Division of Microbiology, Faculty of Medicine, Tohoku Medical and Pharmaceutical University, 1-15-1 Fukumuro, Miyagino-ku, Sendai 983-8536, Japan.
Viruses. 2018 Apr 3;10(4):171. doi: 10.3390/v10040171.
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.
疱疹病毒具有相对较大的 DNA 基因组，超过 150kb，难以克隆和测序。细菌人工染色体 (BAC) 克隆疱疹病毒基因组是一种强大的技术，极大地促进了整个病毒基因组测序以及重建病毒的功能表征。我们描述了最近发明的使用 CRISPR/Cas9 介导的同源定向修复快速 BAC 克隆疱疹病毒基因组的技术。我们专注于最近的 EBV 基因组 BAC 克隆技术，并讨论了与先前 EBV-BAC 克隆策略相比，CRISPR/Cas9 介导策略的可能优势。我们还描述了该技术的设计决策以及未来可能存在的缺陷和需要改进的地方。获得的 EBV-BAC 克隆进行长读测序分析，以确定包括重复区域在内的完整 EBV 基因组序列。快速克隆和确定各种 EBV 株将极大地促进对其全球地理分布的理解。该技术还可用于克隆与疾病相关的 EBV 株，并检验其具有特殊特征从而将其与无症状感染的株区分开来的假设。
