A Simple and Rapid Approach to Manipulate Pseudorabies Virus Genome by CRISPR/Cas9 System

Biotechnol Lett. 2015 Jun;37(6):1265-72. doi: 10.1007/s10529-015-1796-2. Epub 2015 Feb 28.

Abstract

Objectives: The broad host range of pseudorabies virus (PRV) and large capacity for foreign DNA make it a promising vector for the development of vaccines and agents of gene therapy.

Results: We show that up to 100 % viral gene disrupting efficiency was achieved by simple co-transfection of the purified PRV genomes with the clustered regularly-interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) into cells. Furthermore, CRISPR/Cas9-mediated knock-in of >4-kb-long DNA cassettes into the PRV genome at a positive rate of 50 % by a homology-independent DNA repair mechanism without constructing homology arms. This approach requires only a simple plasmid construction and is applicable to knock-in of other foreign genes.

Conclusion: Our studies offered simple and efficient methods to manipulate PRV.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • Drug Carriers
  • Gene Knock-In Techniques / methods*
  • Genetic Vectors
  • Genetics, Microbial / methods*
  • Genome, Viral*
  • Herpesvirus 1, Suid / genetics*
  • Plasmids
  • Recombination, Genetic
  • Time Factors

Substances

  • Drug Carriers