DEVELOPMENT OF A CRISPR-BASED ANTIVIRAL SYSTEM AGAINST BOHV-1

Abstract

<h3>Background</h3> Bovine Herpesvirus type 1 (BoHV-1) infection has no cure and infected animals become lifelong carriers, greatly impacting health and productivity of herds worldwide. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas (CRISPR associated) tool has been widely explored for the development of antiviral strategies due to its ability to precisely edit genetic sequences. In this manner, we have designed a CRISPR-based antiviral system against BoHV-1 through the tetracycline-inducible expression of the Cas9 nuclease and a guide RNA (gRNA) homologous to a viral sequence in genetically modified bovine cells. <h3>Methods</h3> The in silico design of the antiviral system was based on the literature. BoHV-1 and bovine (Bos taurus taurus) genome were obtained from GenBank and basic molecular vectors from Addgene. Sequence analysis of appropriate targets were performed with Snapgene and Serial Cloner. Primers for cloning were obtained using the NCBI tool Primer BLAST. The gRNA for bovine DNA cleavage was selected from literature considering cleavage efficiency and potential off-target effects. The gRNA for the viral genome cleavage was designed using the CRISPR gRNA design tool from ATUM and CHOPCHOP. <h3>Results</h3> The developed molecular vector allows the delivery of the necessary machinery to perform a cleavage in the chromosome 25 of the bovine DNA between the FSCN1 and the ACTB genes, where the sequence of the antiviral mechanism will be inserted by homologous recombination (HR). The cleavage site has already been reported as safe for the insertion of sequences into the bovine genome. The HR fragment has the Cas9 nuclease sequence and a tetracycline-inducible expression system capable of conditioning the expression of the nuclease gene to the presence of the antibiotic. In addition, the fragment has a sequence for the expression of a gRNA targeting an essential sequence for virus replication, the UL52 gene. The expressed Cas9 enzyme and gRNA form a ribonucleoprotein complex capable of cleaving only the virus genome leading to its inactivation and preventing its replication in the genetically modified cells stimulated by tetracycline. <h3>Conclusion</h3> The mechanism and elements developed explore new applications of the CRISPR-Cas tool to combat viral diseases that currently have no cure. The ease of altering the gRNA target makes the proposed system highly flexible and can possibly be applied to different diseases caused by viruses.