CRISPR/Cas9-mediated 2-sgRNA cleavage facilitates pseudorabies virus editing

FASEB J. 2018 Aug;32(8):4293-4301. doi: 10.1096/fj.201701129R. Epub 2018 Mar 6.

Abstract

Several groups have used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) for DNA virus editing. In most cases, one single-guide RNA (sgRNA) is used, which produces inconsistencies in gene editing. In this study, we used a swine herpesvirus, pseudorabies virus, as a model to systematically explore the application of CRISPR/Cas9 in DNA virus editing. In our current report, we demonstrated that cotransfection of 2 sgRNAs and a viral genome resulted in significantly better knockout efficiency than the transfection-infection-based approach. This method could result in 100% knockout of ≤3500 bp of viral nonessential large fragments. Furthermore, knockin efficiency was significantly improved by using 2 sgRNAs and was also correlated with the number of background viruses. We also demonstrated that the background viruses were all 2-sgRNA-mediated knockout mutants. Finally, this study demonstrated that the efficacy of gene knockin is determined by the replicative kinetics of background viruses. We propose that CRISPR/Cas9 coupled with 2 sgRNAs creates a powerful tool for DNA virus editing and offers great potential for future applications.-Tang, Y.-D., Guo, J.-C., Wang, T.-Y., Zhao, K., Liu, J.-T., Gao, J.-C., Tian, Z.-J., An, T.-Q., Cai, X.-H. CRISPR/Cas9-mediated 2-sgRNA cleavage facilitates pseudorabies virus editing.

Keywords: DNA virus; herpesvirus; knockin; knockout; recombination.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics*
  • Cell Line
  • Chlorocebus aethiops
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • DNA Viruses / genetics*
  • Gene Editing / methods
  • Gene Knockout Techniques / methods
  • Genome, Viral / genetics
  • Herpesvirus 1, Suid / genetics
  • RNA, Guide, CRISPR-Cas Systems / genetics*
  • Transfection / methods
  • Vero Cells

Substances

  • RNA, Guide, CRISPR-Cas Systems