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

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, constant expression with plasmid based delivery may result in off-target activity which can be ruled out with short lived in-vitro transcribed sgRNA.

Lately, CRISPR/Cas9 gene-editing technology has emerged as a powerful tool for RNAguided specific genome modifications, which has the potential to develop new generation therapeutics. CRISPR/Cas9 system has been tested for its therapeutic potential (invitro and in-vivo) for various viruses (reviewed in [8]), either through direct disruption of the viral genome or by hampering the host factors helping in the virus replication [8]. To explore a similar strategy for BHV-1, the present study was designed to evaluate the efficiency of the CRISPR/Cas9 system to limit BHV-1 replication.
The essential and non-essential genes required for in-vitro replication of BHV-1 were selected as described by Robinson and co-workers [9]. Six essential genes spread across the three temporal expression zones viz. immediate-early (IE), early (E), and late (L) genes were targeted to achieve maximum inhibition of replication. Glycoprotein E (gE) gene of BHV-1 which is not essential for in-vitro replication was also targeted with CRISPR/Cas9 to evaluate specific genome editing and effects on virus replication. Along with RNP-based expression, the sgRNA target sequences were also expressed via U6 promoter-driven PX459 vector, following the protocol described by Ran and co-workers [11].
Briefly, sgRNA target sequences were cloned in PX459 vector, transformed in the DH5α strain of E. coli. and the plasmids were extracted using an endotoxin-free plasmid extraction kit (Qiagen plasmid midiprep kit). Similar to RNP based expression method, the MDBK cells were seeded and transfected with plasmids (500ng/well in 24 well plate) cloned with each sgRNA target using p3000 reagent (Thermo, USA) as per manufacturer's instructions. For plasmid-based transfection, BHV-1 infection (1MOI) was given 12-14 hours posttransfection (hpt).
BHV-1 infected and mock-infected cells were harvested in triplicates at 06, 12, 24 and 48 hpt for RNP transfected cells and at 72 hpi for plasmid transfected cells. Virus quantification was performed by endpoint dilution method of titration [11]. Plaque-assay was performed for virus quantification and comparison of plaque morphologies of wild type (WT) and CRISPR targeted BHV-1, as described by Baer and Hall [12]. Viral DNA load was quantified by realtime PCR assay using SYBR chemistry. Specific forward primer (TGTGGACCTAAACCT-CACGGT) and reverse primer (GTAGTCGAGCAGACCCGTGTC) were synthesized for quantification using plasmid DNA standards as described elsewhere [13]. In order to ascertain the inhibition of virus replication, virus induced apoptosis [15] was estimated using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay (Promega, USA) as per the manufacturer's instructions.
T7 endonuclease assay was performed to evaluate gene editing in BHV-1 that was targeted for a non-essential gene (gE), using commercially available kit (GeneArt Genome Cleavage detection kit, Thermo, USA) as per the recommendations. Forward primer (FP: CGTGTGTCTTGGTTTCTGCG) and reverse primer (RP: GAAGACCGTGTCGACCGAAG) were designed to amplify CRISPR targeted region to confirm the insertion/deletion. Bhattacharya and Meir, [14] demonstrated that CRISPR/Cas9 induced insertions/deletions can also be detected by high density agarose gel electrophoresis.
Therefore, the amplified (used FP and RP) target gene was also resolved on high density (5%) agarose gel.
Characteristic CPE for BHV-1 appeared after 12hpi and completed by 48hpi ( Fig. 1a- On the other hand, statistically insignificant (p>0.05) inhibition was observed in the BHV-1 treated with sgRNA targeting a non-essential (gE) gene (Fig. 2h), which indicates no harmful effects of the components of CRISPR/cas9 system. Viral gene editing was successfully demonstrated by T7 endonuclease assay which resulted in three amplicons (one; parental and two; cleaved products) in the virus population confirming the presence of mutant as well as WT BHV-1 (Fig. 3c). Similar findings were obtained when PCR amplicons were resolved in high concentration (>5%) agarose gel electrophoresis that showed multiple PCR amplicons in CRISPR targeted gE amplified products (mutants/MT) which were absent in the untreated WT BHV-1 (Fig. 3b).
Inhibition of virus replication was also demonstrated by TUNEL assay where virus induced apoptosis (Fig. 5b) was reduced in the essential gene targeted BHV-1 (Fig. 5c).On the other hand, negligible changes were observed for a non-essential gene targeted BHV-1 (Fig. 5d).
Over the past few years, specific gene editing by CRISPR/Cas9 has exemplified the possibility for the development of new generation antivirals.  [16][17][18][19][20]. In this study, we demonstrated the antiviral potential of CRISPR-Cas9 against BHV-1. It was observed that BHV-1 replication is inhibited by a minimum of 60% on targeting any one of the six essential genes evaluated in this study. BHV-1 genes namely UL52, circ, and UL27 were shown as potential targets to impair BHV-1 replication (Fig. 2a-c). Among all the targeted essential genes, UL27 (encodes for glycoprotein B/ gB) could be the most potential single target because it starts one of the earliest contacts with the host cell, since it acts as a ligand for attachment receptors and also has a role in the fusion of virus envelope with the host cell membrane [4]. We also observed that the RNP complex resulted in significant inhibition only up to 24hpt, and the virus replication again peeked after 24hpt (Fig 2a-g). The plausible reason behind incomplete abrogation could be the limited half-life of the Cas9 enzyme which is approximately 20hrs [21]. Complete abrogation of virus replication has been shown for other viruses using vector-based sgRNA/Cas9 delivery system [16][17][18][19].
Complete abrogation of BHV-1 using a vector-based delivery system against all the six genes. Although plasmid-based delivery showed promising results in the inhibition of virus replication, but it is speculated that with plasmid system, there are chances of integration in host genome and persistent activity of Cas9 enzyme might lead to non-specific gene editing in virus or host genome [22]. Therefore, RNP platform with a short half-life should be preferred for the development of antivirals. Consequently, we attempted the delivery of the second dose of sgRNA/Cas9 after 24hpt and complete viral replication abrogation was observed, that was similar to the vector-based delivery system. Non-specific effects of sgRNA/Cas9 based gene editing on virus replication were evaluated by keeping one control (gE gene) which is not essential for virus replication in the experiment. No significant drop in virus titer was observed after targeting gE (Fig 2h). Successful gE gene editing was achieved by CRISPR-Cas9 (Fig 3b). The mutation(s) was confirmed by T7 endonucleaseassay indicating the specific nature of genome editing by CRISPR/Cas9 (Fig 3c). Off-target activities of the designed sgRNAs were evaluated in-silico and by sequencing the amplified top five off-targets in the host genome wherein we did not find any changes within the amplified regions. Although CRISPR/Cas9 mediated gene editing is highly specific in nature, confirming minimum/no non-specificity is of utmost importance for the development of antivirals, to ensure safety [8].
It is concluded that BHV-1 replication can be inhibited using CRISPR/Cas9 based gene editing. Although constant expression of sgRNA through vector-based system resulted in complete abrogation of replication over RNP-based platform for a longer duration, but the short half-life of the RNP complex has the advantage of eliminating the chances of off-target activity, which is the most crucial concern for developing new generation antivirals. Hence, multiple dosing of short-lived sgRNAs/Cas9 complex may be attempted for complete inhibition of virus with minimum or no off-target activity. However, further studies including host genome-wide screening to detect non-specificity would be required to confirm the specific antiviral potential of CRISPR/cas9 before it is evaluated in the laboratory animals. CRISPR/Cas9 targeting of essential genes inhibits BHV-1 replication (a-g). BHV-1 infected MDBK cells were transfected with sgRNAs against the indicated genes (a-h). To assess the percentage of BHV-1 replication inhibition (relative to virus control), viruses were collected at 6, 12, 24, and 48 hours posttransfection (hpt) and serially diluted 10-fold (10-1 to . Each dilution was replicated six times and the virus titer (TCID50/ml) was calculated using Reed and Muench method and (h) shows the BHV-1 replication inhibition for a non-essential gene Reduction in BHV-1 plaques with inhibition of essential genes by CRISPRcas9 at 24hpi. (a) Non-infected cell control (b) un-treated BHV-1 (WT) in 10-2 dilution (c) BHV-1 treated with pool of sgRNAs against UL52, Circ, UL27, US6, UL18 and UL34 in 10-2 dilution (d) BHV-1 treated with sgRNA against non-essential gene (gE) in 10-2 dilution