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Open AccessArticle

Anti-CRISPR-Based and CRISPR-Based Genome Editing of Sulfolobus islandicus Rod-Shaped Virus 2

Danish Archaea Centre, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark
*
Author to whom correspondence should be addressed.
These authors contributed equally to the work.
Viruses 2018, 10(12), 695; https://doi.org/10.3390/v10120695
Received: 14 November 2018 / Revised: 5 December 2018 / Accepted: 6 December 2018 / Published: 8 December 2018
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)
Genetic engineering of viruses has generally been challenging. This is also true for archaeal rod-shaped viruses, which carry linear double-stranded DNA genomes with hairpin ends. In this paper, we describe two different genome editing approaches to mutate the Sulfolobus islandicus rod-shaped virus 2 (SIRV2) using the archaeon Sulfolobus islandicus LAL14/1 and its derivatives as hosts. The anti-CRISPR (Acr) gene acrID1, which inhibits CRISPR-Cas subtype I-D immunity, was first used as a selection marker to knock out genes from SIRV2M, an acrID1-null mutant of SIRV2. Moreover, we harnessed the endogenous CRISPR-Cas systems of the host to knock out the accessory genes consecutively, which resulted in a genome comprised solely of core genes of the 11 SIRV members. Furthermore, infection of this series of knockout mutants in the CRISPR-null host of LAL14/1 (Δarrays) confirmed the non-essentiality of the deleted genes and all except the last deletion mutant propagated as efficiently as the WT SIRV2. This suggested that the last gene deleted, SIRV2 gp37, is important for the efficient viral propagation. The generated viral mutants will be useful for future functional studies including searching for new Acrs and the approaches described in this case are applicable to other viruses. View Full-Text
Keywords: anti-CRISPR-based genome editing; selection marker; CRISPR-based genome editing; core genome; accessory genes; essentiality; virus-host interaction anti-CRISPR-based genome editing; selection marker; CRISPR-based genome editing; core genome; accessory genes; essentiality; virus-host interaction
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MDPI and ACS Style

Mayo-Muñoz, D.; He, F.; Jørgensen, J.B.; Madsen, P.K.; Bhoobalan-Chitty, Y.; Peng, X. Anti-CRISPR-Based and CRISPR-Based Genome Editing of Sulfolobus islandicus Rod-Shaped Virus 2. Viruses 2018, 10, 695.

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