CRISPR-Csy4-Mediated Editing of Rotavirus Double-Stranded RNA Genome

Overview

Journal Cell Rep

Publisher Cell Press

Specialties Biology
Cell Biology
Molecular Biology

Date 2020 Sep 30

PMID 32997981

Citations 14

Authors

Guido Papa

Luca Venditti

Luca Braga

Edoardo Schneider

Mauro Giacca

Gianluca Petris

Oscar R Burrone

Affiliations

Soon will be listed here.

Abstract

CRISPR-nucleases have been widely applied for editing cellular and viral genomes, but nuclease-mediated genome editing of double-stranded RNA (dsRNA) viruses has not yet been reported. Here, by engineering CRISPR-Csy4 nuclease to localize to rotavirus viral factories, we achieve the nuclease-mediated genome editing of rotavirus, an important human and livestock pathogen with a multisegmented dsRNA genome. Rotavirus replication intermediates cleaved by Csy4 is edited through the formation of precise deletions in the targeted genome segments in a single replication cycle. Using CRISPR-Csy4-mediated editing of rotavirus genome, we label the products of rotavirus secondary transcription made by newly assembled viral particles during rotavirus replication, demonstrating that this step largely contributes to the overall production of viral proteins. We anticipate that the nuclease-mediated cleavage of dsRNA virus genomes will promote an advanced level of understanding of viral replication and host-pathogen interactions, also offering opportunities to develop therapeutics.

Citing Articles

A trigger-inducible split-Csy4 architecture for programmable RNA modulation.

Zhang L, Qiu X, Zhou Y, Luo Z, Zhu L, Shao J Nucleic Acids Res. 2025; 53(2).

PMID: 39817512 PMC: 11734699. DOI: 10.1093/nar/gkae1319.

Biotechnological Interventions for the Production of Subunit Vaccines Against Group A Rotavirus.

Prajapati M, Malik P, Sinha A, Yadav H, Jaiwal Y, Ahlawat Y Cell Biochem Funct. 2024; 42(8):e70031.

PMID: 39707603 PMC: 11662099. DOI: 10.1002/cbf.70031.

Generation of single-round infectious rotavirus with a mutation in the intermediate capsid protein VP6.

Kotaki T, Kanai Y, Onishi M, Minami S, Chen Z, Nouda R J Virol. 2024; 98(7):e0076224.

PMID: 38837379 PMC: 11265344. DOI: 10.1128/jvi.00762-24.

The recruitment of TRiC chaperonin in rotavirus viroplasms correlates with virus replication.

Vetter J, Papa G, Tobler K, Rodriguez J, Kley M, Myers M mBio. 2024; 15(4):e0049924.

PMID: 38470055 PMC: 11005421. DOI: 10.1128/mbio.00499-24.

Rotavirus Particle Disassembly and Assembly In Vivo and In Vitro.

Asensio-Cob D, Rodriguez J, Luque D Viruses. 2023; 15(8).

PMID: 37632092 PMC: 10458742. DOI: 10.3390/v15081750.

References

Schnepf N, Deback C, Dehee A, Gault E, Parez N, Garbarg-Chenon A . Rearrangements of rotavirus genomic segment 11 are generated during acute infection of immunocompetent children and do not occur at random. J Virol. 2008; 82(7):3689-96. PMC: 2268475. DOI: 10.1128/JVI.01770-07. View

Eichwald C, Arnoldi F, Laimbacher A, Schraner E, Fraefel C, Wild P . Rotavirus viroplasm fusion and perinuclear localization are dynamic processes requiring stabilized microtubules. PLoS One. 2012; 7(10):e47947. PMC: 3479128. DOI: 10.1371/journal.pone.0047947. View

Haurwitz R, Jinek M, Wiedenheft B, Zhou K, Doudna J . Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science. 2010; 329(5997):1355-8. PMC: 3133607. DOI: 10.1126/science.1192272. View

Abbott T, Dhamdhere G, Liu Y, Lin X, Goudy L, Zeng L . Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza. Cell. 2020; 181(4):865-876.e12. PMC: 7189862. DOI: 10.1016/j.cell.2020.04.020. View

Lopez T, Camacho M, Zayas M, Najera R, Sanchez R, Arias C . Silencing the morphogenesis of rotavirus. J Virol. 2004; 79(1):184-92. PMC: 538724. DOI: 10.1128/JVI.79.1.184-192.2005. View

Lee H, Haurwitz R, Apffel A, Zhou K, Smart B, Wenger C . RNA-protein analysis using a conditional CRISPR nuclease. Proc Natl Acad Sci U S A. 2013; 110(14):5416-21. PMC: 3619310. DOI: 10.1073/pnas.1302807110. View

Yoshiba T, Saga Y, Urabe M, Uchibori R, Matsubara S, Fujiwara H . CRISPR/Cas9-mediated cervical cancer treatment targeting human papillomavirus E6. Oncol Lett. 2019; 17(2):2197-2206. PMC: 6341785. DOI: 10.3892/ol.2018.9815. View

Borchardt E, Vandoros L, Huang M, Lackey P, Marzluff W, Asokan A . Controlling mRNA stability and translation with the CRISPR endoribonuclease Csy4. RNA. 2015; 21(11):1921-30. PMC: 4604432. DOI: 10.1261/rna.051227.115. View

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna J, Charpentier E . A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337(6096):816-21. PMC: 6286148. DOI: 10.1126/science.1225829. View

10.

Chen Y, Sheng J, Trang P, Liu F . Potential Application of the CRISPR/Cas9 System against Herpesvirus Infections. Viruses. 2018; 10(6). PMC: 6024784. DOI: 10.3390/v10060291. View

11.

Haurwitz R, Sternberg S, Doudna J . Csy4 relies on an unusual catalytic dyad to position and cleave CRISPR RNA. EMBO J. 2012; 31(12):2824-32. PMC: 3380207. DOI: 10.1038/emboj.2012.107. View

12.

Papa G, Venditti L, Arnoldi F, Schraner E, Potgieter C, Borodavka A . Recombinant Rotaviruses Rescued by Reverse Genetics Reveal the Role of NSP5 Hyperphosphorylation in the Assembly of Viral Factories. J Virol. 2019; 94(1). PMC: 6912106. DOI: 10.1128/JVI.01110-19. View

13.

Silvestri L, Taraporewala Z, Patton J . Rotavirus replication: plus-sense templates for double-stranded RNA synthesis are made in viroplasms. J Virol. 2004; 78(14):7763-74. PMC: 434085. DOI: 10.1128/JVI.78.14.7763-7774.2004. View

14.

Gonzalez S, Mattion N, Bellinzoni R, Burrone O . Structure of rearranged genome segment 11 in two different rotavirus strains generated by a similar mechanism. J Gen Virol. 1989; 70 ( Pt 6):1329-36. DOI: 10.1099/0022-1317-70-6-1329. View

15.

Eichwald C, Rodriguez J, Burrone O . Characterization of rotavirus NSP2/NSP5 interactions and the dynamics of viroplasm formation. J Gen Virol. 2004; 85(Pt 3):625-634. DOI: 10.1099/vir.0.19611-0. View

16.

Brinkman E, Chen T, Amendola M, van Steensel B . Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 2014; 42(22):e168. PMC: 4267669. DOI: 10.1093/nar/gku936. View

17.

Davis K, Patton J . Shutdown of interferon signaling by a viral-hijacked E3 ubiquitin ligase. Microb Cell. 2017; 4(11):387-389. PMC: 5695857. DOI: 10.15698/mic2017.11.600. View

18.

Murugan K, Babu K, Sundaresan R, Rajan R, Sashital D . The Revolution Continues: Newly Discovered Systems Expand the CRISPR-Cas Toolkit. Mol Cell. 2017; 68(1):15-25. PMC: 5683099. DOI: 10.1016/j.molcel.2017.09.007. View

19.

Nissim L, Perli S, Fridkin A, Perez-Pinera P, Lu T . Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Mol Cell. 2014; 54(4):698-710. PMC: 4077618. DOI: 10.1016/j.molcel.2014.04.022. View

20.

Sternberg S, Haurwitz R, Doudna J . Mechanism of substrate selection by a highly specific CRISPR endoribonuclease. RNA. 2012; 18(4):661-72. PMC: 3312554. DOI: 10.1261/rna.030882.111. View