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

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.

Principles of RNA recruitment to viral ribonucleoprotein condensates in a segmented dsRNA virus

“…This implies that the unique viroplasmic condensate composition must be maintained through enrichment of cognate RNA-binding viral proteins. Remarkably, targeting the RV RNP condensates by a catalytically active Cas6f/Csy4 endonuclease fused to NSP5 results in the viral RNA genome editing that occurs within these condensates 22 , further confirming the essential role of these RNP condensates in the RV transcript selection and enrichment. The UDEx-FISH analysis of single cells revealed that the apparent stoichiometries of viroplasm-enriched and cytoplasmicallylocalised RV transcripts were different, suggesting that the observed RNA partitioning mechanism may also contribute to the efficient stoichiometric enrichment of eleven non-identical viral transcripts in these condensates.…”

“…MA104-shRNA-NSP2 cell line was generated using the PiggyBac system. Briefly, 10 5 MA104 cells were co-transfected with the plasmid pCMV-HyPBase encoding the hyperactive variant of PiggyBac transposase 56,57,22 along with the plasmid pPB[shRNA]-EGFP:T2A:Puro-U6 harbouring shRNA targeting RVA NSP2 gene using Lipofectamine 3000 (Sigma-Aldrich), following the manufacturer's instructions. The cells were maintained in DMEM supplemented with 10% FBS for 3 days, and then the cells were subjected to selection in the presence of 5 μg/ml puromycin (Sigma-Aldrich) for 4 days, prior to further selection by FACS sorting for EGFP expression.…”

“…Rescue of the recombinant NSP3-2A-EGFP virus (strain SA11) was carried out as previously described 22,31,58,59 . Briefly, monolayers of BHK-T7 cells (4 × 10 5 ) cultured in 12-well plates were co-transfected using 2.5 μL of TransIT-LT1 transfection reagent (Mirus) per microgram of DNA plasmid.…”

“…In principle, the formation of dynamic ribonucleoprotein condensates in RV-infected cells could facilitate selective enrichment of cognate viral transcripts required for a robust and stoichiometric RNA assembly and packaging. Despite the extensive evidence of the importance of viroplasms in RV replication 13,[18][19][20][21][22] , the analysis of their molecular composition have been confounded by both their dynamic and liquid-like nature that precluded successful isolation from the RV-infected cells. Thus, the exact RNA composition of these assemblies has remained enigmatic.…”

A recombinant rotavirus harboring a spike protein with a heterologous peptide reveals a novel role of VP4 in viroplasm stability

“…We next assessed whether these four VP4-BAP proteins could assemble into infectious rotavirus particles and support virus replication. For this purpose, we took advantage of a newly developed reverse genetics system to rescue recombinant rotavirus (rRV) harboring a genetically modified genome segment 4 (gs4) encoding the different VP4-BAP proteins (gs4-BAP) (38)(39)(40)(41).…”