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Viruses 2018, 10(4), 171; https://doi.org/10.3390/v10040171

Rapid CRISPR/Cas9-Mediated Cloning of Full-Length Epstein-Barr Virus Genomes from Latently Infected Cells

Division of Microbiology, Faculty of Medicine, Tohoku Medical and Pharmaceutical University, 1-15-1 Fukumuro, Miyagino-ku, Sendai 983-8536, Japan
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Received: 23 February 2018 / Revised: 28 March 2018 / Accepted: 31 March 2018 / Published: 3 April 2018
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)
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Abstract

Herpesviruses have relatively large DNA genomes of more than 150 kb that are difficult to clone and sequence. Bacterial artificial chromosome (BAC) cloning of herpesvirus genomes is a powerful technique that greatly facilitates whole viral genome sequencing as well as functional characterization of reconstituted viruses. We describe recently invented technologies for rapid BAC cloning of herpesvirus genomes using CRISPR/Cas9-mediated homology-directed repair. We focus on recent BAC cloning techniques of Epstein-Barr virus (EBV) genomes and discuss the possible advantages of a CRISPR/Cas9-mediated strategy comparatively with precedent EBV-BAC cloning strategies. We also describe the design decisions of this technology as well as possible pitfalls and points to be improved in the future. The obtained EBV-BAC clones are subjected to long-read sequencing analysis to determine complete EBV genome sequence including repetitive regions. Rapid cloning and sequence determination of various EBV strains will greatly contribute to the understanding of their global geographical distribution. This technology can also be used to clone disease-associated EBV strains and test the hypothesis that they have special features that distinguish them from strains that infect asymptomatically. View Full-Text
Keywords: Epstein-Barr virus (EBV); latent infection; CRISPR/Cas9; bacterial artificial chromosome (BAC); PacBio sequencing Epstein-Barr virus (EBV); latent infection; CRISPR/Cas9; bacterial artificial chromosome (BAC); PacBio sequencing
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).
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Yajima, M.; Ikuta, K.; Kanda, T. Rapid CRISPR/Cas9-Mediated Cloning of Full-Length Epstein-Barr Virus Genomes from Latently Infected Cells. Viruses 2018, 10, 171.

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