Engineering herpes simplex viruses by infection–transfection methods including recombination site targeting by CRISPR/Cas9 nucleases

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dc.contributor.authorRussell, Tiffany A.
dc.contributor.authorStefanovic, Tijana
dc.contributor.authorTscharke, David C.
dc.date.accessioned2015-03-05T00:54:32Z
dc.date.available2015-03-05T00:54:32Z
dc.date.issued2015
dc.date.updated2015-12-11T09:15:04Z
dc.description.abstractHerpes simplex viruses (HSVs) are frequent human pathogens and the ability to engineer these viruses underpins much research into their biology and pathogenesis. Often the ultimate aim is to produce a virus that has the desired phenotypic change and no additional alterations in characteristics. This requires methods that minimally disrupt the genome and, for insertions of foreign DNA, sites must be found that can be engineered without disrupting HSV gene function or expression. This study advances both of these requirements. Firstly, the use of homologous recombination between the virus genome and plasmids in mammalian cells is a reliable way to engineer HSV such that minimal genome changes are made. This has most frequently been achieved by cotransfection of plasmid and isolated viral genomic DNA, but an alternative is to supply the virus genome by infection in a transfection-infection method. Such approaches can also incorporate CRISPR/Cas9 genome engineering methods. Current descriptions of infection-transfection methods, either with or without the addition of CRISPR/Cas9 targeting, are limited in detail and the extent of optimization. In this study it was found that transfection efficiency and the length of homologous sequences improve the efficiency of recombination in these methods, but the targeting of the locus to be engineered by CRISPR/Cas9 nucleases has an overriding positive impact. Secondly, the intergenic space between UL26 and UL27 was reexamined as a site for the addition of foreign DNA and a position identified that allows insertions without compromising HSV growth in vitro or in vivo.
dc.description.sponsorshipThis work was funded by NHMRC Project Grant APP1005846 and ARC Future Fellowship FT110100310.en_AU
dc.format7 pages
dc.identifier.issn0166-0934
dc.identifier.urihttp://hdl.handle.net/1885/12813
dc.provenanceAuthors pre-print on any website http://www.sherpa.ac.uk/romeo/issn/0166-0934/
dc.publisherElsevier
dc.relationhttp://purl.org/au-research/grants/nhmrc/1005846
dc.relationhttp://purl.org/au-research/grants/arc/ft110100310
dc.rights© 2015 Elsevier
dc.sourceJournal of Virological Methods
dc.subjectCRISPR
dc.subjectCas9
dc.subjectGenome engineering
dc.subjectHerpes simplex virus
dc.subjectRecombinant virus
dc.titleEngineering herpes simplex viruses by infection–transfection methods including recombination site targeting by CRISPR/Cas9 nucleases
dc.typeJournal article
dcterms.accessRightsOpen Access
dcterms.dateAccepted2014-11-25
local.bibliographicCitation.lastpage25en_AU
local.bibliographicCitation.startpage18en_AU
local.contributor.affiliationTscharke, David C., Division of Biomedical Science and Biochemistry, Research School of Biology, The Australian National Universityen_AU
local.contributor.affiliationRussell, Tiffany A., Division of Biomedical Science and Biochemistry, Research School of Biology, The Australian National Universityen_AU
local.contributor.affiliationStefanovic, Tijana, Division of Biomedical Science and Biochemistry, Research School of Biology, The Australian National Universityen_AU
local.contributor.authoremaildavid.tscharke@anu.edu.auen_AU
local.contributor.authoruidu4334102en_AU
local.identifier.absfor060506 - Virology
local.identifier.absseo920109 - Infectious Diseases
local.identifier.ariespublicationU3488905xPUB4867
local.identifier.citationvolume213en_AU
local.identifier.doi10.1016/j.jviromet.2014.11.009en_AU
local.identifier.essn1879-0984en_AU
local.identifier.scopusID2-s2.0-84916912019
local.identifier.uidSubmittedByu4334102en_AU
local.publisher.urlhttp://www.journals.elsevier.com/en_AU
local.type.statusSubmitted Versionen_AU

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