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.
Highlights
Prokaryotes have evolved an anti-viral defense mechanism based on CRISPR loci
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
Summary
Prokaryotes have evolved an anti-viral defense mechanism based on CRISPR loci These regions express pre-CRISPR RNAs (pre-crRNAs) containing short virus-derived sequences. Csy4/Cas6f of Pseudomonas aeruginosa type I-F CRISPR systems (Makarova et al, 2018), is a well-characterized small (21 kDa) and highly specific single-turnover RNA endoribonuclease, processing pre-crRNAs in a 28-nucleotide (nt) sequence (Cy28) to generate the mature crRNAs. Csy binds a 16-nt RNA hairpin within Cy28 with very high affinity (KD = 50 pM) and cleaves directly downstream of the five-base-pair stem element (Haurwitz et al, 2010; Sternberg et al, 2012; Lee et al, 2013). Csy has already been applied to cleave several engineered Cy28-containing RNAs for biotechnological applications including a model human immunodeficiency virus (HIV) (Guo et al, 2015; Nissim et al, 2014; Lee et al, 2013)
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