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.
Highlights
Epstein-Barr virus (EBV) is a human lymphocryptovirus that establishes a life-long latent infection in the majority of healthy adults
Another way to extract EBV DNA from latently infected cells is to clone the EBV genome in a bacterial artificial chromosome (BAC) vector, a cloning vector that can accommodate more than 200 kb of DNA
We focus on our Bacterial artificial chromosome (BAC) cloning strategy of EBV genomes, whereby Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 plasmid and circular donor plasmids are simultaneously transfected
Summary
Epstein-Barr virus (EBV) is a human lymphocryptovirus that establishes a life-long latent infection in the majority of healthy adults. Since some of the repetitive regions affect EBV viral functions, an alternative experimental strategy to determine the complete sequences of various EBV strains is highly desirable Another way to extract EBV DNA from latently infected cells is to clone the EBV genome in a bacterial artificial chromosome (BAC) vector, a cloning vector that can accommodate more than 200 kb of DNA. We focus on our BAC cloning strategy of EBV genomes, whereby CRISPR/Cas plasmid and circular donor plasmids are simultaneously transfected This experimental strategy has greatly facilitated EBV genome cloning and whole viral genome sequencing including the sequencing of repetitive regions. A CRISPR/Cas plasmid and a donor plasmid, are transfected into EBV latently infected cells, and a BAC vector sequence and marker genes are inserted into the EBV genome via homology-directed repair. EBV-BAC clones can readily be obtained by transforming DH10B E. coli with episomal DNA
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