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.
Highlights
Pseudorabies virus (PRV), the causative agent of Aujeszky’s disease, is a member of the family Herpesviridae, the subfamily Alphaherpesvirinae, and the genus Varicellovirus (Matthews, 1982)
Vero cells were cotransfected with pBAC-GFP62 and the extracted PRV HLJ genome DNA (Szpara et al, 2011), and the homologous recombination (HR) efficiency of the group adding additional 2 μg pCas9 vector was regarded as the control
PCas9Us6 was applied, but showed a lower HR efficiency even than the control group, since it incised PRV HLJ genome, and incised the transfer vector pBAC-GFP62, we strongly suggest the target sites should be avoided that both incise the plasmid and genome for higher efficiency
Summary
Pseudorabies virus (PRV), the causative agent of Aujeszky’s disease, is a member of the family Herpesviridae, the subfamily Alphaherpesvirinae, and the genus Varicellovirus (Matthews, 1982). PRV can infect most mammals, except humans and other higher primates, but pigs are the only known natural reservoir (Klupp et al, 1995). Because it is a neurotropic pathogen, effectively invading the peripheral nervous system and establishing a lifelong latent infection in the neurons resident in the peripheral ganglia (Smith, 2012), it is an important model to analyze the virus transportation mechanism in the nerve system. For some fastidious viruses that fail to produce plaques in cells, such as Kaposi’s sarcoma-associated herpesvirus, it can be impossible to isolate the purified recombinant virus (Zhou et al, 2002)
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