Rapid poxvirus engineering using CRISPR/Cas9 as a selection tool

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.

Virus-encoded histone doublets are essential and form nucleosome-like structures

“…Genetic manipulation of giant viruses has been a neglected topic of research since their discovery despite the immense richness in genes with unknown functions encoded by their genome. Homology recombination has been used as a powerful tool to modify the genome of other viruses (Gowripalan et al, 2020), and we show here that it can be efficiently utilized for genetic manipulation of MV. Moreover, the essentiality of genes that encode MV histones, like mel_369 and mel_368, can be overcome by trans-complementation, introducing ectopic copies of the gene in the amoeba genome.…”

Advancements in the Growth and Construction of Recombinant Lumpy Skin Disease Virus (LSDV) for Use as a Vaccine Vector

“…Selection of poxvirus recombinants by homologous recombination is a long and laborious process and so alternatives are needed. Recent improvements have included the synthesis of horsepox virus from chemically synthesized DNA, which could be used to make recombinants [ 52 ], and the use of CRISPR/Cas9 technology to target the parent poxvirus genome [ 53 ]. Both these approaches are relatively expensive.…”

MAVERICC: Marker-free Vaccinia Virus Engineering of Recombinants through in vitro CRISPR/Cas9 Cleavage

“…Thus, these workflows still required the use of selectable markers and multiple rounds of plaque purification. A more recent study demonstrated that Cas9 could rapidly and efficiently cleave vDNA in cells, but found that cleavage reduced viral replication and multiplication while having no stimulating effect on genome repair through homologous recombination [13]. This may be the case because (i) the cytoplasmic vDNA cannot be repaired by cellular HDR, which requires components that are localized to the nucleus; and (ii) efficient cleavage of vDNA by Cas9 soon after viral entry may inhibit VACV DNA replication, thereby also inhibiting homologous recombination, to which it is coupled.…”

“…Recently, CRISPR/Cas9 engineering [10] has been employed in attempts to streamline the rVACV rescue process [11][12][13][14][15]. Yuan and co-workers [11] introduced Streptococcus pyogenes Cas9 and vDNA-specific single-guide RNAs (sgRNAs) into cells to cleave the vDNA from infecting VACVs and enhance its homologous recombination with a transfected transfer plasmid.…”

“…This approach marginally increased the efficiency of rVACV formation over the traditional method but still required the insertion, and possible subsequent removal, of a marker gene. An elaboration of this strategy used intracellular vDNA cleavage by Cas9 to selectively inhibit the replication of parental genomes, thereby affording the marker-free recovery of rVACVs [13].…”