Anti-CRISPR-Based and CRISPR-Based Genome Editing of Sulfolobus islandicus Rod-Shaped Virus 2
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Anti-CRISPR-Based and CRISPR-Based Genome Editing of Sulfolobus islandicus Rod-Shaped Virus 2. / Mayo-Muñoz, David; He, Fei; Jørgensen, Jacob Bruun; Madsen, Poul Kári; Bhoobalan-Chitty, Yuvaraj; Peng, Xu.
I: Viruses, Bind 10, Nr. 12, 695, 2018, s. 1-17.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - Anti-CRISPR-Based and CRISPR-Based Genome Editing of Sulfolobus islandicus Rod-Shaped Virus 2
AU - Mayo-Muñoz, David
AU - He, Fei
AU - Jørgensen, Jacob Bruun
AU - Madsen, Poul Kári
AU - Bhoobalan-Chitty, Yuvaraj
AU - Peng, Xu
PY - 2018
Y1 - 2018
N2 - 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 (∆arrays) 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.
AB - 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 (∆arrays) 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.
KW - Accessory genes
KW - Anti-CRISPR-based genome editing
KW - Core genome
KW - CRISPR-based genome editing
KW - Essentiality
KW - Selection marker
KW - Virus-host interaction
U2 - 10.3390/v10120695
DO - 10.3390/v10120695
M3 - Journal article
C2 - 30544778
AN - SCOPUS:85058296457
VL - 10
SP - 1
EP - 17
JO - Viruses
JF - Viruses
SN - 1999-4915
IS - 12
M1 - 695
ER -
ID: 211855715