Multiplexed editing of a begomovirus genome restricts escape mutant formation and disease development
Autoři:
Anirban Roy aff001; Ying Zhai aff001; Jessica Ortiz aff003; Michael Neff aff003; Bikash Mandal aff002; Sunil Kumar Mukherjee aff002; Hanu R. Pappu aff001
Působiště autorů:
Department of Plant Pathology, Washington State University, Pullman, WA, United States of America
aff001; Advanced Centre for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi, India
aff002; Department of Crop and Soil Sciences, Washington State University, Pullman, WA, United States of America
aff003
Vyšlo v časopise:
PLoS ONE 14(10)
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pone.0223765
Souhrn
Whitefly-transmitted begomoviruses cause serious damage to many economically important food, feed, and fiber crops. Numerous vegetable crops are severely affected and chilli leaf curl virus (ChiLCV) is the most dominant and widely distributed begomovirus in chilli (Capsicum annuum) throughout the Indian subcontinent. Recently, CRISPR-Cas9 technology was used as a means to reduce geminivirus replication in infected plants. However, this approach was shown to have certain limitations such as the evolution of escape mutants. In this study, we used a novel, multiplexed guide RNA (gRNA) based CRISPR-Cas9 approach that targets the viral genome at two or more sites simultaneously. This tactic was effective in eliminating the ChiLCV genome without recurrence of functional escape mutants. Six individual gRNA spacer sequences were designed from the ChiLCV genome and in vitro assays confirmed the cleavage behaviour of these spacer sequences. Multiplexed gRNA expression clones, based on combinations of the above-mentioned spacer sequences, were developed. A total of nine-duplex and two-triplex CRISPR-Cas9 constructs were made. The efficacy of these constructs was tested for inhibition of ChiLCV infection in Nicotiana benthamiana. Results indicated that all the constructs caused a significant reduction in viral DNA accumulation. In particular, three constructs (gRNA5+4, gRNA5+2 and gRNA1+2) were most effective in reducing the viral titer and symptoms. T7E1 assay and sequencing of the targeted viral genome did not detect any escape mutants. The multiplexed genome-editing technique could be an effective way to trigger a high level of resistance against begemoviruses. To our knowledge, this is the first report of demonstrating the effectiveness of a multiplexed gRNA-based plant virus genome editing to minimize and eliminate escape mutant formation.
Klíčová slova:
Sequence motif analysis – Viral replication – Leaves – Viral genomics – Viral genome – CRISPR – Guide RNA – Ribonucleoproteins
Zdroje
1. Mansoor S, Briddon RW, Zafar Y, Stanley J. Geminivirus disease complexes: an emerging threat. Trends Plant Sci. 2003; 8: 128–134. doi: 10.1016/S1360-1385(03)00007-4 12663223
2. Varma A, Mandal B, Singh MK. Globale and spread of whitefly (Bemisiatabaci) transmitted Geminiviruses. In: Thompson W, Editor. The Whitefly, Bemisiatabaci (Homoptera: Aleyrodidae) Interaction with Geminivirus-Infected Host Plants; 2011. pp. 205–292.
3. Malathi VG, Renukadevi P, Chakraborty S, Biswas KK, Roy A, Sivalingam PN, et al. Begomoviruses and Their Satellites Occurring in India: Distribution, Diversity and Pathogenesis. In: Mandal B, Rao G, Baranwal V, Jain R, edtiors. A Century of Plant Virology in India; 2017. pp. 75–177.
4. Zehra SB, Ahmad A, Sharma A, Sofi S, Lateef A, Bashir Z, et al. Chilli Leaf Curl Virus an Emerging Threat to Chilli in India. Int. J. Pure App. Biosci. 2017; 5: 404–414.
5. Senanayake DMJB, Varma A, Mandal B. Virus–vector Relationships, Host Range, Detection and Sequence Comparison of Chilli leaf curl virus Associated with an Epidemic of Leaf Curl Disease of Chilli in Jodhpur, India. J. Phytopathol. 2012; 160: 146–155.
6. Zhou X. Advances in understanding begomovirus satellites. Annu. Rev. Phytopathol. 2013; 51: 357–381. doi: 10.1146/annurev-phyto-082712-102234 23915133
7. Lazarowitz S. Geminiviruses: Genome structure andgene function. Crit. Rev. Plant Sci. 1992; 11: 327–349.
8. Hanley-Bowdoin L, Settlage SB, Orozco BM, Nagar S, Robertson D. Geminiviruses: models for plant DNA replication, transcription, and cell cycle regulation. Crit. Rev. Biochem. Mol. Biol. 2000; 35: 105–140. 10821479
9. Fondong VN. Geminivirus protein structure and function. Mol. Plant Pathol. 2013; 14: 635–649. doi: 10.1111/mpp.12032 23615043
10. Lapidot M, Legg JP, Wintermantel WM, Polston JE. Management of whitefly-transmitted viruses in open-field production systems. Adv. Virus Res. 2014; 90: 147–206. doi: 10.1016/B978-0-12-801246-8.00003-2 25410102
11. Vu TV, Choudhury NR, Mukherjee SK. Transgenic tomato plants expressing artificial microRNAs for silencing pre-coat and coat proteins of a begomovirus ToLCNDV, shows tolerance to virus infection. Virus Res. 2013; 172: 35–45. doi: 10.1016/j.virusres.2012.12.008 23276684
12. Singh A, Taneja J, Dasgupta I, Mukherjee SK. Development of plants resistant to tomato geminiviruses using artificial transacting small interfering RNA. Mol. Plant Pathol. 2015; 16: 724–734. doi: 10.1111/mpp.12229 25512230
13. Rojas MR, Macedo MA, Maliano MR, Soto-Aguilar M, Souza JO, Briddon RW, et al. World Management of Geminiviruses. Annu. Rev.Phytopathol. 2018; 56: 637–677. doi: 10.1146/annurev-phyto-080615-100327 30149794
14. Gill U, Scott JW, Shekasteband R, Ogundiwin E, Schuit C, Francis DM, et al. Ty-6, a major begomovirus resistance gene on chromosome 10, is effective against Tomato yellow leaf curl virus and Tomato mottle virus. Theor. Appl. Genet. 2019; 132: 1543–1554. doi: 10.1007/s00122-019-03298-0 30758531
15. Bonfim K, Faria JC, Nogueira EOPL, Mendes EA, Arago FJL. RNAi mediated resistance to Bean Golden Mosaic Virus in Genetically engineered common bean (Phaseolus vulguris). Mol. Plant Microbe Interact. 2007; 20: 717–726. doi: 10.1094/MPMI-20-6-0717 17555279
16. Voytas DF, Gao C. Precision genome engineering and agriculture: opportunities and regulatory challenges. PLoS Biol. 2014; 12: e1001877. doi: 10.1371/journal.pbio.1001877 24915127
17. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 2007; 315: 1709–1712. doi: 10.1126/science.1138140 17379808
18. Wright AV, Nunez JK, Doudna JA. Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell 2016; 164: 29–44. doi: 10.1016/j.cell.2015.12.035 26771484
19. Aouida M, Eid A, Ali Z, Cradick T, Lee C, Deshmukh H, et al. Efficient fdCas9 synthetic endonuclease with improved specificity for precise genome engineering. PLoS One 2015; 10: e0133373. doi: 10.1371/journal.pone.0133373 26225561
20. Piatek A, Mahfouz MM. Targeted genome regulation via synthetic programmable transcriptional regulators. Crit. Rev.Biotechnol. 2016; 37: 429–440. doi: 10.3109/07388551.2016.1165180 27093352
21. Chaparro-Garcia A, Kamoun S, Nekrasov V. Boosting plant immunity with CRISPR/Cas. Genome Biol. 2015; 16: 254–257. doi: 10.1186/s13059-015-0829-4 26585913
22. Hadidi A, Flores R, Candresse T, Barba M. Next-Generation Sequencing and Genome Editing in Plant Virology. Front. Microbiol. 2016; 7: 1325. doi: 10.3389/fmicb.2016.01325 27617007
23. Ali Z, Abulfaraj A, Idris A, Ali S, Tashkandi M, Mahfouz MM. CRISPR/Cas9-mediated viral interference in plants. Genome Biol. 2015; 16: 238. doi: 10.1186/s13059-015-0799-6 26556628
24. Ali Z, Ali S, Tashkandi M, Zaidi SS, Mahfouz MM. CRISPR/Cas9-mediated immunity to geminiviruses: differential interference and evasion. Sci. Rep. 2016; 6: 26912. doi: 10.1038/srep26912 27225592
25. Baltes NJ, Hummel AW, Konecna E, Cegan R, Bruns AN, Bisaro DM, et al. Conferring resistance to geminiviruses with the CRISPR–Cas prokaryotic immune system. Nature Plants 2015; 1: 15145.
26. Ji X, Zhang H, Zhang Y, Wang Y, Gao C. Establishing a CRISPR–Cas-like immune system conferring DNA virus resistance in plants. Nature Plants 2015; 1: 15144. doi: 10.1038/nplants.2015.144 27251395
27. Liu HJ, Soyars CL, Li JH, Fei QL, He GJ, Fei Q, et al. CRISPR/Cas9-mediated resistance to cauliflower mosaic virus. Plant Direct. 2018; 2: e00047. doi: 10.1002/pld3.47 31245713
28. Mehta D, Stürchler A, Anjanappa RB, Zaidi SS, Hirsch-Hoffmann M, Gruissem W, et al. Linking CRISPR-Cas9 interference in cassava to the evolution of editing-resistant geminiviruses. Genome Biol. 2019; 20: 80. doi: 10.1186/s13059-019-1678-3 31018865
29. Rybicki EP. CRISPR–Cas9 strikes out in cassava. Nature Biotechnol. 2019; 37: 727–728.
30. Zaidi SSA, Tashkandi M, Mahfouz MM. Engineering molecular immunity against plant viruses. Prog. Mol. Biol. Transl. 2017; 149: 167–186.
31. Shilpi S, Kumar A, Biswas S, Roy A, Mandal B. A recombinant Tobacco curly shoot virus causes leaf curl disease in tomato in a north-eastern state of India and has potentiality to trans-replicate a non-cognate betasatellite. Virus Genes 2015; 50: 87–96. doi: 10.1007/s11262-014-1141-1 25410052
32. Lowder LG, Zhang D, Baltes NJ, Paul JW, Tang X, Zheng X, et al. A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol. 2015; 169: 971–985. doi: 10.1104/pp.15.00636 26297141
33. Liu D, Shi L, Han C, Yu J, Li D, Zhang Y. Validation of reference genes for gene expression studies in virus-infected Nicotiana benthamiana using quantitative real-time PCR. PLoS One 2012; 7: e46451. doi: 10.1371/journal.pone.0046451 23029521
34. Brinkman EK, Chen T, Amendola M, van Steensel B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Rec. 2014; 42: e168. doi: 10.1093/nar/gku936 25300484
35. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 2014; 157: 1262–1278. doi: 10.1016/j.cell.2014.05.010 24906146
36. Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, et al. A CRISPR/Cas9 tool kit for multiplex genome editing in plants. BMC PlantBiol. 2014; 14: 327–339.
37. Xie K, Minkenberg B, Yang Y. Boosting CRISPR/Cas9 multiplex editing capability with the endogenoust RNA-processing system. Proc. Natl. Acad. Sci. U.S.A. 2015; 112: 3570–3575. doi: 10.1073/pnas.1420294112 25733849
38. Brinkman EK, van Steensel B. Rapid Quantitative Evaluation of CRISPR Genome Editing by TIDE and TIDER. In: Luo Y. (eds) CRISPR Gene Editing. Methods in Molecular Biology, vol 1961. Humana Press, New York, NY. 2019.
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