Abstract
In standard uses of CRISPR/Cas9 technology, the cutting of genomes and their efficient repair are considered to go hand-in-hand to achieve desired genetic changes. This includes the current approach for engineering genomes of large dsDNA viruses. However, for poxviruses we show that Cas9-guide RNA complexes cut viral genomes soon after their entry into cells, but repair of these breaks is inefficient. As a result, Cas9 targeting makes only modest, if any, improvements to basal rates of homologous recombination between repair constructs and poxvirus genomes. Instead, Cas9 cleavage leads to inhibition of poxvirus DNA replication thereby suppressing virus spread in culture. This unexpected outcome allows Cas9 to be used as a powerful tool for selecting conventionally generated poxvirus recombinants, which are otherwise impossible to separate from a large background of parental virus without the use of marker genes. This application of CRISPR/Cas9 greatly speeds up the generation of poxvirus-based vaccines, making this platform considerably more attractive in the context of personalised cancer vaccines and emerging disease outbreaks.
Highlights
In standard uses of CRISPR/Cas[9] technology, the cutting of genomes and their efficient repair are considered to go hand-in-hand to achieve desired genetic changes
We have previously described a successful protocol for editing of herpes simplex virus (HSV) genomes with CRISPR/Cas[934–36]
We set up fluorescent virus systems to quantify the efficiency of any CRISPR/Cas[9] activity, starting without a repair template to examine Vaccinia virus (VACV) genome editing by non-homologous end joining (NHEJ)
Summary
In standard uses of CRISPR/Cas[9] technology, the cutting of genomes and their efficient repair are considered to go hand-in-hand to achieve desired genetic changes. Genome editing of VACV by homologous recombination is relatively efficient compared with other DNA viruses, such as herpesviruses, which rely on an array of host repair enzymes[6,29] This efficiency remains in the range of 0.1–1% for VACV, meaning marker genes are inevitably required to enable selection or screening of desired recombinants from a large background of unmodified parent virus. Inefficient repair leaves the cut genomes unable to be replicated With this insight we go on to show that the utility of CRISPR/Cas[9] lies in its use as a highly versatile and efficient selection method for recombinant VACVs, allowing for generation of marker-free viruses in under 2 weeks
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