CRISPR/Cas9 assisted gene targeting efficiently inhibits bovine herpesvirus-1 replication

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein (cas) are now being accepted as a highly specific method of gene editing. Among many other applications, CRISPR/cas has an immense potential to be used as antivirals. In this study, we successfully demonstrated CRISPR/Cas9 mediated inhibition of Bovine Herpes virus -1 (BHV-1) replication. BHV-1 causes economically important diseases in bovines with establishment of latency. Six essential genes and one non-essential gene of BHV-1 were targeted to assess the impact on virus replication. Inhibition of UL52, circ, and UL27 genes showed promising results, whereas the other three genes US6, UL18, and UL34 resulted in lower level of inhibition. Non-specific gene editing in host and virus was in-silico evaluated and was demonstrated by inhibition of virus induced apoptosis. Successful editing of one viral non-essential gene without any alterations in virus replication demonstrated the potential of CRISPR/Cas9 in replicating viral genome. Complete abrogation of virus replication was observed transiently (~24 hours post-transfection/hpt) when transfected with short lived in-vitro transcribed sgRNAs. Whereas, under constant expression of sgRNAs through plasmid, complete inhibition of virus replication was observed till ~72 hours post-infection. Complete inhibition of replication was also observed with in-vitro transcribed sgRNA when booster dose of sgRNA was trasnfected at 24hpt. It has been speculated that constant expression with plasmid based delivery may result in off-target activity which can be ruled out with short lived in-vitro transcribed sgRNA.

Modulation of Equid Herpesvirus-1 Replication Dynamics In Vitro Using CRISPR/Cas9-Assisted Genome Editing

“…Targeting ORF74 in the present study did not affect, or had a low effect on, viral replication dynamics, although the cleavage of the viral genome was confirmed by the cleavage detection kit. This result is in accordance with a recent study conducted on BHV-1, where the gE gene was used as a control to evaluate the non-specific effects of CRISPR components on virus replication [83].…”