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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 localise to rotavirus viral factories, we achieved the first nuclease-mediated genome editing of rotavirus, an important human and livestock pathogen with a multi-segmented dsRNA genome. Rotavirus replication intermediates cleaved by Csy4 were repaired through the formation of defined deletions in the targeted genome segments in a single replication cycle. Using CRISPR-Csy4-mediated editing of rotavirus genome, we labelled for the first time 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 a new level of understanding of viral replication and host-pathogen interactions, offering the opportunity to develop new therapeutics.

This paper addresses a practical scheduling problem arising in the packaging department of a pharmaceutical industrial plant. The problem is modeled as a multi-purpose machine scheduling problem with setup and removal times, release and due dates and additional constraints related to the scarce availability of tools and human operators. The objective functions are minimization of makespan and maximum tardiness in lexicographic order. Representing a solution with a directed graph allows us to devise an effective tabu search algorithm to solve the problem. Computational experiments, carried on real and randomly generated instances, show the effectiveness of this approach.