scholarly journals Herpesvirus BACs: Past, Present, and Future

2011 ◽  
Vol 2011 ◽  
pp. 1-16 ◽  
Author(s):  
Charles Warden ◽  
Qiyi Tang ◽  
Hua Zhu
Keyword(s):  
Vaccine Development ◽  
Genetic Manipulation ◽  
Large Family ◽  
Site Directed Mutagenesis ◽  
Bacterial Artificial Chromosomes ◽  
Dna Viruses ◽  
Recombinant Viruses ◽  
Artificial Chromosomes ◽  
Viral Genes ◽  
Functional Analyses

The herpesviridae are a large family of DNA viruses with large and complicated genomes. Genetic manipulation and the generation of recombinant viruses have been extremely difficult. However, herpesvirus bacterial artificial chromosomes (BACs) that were developed approximately 10 years ago have become useful and powerful genetic tools for generating recombinant viruses to study the biology and pathogenesis of herpesviruses. For example, BAC-directed deletion mutants are commonly used to determine the function and essentiality of viral genes. In this paper, we discuss the creation of herpesvirus BACs, functional analyses of herpesvirus mutants, and future applications for studies of herpesviruses. We describe commonly used methods to create and mutate herpesvirus BACs (such as site-directed mutagenesis and transposon mutagenesis). We also evaluate the potential future uses of viral BACs, including vaccine development and gene therapy.

scholarly journals Viral Bacterial Artificial Chromosomes: Generation, Mutagenesis, and Removal of Mini-F Sequences

2012 ◽  
Vol 2012 ◽  
pp. 1-14 ◽  
Author(s):  
B. Karsten Tischer ◽  
Benedikt B. Kaufer
Keyword(s):  
Dna Sequences ◽  
Rna Virus ◽  
Virus Genome ◽  
Virus Species ◽  
Bacterial Artificial Chromosomes ◽  
Nucleotide Substitutions ◽  
Recombinant Viruses ◽  
Artificial Chromosomes ◽  
Dna And Rna ◽  
Virus Genomes

Maintenance and manipulation of large DNA and RNA virus genomes had presented an obstacle for virological research. BAC vectors provided a solution to both problems as they can harbor large DNA sequences and can efficiently be modified using well-established mutagenesis techniques inEscherichia coli. Numerous DNA virus genomes of herpesvirus and pox virus were cloned into mini-F vectors. In addition, several reverse genetic systems for RNA viruses such as members ofCoronaviridaeandFlaviviridaecould be established based on BAC constructs. Transfection into susceptible eukaryotic cells of virus DNA cloned as a BAC allows reconstitution of recombinant viruses. In this paper, we provide an overview on the strategies that can be used for the generation of virus BAC vectors and also on systems that are currently available for various virus species. Furthermore, we address common mutagenesis techniques that allow modification of BACs from single-nucleotide substitutions to deletion of viral genes or insertion of foreign sequences. Finally, we review the reconstitution of viruses from BAC vectors and the removal of the bacterial sequences from the virus genome during this process.

scholarly journals Targeted Mutagenesis of Guinea Pig Cytomegalovirus Using CRISPR/Cas9-Mediated Gene Editing

2016 ◽  
Vol 90 (15) ◽  
pp. 6989-6998 ◽  
Author(s):  
Craig J. Bierle ◽  
Kaitlyn M. Anderholm ◽  
Jian Ben Wang ◽  
Michael A. McVoy ◽  
Mark R. Schleiss
Keyword(s):  
Guinea Pig ◽  
Viral Genome ◽  
Genetic Manipulation ◽  
Spontaneous Mutation ◽  
Lytic Replication ◽  
Targeted Mutagenesis ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes ◽  
Guinea Pig Cytomegalovirus ◽  
Infected Cells

ABSTRACTThe cytomegaloviruses (CMVs) are among the most genetically complex mammalian viruses, with viral genomes that often exceed 230 kbp. Manipulation of cytomegalovirus genomes is largely performed using infectious bacterial artificial chromosomes (BACs), which necessitates the maintenance of the viral genome inEscherichia coliand successful reconstitution of virus from permissive cells after transfection of the BAC. Here we describe an alternative strategy for the mutagenesis of guinea pig cytomegalovirus that utilizes clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing to introduce targeted mutations to the viral genome. Transient transfection and drug selection were used to restrict lytic replication of guinea pig cytomegalovirus to cells that express Cas9 and virus-specific guide RNA. The result was highly efficient editing of the viral genome that introduced targeted insertion or deletion mutations to nonessential viral genes. Cotransfection of multiple virus-specific guide RNAs or a homology repair template was used for targeted, markerless deletions of viral sequence or to introduce exogenous sequence by homology-driven repair. As CRISPR/Cas9 mutagenesis occurs directly in infected cells, this methodology avoids selective pressures that may occur during propagation of the viral genome in bacteria and may facilitate genetic manipulation of low-passage or clinical CMV isolates.IMPORTANCEThe cytomegalovirus genome is complex, and viral adaptations to cell culture have complicated the study of infectionin vivo. Recombineering of viral bacterial artificial chromosomes enabled the study of recombinant cytomegaloviruses. Here we report the development of an alternative approach using CRISPR/Cas9-based mutagenesis in guinea pig cytomegalovirus, a small-animal model of congenital cytomegalovirus disease. CRISPR/Cas9 mutagenesis can introduce the same types of mutations to the viral genome as bacterial artificial chromosome recombineering but does so directly in virus-infected cells. CRISPR/Cas9 mutagenesis is not dependent on a bacterial intermediate, and defined viral mutants can be recovered after a limited number of viral genome replications, minimizing the risk of spontaneous mutation.

High-efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificial chromosomes

2011 ◽  
Vol 9 (1) ◽  
pp. 103-109 ◽  
Author(s):  
Alexander W Bird ◽  
Axel Erler ◽  
Jun Fu ◽  
Jean-Karim Hériché ◽  
Marcello Maresca ◽  
...  
Keyword(s):  
High Efficiency ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes

scholarly journals HHi-FiVe: A high-fidelity genetic engineering pipeline for construction of herpesvirus-based vaccines

2021 ◽  
Author(s):  
Michael Jarvis ◽  
Thekla Mauch ◽  
Eleonore Ostermann ◽  
Yvonne Wezel ◽  
Jenna Nichols ◽  
...  
Keyword(s):  
Ebola Virus ◽  
Genetic Manipulation ◽  
Rhesus Macaques ◽  
High Fidelity ◽  
Bacterial Artificial Chromosomes ◽  
Virus Challenge ◽  
Artificial Chromosomes ◽  
Vector Construction ◽  
Animal Populations ◽  
Cellular Tropism

Abstract Herpesvirus-based vectors are attractive for use both as conventional and as transmissible vaccines against emerging zoonoses in hard-to-reach animal populations. However, the threat of off-site mutations during genetic manipulation of vector genomes poses a significant challenge to vaccine construction. Herein, we present the HHi-FiVe (herpesvirus high-fidelity vector) construction pipeline for generating herpesvirus-based vectors by modifying bacterial artificial chromosomes (BACs) and monitoring integrity at each stage by complete genome sequencing. We used this pipeline to repair a highly mutated rhesus cytomegalovirus BAC containing an Ebola virus transgene. The vector derived from this BAC had been shown previously to protect rhesus macaques from lethal Ebola virus challenge by conventional vaccination. Repair of this BAC restored wild-type cellular tropism to the vector, which is essential for transmissible vaccination. Construction of this candidate transmissible vaccine against Ebola virus demonstrates the utility of the HHi-FiVe pipeline for creating precision-made herpesvirus-based vectors.

scholarly journals Latest Advances of Virology Research Using CRISPR/Cas9-Based Gene-Editing Technology and Its Application to Vaccine Development

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 779
Author(s):  
Man Teng ◽  
Yongxiu Yao ◽  
Venugopal Nair ◽  
Jun Luo
Keyword(s):  
Biological Sciences ◽  
Gene Therapy ◽  
Genetic Engineering ◽  
Gene Function ◽  
Viral Genome ◽  
Gene Editing ◽  
Vaccine Development ◽  
Future Prospects ◽  
Dna Viruses ◽  
Viral Genomes

In recent years, the CRISPR/Cas9-based gene-editing techniques have been well developed and applied widely in several aspects of research in the biological sciences, in many species, including humans, animals, plants, and even in viruses. Modification of the viral genome is crucial for revealing gene function, virus pathogenesis, gene therapy, genetic engineering, and vaccine development. Herein, we have provided a brief review of the different technologies for the modification of the viral genomes. Particularly, we have focused on the recently developed CRISPR/Cas9-based gene-editing system, detailing its origin, functional principles, and touching on its latest achievements in virology research and applications in vaccine development, especially in large DNA viruses of humans and animals. Future prospects of CRISPR/Cas9-based gene-editing technology in virology research, including the potential shortcomings, are also discussed.

scholarly journals Cross-Species RNAi Rescue Platform in Drosophila melanogaster

Genetics ◽  
2009 ◽  
Vol 183 (3) ◽  
pp. 1165-1173 ◽  
Author(s):  
Shu Kondo ◽  
Matthew Booker ◽  
Norbert Perrimon
Keyword(s):  
Cell Culture ◽  
Drosophila Melanogaster ◽  
Large Scale ◽  
Transgenic Animals ◽  
Selection Marker ◽  
Bacterial Artificial Chromosomes ◽  
Loss Of Function ◽  
Artificial Chromosomes ◽  
Target Effects

RNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.

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