Inhibition Of Rabies Virus Replication Using CRISPR-Cas9 Technology

Rabies caused by rabies virus(RABV)infection with a mortality rate of 100%.According to the World Health Organization,about 59,000 people worldwide die from rabies every year,most of them from developing countries in Asia and Africa.In Asia,the rabies is major happened in India and China.Although rabies vaccine can prevent the spread of rabies virus,the treatment of rabies still faces many problems.Currently,various antiviral therapies commonly used have failed to therapy rabies.The recently introduced CRISPR(Clustered regular interspaced short palindromic repeats)-Cas9 system can edited genetic point on the genome of any species,which is simpler,faster and more efficient than previous gene editing techniques.And it is provided a treatment of diseases such as rabies and chromosomal defects.G protein is the only membraneprotein of RABV which induce viral entry into cells and play a key role in virulence.Here we use CRISPR-Cas9 technology to slice the G protein of rabies virus to inhibit viral replication.In the present study,we designed four pair of gRNA target G.We constructed CRISPR-Cas9 vector for rabies virus glycoprotein and screened high-efficiency gRNA by flow cytometry.Then,the selected gRNA was used for rabies virus of different strains such as SAD strain,DRV strain,CVS strain,and B2 cstrain.Furthermore,the site where the mutated targetsequence is detected by sequencing.Finally,in order to simulate the environment in which the drug can be administered multiple times,we continuously applied the CRISPR-Cas9 system to the rabies virus twice to determine the change of virus titer.Thus,these results showed that the PX459-gRNA plasmid was successfully constructed,and PX459-G223 was the highest slicing gRNA target RABVs genome.The effectiveness of the CRISPR-Cas9 system on imbibing rabies virus replication was verified,and multiple treatment of CRISPR-Cas9 could achieve better results.The results suggest that CRISPR-Cas9 can disrupt the rabies G protein genome during the RABV infection and inhibit rabies virus replication at the cellular level,which suggests that CRISPR-Cas9-mediated G protein gene editing offers the possibility of rabies gene therapy.