70 ISSN 0026-8933, Molecular Biology, 2019, Vol. 53, No. 1, pp. 70–78. © Pleiades Publishing, Inc., 2019. Russian Text © D.S. Karpov, V.L. Karpov, R.R. Klimova, N.A. Demidova, A.A. Kushch, 2019, published in Molekulyarnaya Biologiya, 2019, Vol. 53, No. 1, pp. 80–89. A Plasmid-Expressed CRISPR/Cas9 System Suppresses Replication of HSV Type I in a Vero Cell Culture D. S. Karpov a, b *, V. L. Karpov a , R. R. Klimova c , N. A. Demidova c , and A. A. Kushch c a Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia b Orekhovich Institute of Biomedical Chemistry, Moscow, 119121 Russia c Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, Moscow, 123098 Russia *e-mail: aleom@yandex.ru Received August 28, 2018; revised August 29, 2018; accepted September 17, 2018 Abstract—Herpesviruses are widespread in the human population. Herpes simplex virus type 1 (HSV1) alone infects more than 3.7 billion people. In most of these, the virus establishes a latent form resistant to the action of all antiviral drugs. Moreover, completely drug-resistant strains of herpesviruses are known, which has prompted the search for alternative approaches to the treatment of herpesviruses, including genome editing with prokaryotic CRISPR/Cas. The CRISPR/Cas9 system of Streptococcus pyogenes effectively suppresses HSV1 infection when expressed from genome-integrated lentiviral vectors. However, there are concerns about the safety of this approach. Here we describe the system built upon the plasmid-encoded CRISPR/Cas9 targeted against UL52 and UL29 genes of the HSV1 primase–helicase complex. The con- struct was transfected into Vero cells with no significant cytotoxic effects detected. Complete suppression of HSV1 infection within two days was observed, raising the possibility that the proposed plasmid-expressed CRISPR/Cas9 system may be used for the screening of genes important for the HSV1 life cycle and for devel- opment of novel strategies for targeted therapy of herpesvirus infections. Keywords: CRISPR/Cas9, herpes simplex virus type 1, herpesvirus infection, Vero cells DOI: 10.1134/S0026893319010059 INTRODUCTION Viruses of the family Herpesviridae are among the most widespread pathogens in the human population. In particular, 80–90% of people have antibodies to herpes simplex virus type 1 and 2 (HSV1 and HSV2). According to the data from 2012, more than 3.7 billion people aged under 50 years, or 67% of the total popula- tion, were infected with HSV1 [1]. HSV infection is par- ticularly dangerous for individuals with compromised immunity: pregnant women, newborns, HIV-infected patients, and transplant recipients. In newborns, HSV infection may cause chorioretinitis, microcephaly, respiratory dysfunction, developmental retardation, disability, and even death [2]. Herpesvirus infections of the urogenital system are considered a possible cause of infertility [3]. Primary infection is usually rel- atively mild (labial herpes), but it may also take a severe clinical course (recurrent genital herpes, or encephalitis) with a possibility of death. In most cases, after a primary infection, inactive herpesvirus remains in host cells, establishing latent infection. Multiple factors of a different nature may cause HSV reactiva- tion, inducing virus replication and clinical manifesta- tion of the disease, frequently in the recurrent form. One of the principal problems of herpesvirus control is that most patent drugs used for herpesvirus manage- ment are designed to treat the acute, clinically mani- fest disease, but cannot eliminate the latent virus which is capable of lifelong presence in the organism. Another problem is the appearance of strains resistant to chemical agents used in antiviral therapy (e.g., acy- clovir) [4, 5]. The search for novel therapeutic approaches is therefore a topical issue in this field. One of the most recent advances is the genome editing technique using CRISPR/Cas9 systems [6–8]. A pio- neering study in cells infected with Epstein–Barr virus demonstrated that it was possible and promising to employ the CRISPR/Cas9 system against viruses with DNA genomes [9]. More recent works showed that the CRISPR/Cas9 system incorporated into a mamma- lian genome using lentivirus-based constructs effi- ciently suppressed HSV1 infection [10–13]. However, although the CRISPR/Cas9 system within genome- integrated constructs can efficiently suppress herpes- virus infection, this means of its delivery into human cells raises serious safety concerns. Among the possi- Abbreviations: HSV1, herpes simplex virus type 1; MOI, multi- plicity of infection; CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; Cas9, CRISPR-associated protein 9. MOLECULAR CELL BIOLOGY UDC 578.242.2,577.29