ArticleFrontiers in microbiology2016
Highly Efficient CRISPR/Cas9-Mediated Homologous Recombination Promotes the Rapid Generation of Bacterial Artificial Chromosomes of Pseudorabies Virus.
Article in Frontiers in microbiology, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed, 24 citations in OpenAlex.
- Development of viral infectious clones and their applications based on yeast and bacterial artificial chromosome platforms.Molecular biomedicine · 2025Review
- CRISPR-based functional genomics for schistosomes and related flatworms.Trends in parasitology · 2024Review
- Targeted insertion and reporter transgene activity at a gene safe harbor of the human blood fluke,Cell reports methods · 2023Article
- Recombinant Pseudorabies Virus Usage in Vaccine Development against Swine Infectious Disease.Viruses · 2023Review
- CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo.Veterinary research · 2021Article
- CRISPR/Cas9-Constructed Pseudorabies Virus Mutants Reveal the Importance of UL13 in Alphaherpesvirus Escape from Genome Silencing.Journal of virology · 2021Article
- Modeling pediatric AML FLT3 mutations using CRISPR/Cas12a- mediated gene editing.Leukemia & lymphoma · 2020Article
- Identification of ACTB Gene as a Potential Safe Harbor Locus in Pig Genome.Molecular biotechnology · 2020Article
- Glycoproteins C and D of PRV Strain HB1201 Contribute Individually to the Escape From Bartha-K61 Vaccine-Induced Immunity.Frontiers in microbiology · 2020Article
- Generation and characterization of UL41 null pseudorabies virus variant in vitro and in vivo.Virology journal · 2018Article
- Rapid CRISPR/Cas9-Mediated Cloning of Full-Length Epstein-Barr Virus Genomes from Latently Infected Cells.Viruses · 2018Review
- CRISPR-Cas9, a tool to efficiently increase the development of recombinant African swine fever viruses.Scientific reports · 2018Article
- Establishment of an Efficient and Flexible Genetic Manipulation Platform Based on a Fosmid Library for Rapid Generation of Recombinant Pseudorabies Virus.Frontiers in microbiology · 2018Article
- Comparison of Pathogenicity-Related Genes in the Current Pseudorabies Virus Outbreak in China.Scientific reports · 2017Article
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Authors and funding
11 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Bacterial artificial chromosomes (BACs) are powerful tools for the manipulation of the large genomes of DNA viruses, such as herpesviruses. However, the methods currently used to construct the recombinant viruses, an important intermediate link in the generation of BACs, involve the laborious process of multiple plaque purifications. Moreover, some fastidious viruses may be lost or damaged during these processes, making it impossible to generate BACs from these large-genome DNA viruses. Here, we introduce the CRISPR/Cas9 as a site-specific gene knock-in instrument that promotes the homologs recombination of a linearized transfer vector and the Pseudorabies virus genome through double incisions. The efficiency of recombination is as high as 86%. To our knowledge, this is the highest efficiency ever reported for Pseudorabies virus recombination. We also demonstrate that the positions and distances of the CRISPR/Cas9 single guide RNAs from the homology arms correlate with the efficiency of homologous recombination. Our work show a simple and fast cloning method of BACs with large genome inserted by greatly enhancing the HR efficiencies through CRISPR/Cas9-mediated homology-directed repair mechanism, and this method could be of helpful for manipulating large DNA viruses, and will provide a successful model for insertion of large DNA fragments into other viruses.
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