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Screening Method for CRISPR/Cas9 Inhibition of a Human DNA Virus: Herpes Simplex Virus.

ABSTRACT: The efficiency of cleavage of individual CRISPR/Cas9-sgRNAs remains difficult to predict based on the CRISPR target sequence alone. Different intracellular environments (dependent on cell type or cell cycle state for example) may affect sgRNA efficiency by altering accessibility of genomic DNA through DNA modifications such as epigenetic marks and DNA-binding proteins (e.g., histones) as well as alteration of the chromatin state of genomic DNA within the nucleus. We recently reported a multi-step screening method for the identification of efficient sgRNAs targeting the Herpes simplex virus (HSV-1) genome and reported a differential mechanism for viral inhibition by CRISPR-Cas9 in the latent versus lytic phase. The screening platform detailed in this protocol allows step-by-step testing of the efficiency of cleavage in a cell-free system and in the context of viral target cells such as human foreskin fibroblasts followed by functional testing of the effects of CRISPR/sgRNA on viral protein expression, replication, and reactivation. This strategy could be readily applied to other target cells such as pluripotent stem cell-derived human sensory neurons or other human DNA viruses.

SUBMITTER: Neuhausser WM

PROVIDER: S-EPMC7842287 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Complete and Prolonged Inhibition of Herpes Simplex Virus Type 1 Infection In Vitro by CRISPR/Cas9 and CRISPR/CasX Systems.

Project description:Almost all people become infected with herpes viruses, including herpes simplex virus type 1 (HSV-1), during their lifetime. Typically, these viruses persist in a latent form that is resistant to all available antiviral medications. Under certain conditions, such as immunosuppression, the latent forms reactivate and cause disease. Moreover, strains of herpesviruses that are drug-resistant have rapidly emerged. Therefore, it is important to develop alternative methods capable of eradicating herpesvirus infections. One promising direction is the development of CRISPR/Cas systems for the therapy of herpesvirus infections. We aimed to design a CRISPR/Cas system for relatively effective long-term and safe control of HSV-1 infection. Here, we show that plasmids encoding the CRISPR/Cas9 system from Streptococcus pyogenes with a single sgRNA targeting the UL30 gene can completely suppress HSV-1 infection of the Vero cell line within 6 days and provide substantial protection within 9 days. For the first time, we show that CRISPR/CasX from Deltaproteobacteria with a single guide RNA against UL30 almost completely suppresses HSV-1 infection of the Vero cell line for 3 days and provides substantial protection for 6 days. We also found that the Cas9 protein without sgRNAs attenuates HSV-1 infection. Our results show that the developed CRISPR/Cas systems are promising therapeutic approaches to control HSV-1 infections.

| S-EPMC9738314 | biostudies-literature

Targeting herpes simplex virus with CRISPR-Cas9 cures herpetic stromal keratitis in mice.

Project description:Herpes simplex virus type 1 (HSV-1) is a leading cause of infectious blindness. Current treatments for HSV-1 do not eliminate the virus from the site of infection or latent reservoirs in the trigeminal ganglia. Here, we target HSV-1 genomes directly using mRNA-carrying lentiviral particles that simultaneously deliver SpCas9 mRNA and viral-gene-targeting guide RNAs (designated HSV-1-erasing lentiviral particles, termed HELP). We show that HELP efficiently blocks HSV-1 replication and the occurrence of herpetic stromal keratitis (HSK) in three different infection models. HELP was capable of eliminating the viral reservoir via retrograde transport from corneas to trigeminal ganglia. Additionally, HELP inhibited viral replication in human-derived corneas without causing off-target effects, as determined by whole-genome sequencing. These results support the potential clinical utility of HELP for treating refractory HSK.

| S-EPMC7611178 | biostudies-literature

Developing oncolytic <i>Herpes simplex virus type 1</i> through UL39 knockout by CRISPR-Cas9.

Project description:ObjectivesOncolytic Herpes simplex virus type 1 (HSV-1) has emerged as a promising strategy for cancer therapy. However, development of novel oncolytic mutants has remained a major challenge owing to low efficiency of conventional genome editing methods. Recently, CRISPR-Cas9 has revolutionized genome editing.Materials and methodsIn this study, we aimed to evaluate the capability of CRISPR-Cas9 to manipulate the UL39 gene to create oncolytic HSV-1. Herein, three sgRNAs were designed against the UL39 gene and transfected into HEK-293 cell line followed by infection with HSV-1 KOS.ResultsAfter three rounds of plaque purification, several HSV-1 mutants were identified by PCR analysis and sequencing. One of these mutations in which 55 nucleotides were deleted resulted in a frameshift mutation that in turn produced a truncated protein with only 167 amino acids from 1137 amino acids. Functional analysis in Vero and primary fibroblast cells revealed that viral replication was significantly lower and plaque size was smaller in the HSV-1 mutant compared with HSV-1 KOS. Moreover, the relative amount of viral genome present in the supernatants of infected cells (Vero and primary fibroblast cells) with HSV-1 mutant was significantly decreased compared with those of HSV-1 KOS.ConclusionOur data revealed that targeting UL39 with CRISPR-Cas9 could develop oncolytic HSV-1.

| S-EPMC7395182 | biostudies-literature

Herpes simplex virus eliminates host mitochondrial DNA.

Project description:Mitochondria have crucial roles in the life and death of mammalian cells, and help to orchestrate host antiviral defences. Here, we show that the ubiquitous human pathogen herpes simplex virus (HSV) induces rapid and complete degradation of host mitochondrial DNA during productive infection of cultured mammalian cells. The depletion of mitochondrial DNA requires the viral UL12 gene, which encodes a conserved nuclease with orthologues in all herpesviruses. We show that an amino-terminally truncated UL12 isoform-UL12.5-localizes to mitochondria and triggers mitochondrial DNA depletion in the absence of other HSV gene products. By contrast, full-length UL12, a nuclear protein, has little or no effect on mitochondrial DNA levels. Our data document that HSV inflicts massive genetic damage to a crucial host organelle and show a novel mechanism of virus-induced shutoff of host functions, which is likely to contribute to the cell death and tissue damage caused by this widespread human pathogen.

| S-EPMC1796774 | biostudies-literature

Inhibition of Herpes Simplex Virus-1 Entry into Human Cells by Nonsaccharide Glycosaminoglycan Mimetics.

Project description:Although heparan sulfate (HS) has been implicated in facilitating entry of enveloped viruses including herpes simplex virus (HSV), small molecules that effectively compete with this abundant, cell surface macromolecule remain unknown. We reasoned that entry of HSV-1 involving its glycoprotein D (gD) binding to HS could be competitively targeted through small, synthetic, nonsaccharide glycosaminoglycan mimetics (NSGMs). Screening a library of NSGMs identified a small, distinct group that bound gD with affinities of 8-120 nM. Studies on HSV-1 entry into HeLa, HFF-1, and VK2/E6E7 cells identified inhibitors with potencies in the range of 0.4-1.0 ?M. These synthetic NSGMs are likely to offer promising chemical biology probes and/or antiviral drug discovery opportunities.

| S-EPMC6088358 | biostudies-literature

Herpes simplex virus suppresses necroptosis in human cells.

Project description:Herpes simplex virus (HSV)-1 and HSV-2 are significant human pathogens causing recurrent disease. During infection, HSV modulates cell death pathways using the large subunit (R1) of ribonucleotide reductase (RR) to suppress apoptosis by binding to and blocking caspase-8. Here, we demonstrate that HSV-1 and HSV-2 R1 proteins (ICP6 and ICP10, respectively) also prevent necroptosis in human cells by inhibiting the interaction between receptor-interacting protein kinase 1 (RIP1) and RIP3, a key step in tumor necrosis factor (TNF)-induced necroptosis. We show that suppression of this cell death pathway requires an N-terminal RIP homotypic interaction motif (RHIM) within R1, acting in concert with the caspase-8-binding domain, which unleashes necroptosis independent of RHIM function. Thus, necroptosis is a human host defense pathway against two important viral pathogens that naturally subvert multiple death pathways via a single evolutionarily conserved gene product.

| S-EPMC4382104 | biostudies-literature

Herpes Simplex Virus 1

Project description:The Latency-Associated Transcript Locus of Herpes Simplex Virus 1 is a Virulence Determinant in Human Skin

| PRJNA338711 | ENA

Efficient inhibition of duck hepatitis B virus DNA by the CRISPR/Cas9 system.

Project description:Current therapeutic strategies cannot eradicate hepatitis B virus covalently closed circular DNA (HBV cccDNA), which accounts for the persistence of HBV infection. Very recently, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‑associated protein 9 (Cas9) system has been used as an efficient and powerful tool for viral genome editing. Given that the primary duck hepatocyte (PDH) infected with duck hepatitis B virus (DHBV) has been widely used to study human HBV infection in vitro, the present study aimed to demonstrate the targeted inhibition of DHBV DNA, especially cccDNA, by the CRISPR/Cas9 system using this model. We designed six single‑guide RNAs (sgRNA1‑6) targeting the DHBV genome. The sgRNA/Cas9 plasmid was transfected into DHBV‑infected PDHs, and then DHBV total DNA (in culture medium and PDHs) and cccDNA were quantified by reverse transcription‑quantitative polymerase chain reaction. The combined inhibition of CRISPR/Cas9 system and entecavir (ETV) was also assessed. Two sgRNAs, sgRNA4 and sgRNA6, exhibited efficient inhibition on DHBV total DNA (77.23 and 86.51%, respectively), cccDNA (75.67 and 85.34%, respectively) in PDHs, as well as DHBV total DNA in the culture medium (62.17 and 59.52%, respectively). The inhibition remained or enhanced from day 5 to day 9 following transfection. The combination of the CRISPR/Cas9 system and ETV further increased the inhibitory effect on DHBV total DNA in PDHs and culture medium, but not cccDNA. The CRISPR/Cas9 system has the potential to be a useful tool for the suppression of DHBV DNA.

| S-EPMC5865846 | biostudies-literature

Detection of Herpes simplex virus DNA by real-time PCR.

Project description:Molecular detection of herpes simplex virus (HSV) DNA is recognized as the reference standard assay method for the sensitive and specific diagnosis of central nervous system infections caused by HSV. In this study, a molecular assay based on real-time PCR on the LightCycler (LC) instrument was evaluated and compared with a home-brew molecular assay. The detection limit of the LC assay was determined with 10-fold dilutions of plasmid pS4 with the SalI restriction fragment of the DNA polymerase gene and with the First European Union Concerted Action HSV Proficiency Panel. A total of 59 cerebrospinal fluid (CSF) specimens were investigated for the comparative study. With plasmid pS4, the detection limit of the LC assay was found to be 10(4) copies per ml, i.e., 12.5 copies per run. When samples of the First European Union Concerted Action HSV Proficiency Panel were tested, 2x10(3) to 5x10(3) HSV type 1 genome equivalents (GE) per ml, i.e., 2.5 to 6.3 GE per run, could consistently be detected. There was a correlation between the LC assay and the home-brew assay in 55 of 59 specimens. In conclusion, the LC assay allows very rapid detection of HSV DNA in CSF. It was found to be laborsaving and showed sufficient sensitivity.

| S-EPMC86985 | biostudies-literature

Inhibition of herpes simplex virus type 1 by small interfering RNA.

Project description:BackgroundRNA interference, a conserved mechanism in which a sequence-specific gene-silencing process is mediated by small interfering RNA (siRNA), is a promising method of gene therapy in treating a variety of viral diseases.AimTo investigate the antiviral effects of siRNA on herpes simplex virus type 1 (HSV-1) replication in Vero cells.MethodsThe antiviral effects of siRNA duplexes targeting the VP16 and DNA polymerase genes of HSV-1 were evaluated by yield-reduction and plaque-reduction assays. The effect of siRNA on the expression of target genes was measured by real-time quantitative reverse transcription PCR.ResultsTwo siRNA duplexes (siRNA-1, targeting VP16, and siRNA-4, targeting DNA polymerase), were found to be highly effective in inhibiting HSV-1 replication. siRNA-1 and siRNA-4 reduced HSV-1 replication by around 2 log(10) and 1 log(10) in the yield--reduction assay and by approximately 85% and approximately 70% in the plaque-reduction assay, respectively. Significant decreases in the mRNA level of VP16 and DNA polymerase genes were detected after viral infection in the Vero cells pretreated with siRNA-1 and siRNA-4, respectively.Conclusion:These results indicate that siRNA can potently inhibit HSV-1 replication in vitro, suggesting that siRNA-based antiviral therapy may be a potential effective therapeutic alternative for patients with HSV-1 infection.

| S-EPMC7162058 | biostudies-literature

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