSB induced gene targeting in plants can be improved by activation of the targeting vector by cutting it out of the plant genome.
49. 49 Zhao, Y., Zhang, C., Liu, W., Gao, W., Liu, C., Song, G., Li, W.-X., Mao, L., Chen, B., Xu, Y., et al. An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design. Sci Rep, 6, 2016, 23890, 10.1038/srep23890.
50. 50• Baltes, N.J., Gil-Humanes, J., Cermak, T., Atkins, P.A., Voytas, D.F., DNA replicons for plant genome engineering. Plant Cell 26 (2014), 151–163, 10.1105/tpc.113.119792 Shows that DSB induced gene targeting in plants can be enhanced by activation of the targeting vector by a viral replicon.
51. 51 Cermak, T., Baltes, N.J., Cegan, R., Zhang, Y., Voytas, D.F., High-frequency, precise modification of the tomato genome. Genome Biol, 16, 2015, 232, 10.1186/s13059-015-0796-9.
52. 52 Nishizawa-Yokoi, A., Cermak, T., Hoshino, T., Sugimoto, K., Saika, H., Mori, A., Osakabe, K., Hamada, M., Katayose, Y., Starker, C., et al. A defect in DNA Ligase4 enhances the frequency of TALEN-mediated targeted mutagenesis in rice. Plant Physiol 170 (2016), 653–666, 10.1104/pp.15.01542.
53. 53 Sauer, N.J., Narvaez-Vasquez, J., Mozoruk, J., Miller, R.B., Warburg, Z.J., Woodward, M.J., Mihiret, Y.A., Lincoln, T.A., Segami, R.E., Sanders, S.L., et al. Oligonucleotide-mediated genome editing provides precision and function to engineered nucleases and antibiotics in plants. Plant Physiol 170 (2016), 1917–1928, 10.1104/pp.15.01696.
54. 54 Svitashev, S., Young, J.K., Schwartz, C., Gao, H., Falco, S.C., Cigan, A.M., Targeted mutagenesis, precise gene editing, and site-specific gene insertion in maize using Cas9 and guide RNA. Plant Physiol 169 (2015), 931–945, 10.1104/pp.15.00793.
55. 55• Li, J., Meng, X., Zong, Y., Chen, K., Zhang, H., Liu, J., Li, J., Gao, C., Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9. Nat Plants, 2, 2016, 16139, 10.1038/nplants.2016.139 Shows that site-specific gene insertion can be achieved by Ca9-induced NHEJ in plants.
56. 56 Gilbert, L.A., Larson, M.H., Morsut, L., Liu, Z., Brar, G.A., Torres, S.E., Stern-Ginossar, N., Brandman, O., Whitehead, E.H., Doudna, J.A., et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154 (2013), 442–451, 10.1016/j.cell.2013.06.044.
57. 57 Konermann, S., Brigham, M.D., Trevino, A.E., Joung, J., Abudayyeh, O.O., Barcena, C., Hsu, P.D., Habib, N., Gootenberg, J.S., Nishimasu, H., et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517 (2015), 583–588, 10.1038/nature14136.
58. 58 Thakore, P.I., Black, J.B., Hilton, I.B., Gersbach, C.A., Editing the epigenome: technologies for programmable transcription and epigenetic modulation. Nat Methods 13 (2016), 127–137, 10.1038/nmeth.3733.
59. 59 Hilton, I.B., D'Ippolito, A.M., Vockley, C.M., Thakore, P.I., Crawford, G.E., Reddy, T.E., Gersbach, C.A., Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol 33 (2015), 510–517, 10.1038/nbt.3199.
60. 60 Thakore, P.I., D'Ippolito, A.M., Song, L., Safi, A., Shivakumar, N.K., Kabadi, A.M., Reddy, T.E., Crawford, G.E., Gersbach, C.A., Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nat Methods 12 (2015), 1143–1149, 10.1038/nmeth.3630.
61. 61 Choudhury, S.R., Cui, Y., Lubecka, K., Stefanska, B., Irudayaraj, J., CRISPR-dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter. Oncotarget, 2016, 10.18632/oncotarget.10234.
62. 62 Agius, F., Kapoor, A., Zhu, J.-K., Role of the Arabidopsis DNA glycosylase/lyase ROS1 in active DNA demethylation. Proc Natl Acad Sci U S A 103 (2006), 11796–11801, 10.1073/pnas.0603563103.
63. 63 Vojta, A., Dobrinic, P., Tadic, V., Bockor, L., Korac, P., Julg, B., Klasic, M., Zoldos, V., Repurposing the CRISPR-Cas9 system for targeted DNA methylation. Nucleic Acids Res 44 (2016), 5615–5628, 10.1093/nar/gkw159.
64. 64• Piatek, A., Ali, Z., Baazim, H., Li, L., Abulfaraj, A., Al-Shareef, S., Aouida, M., Mahfouz, M.M., RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors. Plant Biotechnol J 13 (2015), 578–589, 10.1111/pbi.12284 The first report that dCas9 for transcriptional regulation in plants.
65. 65 Lowder, L.G., Zhang, D., Baltes, N.J., Paul, J.W., Tang, X., Zheng, X., Voytas, D.F., Hsieh, T.-F., Zhang, Y., Qi, Y., A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol 169 (2015), 971–985, 10.1104/pp.15.00636.
66. 66 Nelles, D.A., Fang, M.Y., O'Connell, M.R., Xu, J.L., Markmiller, S.J., Doudna, J.A., Yeo, G.W., Programmable RNA tracking in live cells with CRISPR/Cas9. Cell 165 (2016), 488–496, 10.1016/j.cell.2016.02.054.
67. 67 Ma, H., Tu, L.-C., Naseri, A., Huisman, M., Zhang, S., Grunwald, D., Pederson, T., Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat Biotechnol 34 (2016), 528–530, 10.1038/nbt.3526.
68. 68 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 (2016), 420–424, 10.1038/nature17946.
69. 69 Nishida, K., Arazoe, T., Yachie, N., Banno, S., Kakimoto, M., Tabata, M., Mochizuki, M., Miyabe, A., Araki, M., Hara, K.Y., et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science (New York, NY), 2016, 10.1126/science.aaf8729.
70. 70 Louwen, R., Staals, R.H.J., Endtz, H.P., van Baarlen, P., van der Oost, J., The role of CRISPR-Cas systems in virulence of pathogenic bacteria. Microbiol Mol Biol Rev MMBR 78 (2014), 74–88, 10.1128/MMBR.00039-13.
71. 71 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 Cell Mol Biol 84 (2015), 1295–1305, 10.1111/tpj.13078.
72. 72 Kaya, H., Mikami, M., Endo, A., Endo, M., Toki, S., Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9. Sci Rep, 6, 2016, 26871, 10.1038/srep26871.
73. 73 Karvelis, T., Gasiunas, G., Young, J., Bigelyte, G., Silanskas, A., Cigan, M., Siksnys, V., Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements. Genome Biol, 16, 2015, 253, 10.1186/s13059-015-0818-7.
74. 74 Puchta, H., Using CRISPR/Cas in three dimensions: towards synthetic plant genomes, transcriptomes and epigenomes. Plant J Cell Mol Biol 87 (2016), 5–15, 10.1111/tpj.13100.
75. 75•• Zetsche, B., Gootenberg, J.S., Abudayyeh, O.O., Slaymaker, I.M., Makarova, K.S., Essletzbichler, P., Volz, S.E., Joung, J., van der Oost, J., Regev, A., et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163 (2015), 759–771, 10.1016/j.cell.2015.09.038 Fist characterization of Cpf1 which will become one of the main tools for genome engineering.
76. 76 Fonfara, I., Richter, H., Bratovic, M., Le Rhun, A., Charpentier, E., The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA. Nature 532 (2016), 517–521, 10.1038/nature17945.
77. 77 Kim, D., Kim, J., Hur, J.K., Been, K.W., Yoon, S.-H., Kim, J.-S., Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol 34 (2016), 863–868, 10.1038/nbt.3609.
78. 78 Kleinstiver, B.P., Tsai, S.Q., Prew, M.S., Nguyen, N.T., Welch, M.M., Lopez, J.M., McCaw, Z.R., Aryee, M.J., Joung, J.K., Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat Biotechnol 34 (2016), 869–874, 10.1038/nbt.3620.
79. 79 Abudayyeh, O.O., Gootenberg, J.S., Konermann, S., Joung, J., Slaymaker, I.M., Cox, D.B.T., Shmakov, S., Makarova, K.S., Semenova, E., Minakhin, L., et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science (New York, NY), 353, 2016, aaf5573, 10.1126/science.aaf5573.