Research Article: CRISPR/Cas9-Mediated Genome Editing of Herpesviruses Limits Productive and Latent Infections

Date Published: June 30, 2016

Publisher: Public Library of Science

Author(s): Ferdy R. van Diemen, Elisabeth M. Kruse, Marjolein J. G. Hooykaas, Carlijn E. Bruggeling, Anita C. Schürch, Petra M. van Ham, Saskia M. Imhof, Monique Nijhuis, Emmanuel J. H. J. Wiertz, Robert Jan Lebbink, Jay A. Nelson.


Herpesviruses infect the majority of the human population and can cause significant morbidity and mortality. Herpes simplex virus (HSV) type 1 causes cold sores and herpes simplex keratitis, whereas HSV-2 is responsible for genital herpes. Human cytomegalovirus (HCMV) is the most common viral cause of congenital defects and is responsible for serious disease in immuno-compromised individuals. Epstein-Barr virus (EBV) is associated with infectious mononucleosis and a broad range of malignancies, including Burkitt’s lymphoma, nasopharyngeal carcinoma, Hodgkin’s disease, and post-transplant lymphomas. Herpesviruses persist in their host for life by establishing a latent infection that is interrupted by periodic reactivation events during which replication occurs. Current antiviral drug treatments target the clinical manifestations of this productive stage, but they are ineffective at eliminating these viruses from the infected host. Here, we set out to combat both productive and latent herpesvirus infections by exploiting the CRISPR/Cas9 system to target viral genetic elements important for virus fitness. We show effective abrogation of HCMV and HSV-1 replication by targeting gRNAs to essential viral genes. Simultaneous targeting of HSV-1 with multiple gRNAs completely abolished the production of infectious particles from human cells. Using the same approach, EBV can be almost completely cleared from latently infected EBV-transformed human tumor cells. Our studies indicate that the CRISPR/Cas9 system can be effectively targeted to herpesvirus genomes as a potent prophylactic and therapeutic anti-viral strategy that may be used to impair viral replication and clear latent virus infection.

Partial Text

Herpesviruses are large DNA viruses that cause widespread, lifelong infections; most adults carry multiple herpesviruses [1]. The herpesvirus family is divided into three subfamilies, the Alpha-, Beta- and Gammaherpesvirinae. The subfamily of Alphaherpesvirinae includes the herpes simplex virus type 1 and type 2 (HSV-1 and 2) and varicella zoster virus (VZV). HSV-1 causes cold sores and herpes simplex keratitis, a common cause of corneal blindness [2, 3]. HSV-2 is responsible for genital herpes. Primary infection with VZV results in chickenpox; reactivation may lead to herpes zoster or shingles [4]. The subfamily of Betaherpesvirinae includes the human cytomegalovirus (HCMV), which gives rise to serious complications in immuno-compromised individuals [5, 6]. Additionally, HCMV is the most common viral cause of congenital defects. The Gammaherpesvirinae include Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV). EBV induces infectious mononucleosis and is strongly associated with multiple malignancies, including nasopharyngeal carcinoma, Burkitt’s lymphoma, Hodgkin’s lymphoma, gastric carcinoma, and post-transplant lymphoproliferative disorders (PTLD) [7]. KSHV is a human tumor virus that is associated with Kaposi’s sarcoma and two lymphoproliferative disorders occurring in AIDS patients: primary effusion lymphoma and multicentric Castleman disease [8].

Herpesviruses such as HSV-1, HSV-2, HCMV and EBV are a frequent cause of disease and treating these herpesvirus infections remains a major challenge. Current therapies are mainly geared towards limiting productive infections by targeting virus replication via antiviral drugs such as the guanosine analogues acyclovir and ganciclovir. Although these compounds effectively inhibit the viral DNA polymerase during replication, they have limited impact on the latent stage of herpesvirus infections that relies on host polymerases for viral genome maintenance in dividing cells [7]. Strategies to impact the latent stage of infection often depend on reactivation of a latent infection by using chemotherapeutic agents and subsequent targeting of these cells by e.g. ganciclovir [49]. Such strategies are not always effective and may result in loss of the target cell. Although this approach may be beneficial to clear EBV-driven cancer cells, other instances ask for sparing of the infected cell, for example in HSV-1, HSV-2 and VZV-infected neurons. Hence, there is a need for a direct and potent strategy to limit herpesvirus infections by specifically targeting the viral genome or its maintenance. Preferably, such approaches should impact both lytic and latent herpesvirus infections. Indeed, recently, the HSV-1 genome has been targeted by using meganucleases [50, 51]. Here, we performed proof-of-concept studies to show that the CRISPR/Cas9 genome engineering system can be efficiently used to limit or eradicate three of the most prevalent herpesviruses from human cells: HSV-1, HCMV and EBV.




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