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. Graphical Abstract Highlights • CRISPR-Csy4 fused with NSP5 can cleave rotavirus (+)ssRNA inside viroplasms• Csy4-cleaved (+)ssRNA replication intermediates are repaired as edited viral dsRNA• Csy4 editing allows detection of products of secondary transcription• Secondary transcription is the main source of rotavirus proteins in infected cells Papa et al. engineer the CRISPR-Csy4 nuclease to localize into rotavirus viral factories and cleave (+)ssRNA replication intermediates, producing edits of the targeted dsRNA genome segment. This allows for the detection of secondary transcription-derived proteins made by the newly assembled viruses, demonstrating that they largely contribute to overall viral protein production.

SUBMITTER: Papa G 

PROVIDER: S-EPMC7523552 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature