Highly Efficient CRISPR/Cas9-Mediated Homologous Recombination Promotes the Rapid Generation of Bacterial Artificial Chromosomes of Pseudorabies Virus

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.

CRISPR/Cas9‐mediated 2‐sgRNA cleavage facilitates Pseudorabies virus editing

“…Second, the linear or circular donor plasmids might also influence HR efficiency. In our study, we found that a linear donor plasmid is more effective than a circular donor plasmid (17). In another study, however, BAC clones of Epstein-Barr virus utilizing a circular donor plasmid were generated to enabled a higher efficiency (21).…”

“…Target sequences were synthesized and cloned into the PX330 vector (4), as previously reported (14). The sequences and incision efficiencies of pCas9-Us6, pCas9-Us8, pCas9-Us7, pCas9-Us9, and pCas9-Us2 have been previously described (17). The pCas9unique long (UL) region 24 (UL24) targeting site was 59-GCGGCACCGGCAAGAGCACCA-39, and the pCas9-UL24 targeting site was 59-GTGCGCCTTCACGTCGGAGAT-39.…”

“…The cotransfection procedure for the PRV HLJ genome and pBAC-GFP62 with different pCas9s has been previously reported (17). Briefly, 3 mg of the PRV HLJ genome and 3 mg of linearized pBAC-GFP62 were added to each individual well.…”

“…The use of CRISPR/Cas9 has been reported in studies around manipulating large-genome DNA viruses, including adenovirus (5), herpes simplex virus 1 (2, 5-7), hepatitis B virus (8)(9)(10)(11)(12)(13), pseudorabies virus (PRV) (14)(15)(16)(17)(18)(19), and Epstein-Barr virus (20)(21)(22). However, the effects of virus editing by CRISPR/Cas9 have always been inconsistent, and the rate of success in gene editing, such as knocking in foreign genes, has been consistently low.…”

Glycoproteins C and D of PRV Strain HB1201 Contribute Individually to the Escape From Bartha-K61 Vaccine-Induced Immunity

“…So far, it has been widely used in genome editing of many viruses, such as herpes simplex virus (Lin et al, 2016;Roehm et al, 2016), adenovirus (Bi et al, 2014), hepatitis B virus (Liu et al, 2018), African swine fever virus (Borca et al, 2018;Hubner et al, 2018), and so on. However, most of manipulations were focuses on viral non-essential genes and on insertion of a foreign gene (Xu et al, 2015;Guo et al, 2016;Liang et al, 2016;Tang et al, 2016Tang et al, , 2017Tang et al, , 2018. There is no good approach to replace essential genes among the different PRV strains.…”