Potential Application of the CRISPR/Cas9 System against Herpesvirus Infections

Chen, Yuan‐Chuan; Sheng, Jingxue; Trang, Phong; Liu, Fenyong

doi:10.3390/v10060291

Cited by 63 publications

(54 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Numerous studies showed successful use of CRISPR/Cas9 in genetic studies of herpesviruses. However, as of today, there is no report on genetic engineering of KSHV genome in PEL cells by CRISPR/Cas9 (13,14).…”

mentioning

confidence: 99%

CRISPR/Cas9-Mediated Knockout and In Situ Inversion of the ORF57 Gene from All Copies of the Kaposi's Sarcoma-Associated Herpesvirus Genome in BCBL-1 Cells

BeltCappellino

Majerčiak

Lobanov

et al. 2019

J Virol

View full text Add to dashboard Cite

Kaposi’s sarcoma-associated herpesvirus (KSHV)-transformed primary effusion lymphoma cell lines contain ∼70 to 150 copies of episomal KSHV genomes per cell and have been widely used for studying the mechanisms of KSHV latency and lytic reactivation. Here, we report the first complete knockout (KO) of viral ORF57 gene from all ∼100 copies of KSHV genome per cell in BCBL-1 cells. This was achieved by a modified CRISPR/Cas9 technology to simultaneously express two guide RNAs (gRNAs) and Cas9 from a single expression vector in transfected cells in combination with multiple rounds of cell selection and single-cell cloning. CRISPR/Cas9-mediated genome engineering induces the targeted gene deletion and inversion in situ. We found the inverted ORF57 gene in the targeted site in the KSHV genome in one of two characterized single cell clones. Knockout of ORF57 from the KSHV genome led to viral genome instability, thereby reducing viral genome copies and expression of viral lytic genes in BCBL-1-derived single-cell clones. The modified CRISPR/Cas9 technology was very efficient in knocking out the ORF57 gene in iSLK/Bac16 and HEK293/Bac36 cells, where each cell contains only a few copies of the KSHV genome. The ORF57 KO genome was stable in iSLK/Bac16 cells, and, upon lytic induction, was partially rescued by ectopic ORF57 to express viral lytic gene ORF59 and produce infectious virions. Together, the technology developed in this study has paved the way to express two separate gRNAs and the Cas9 enzyme simultaneously in the same cell and could be efficiently applied to any genetic alterations from various genomes, including those in extreme high copy numbers. IMPORTANCE This study provides the first evidence that CRISPR/Cas9 technology can be applied to knock out the ORF57 gene from all ∼100 copies of the KSHV genome in primary effusion lymphoma (PEL) cells by coexpressing two guide RNAs (gRNAs) and Cas9 from a single expression vector in combination with single-cell cloning. The gene knockout efficiency in this system was evaluated rapidly using a direct cell PCR screening. The current CRISPR/Cas9 technology also mediated ORF57 inversion in situ in the targeted site of the KSHV genome. The successful rescue of viral lytic gene expression and infectious virion production from the ORF57 knockout (KO) genome further reiterates the essential role of ORF57 in KSHV infection and multiplication. This modified technology should be useful for knocking out any viral genes from a genome to dissect functions of individual viral genes in the context of the virus genome and to understand their contributions to viral genetics and the virus life cycle.

show abstract

mentioning

confidence: 99%

CRISPR/Cas9-Mediated Knockout and In Situ Inversion of the ORF57 Gene from All Copies of the Kaposi's Sarcoma-Associated Herpesvirus Genome in BCBL-1 Cells

BeltCappellino

Majerčiak

Lobanov

et al. 2019

J Virol

View full text Add to dashboard Cite

show abstract

“…The viral capsid decomposes to release viral DNA genome to manipulates host enzyme systems to make new virions. During symptomatic infection, infected cells express lytic genes to demonstrate a lytic pathway [12]. Nevertheless, instead of this, some viral genes may transcribe latency associated transcripts to accumulate in host cells.…”

Section: Life Cyclementioning

confidence: 99%

“…For the lysogenic pathway, the viruses are persistent in the host, not causing any adverse reactions, but can be transmitted to other hosts by direct contact. When CMV are stimulated by explanation or their host immune system is suppressed, the dormant viruses can reactivate to begin generating large number of viral progenies to cause symptoms and diseases, described as the lytic life cycle [12,13].…”

Section: Life Cyclementioning

confidence: 99%

“…In CRISPR/Cas9 system, CRISPR is used to build RNA-guided genes drives to target a specific DNA sequence. By the Cas proteins and a specifically designed single-guiding RNA (sgRNA), the genome can be cut at most locations with only the limitation of a protospacer adjacent motif (PAM) sequence (NGG) existing in the target site [12]. CRISPR/Cas9 has been extensively used as an effective technique of gene editing for engineering or modifying specific genes.…”

Section: Crispr/cas9mentioning

confidence: 99%

See 1 more Smart Citation

Antiviral Agents as Therapeutic Strategies Against Cytomegalovirus Infections

Chen

Wang

Chen

2019

Viruses

Self Cite

View full text Add to dashboard Cite

Cytomegalovirus (CMV) is a threat to human health in the world, particularly for immunologically weak patients. CMV may cause opportunistic infections, congenital infections and central nervous system infections. CMV infections are difficult to treat due to their specific life cycles, mutation, and latency characteristic. Despite recent advances, current drugs used for treating active CMV infections are limited in their efficacy, and the eradication of latent infections is impossible. Current antiviral agents which target the UL54 DNA polymerase are restricted because of nephrotoxicity and viral resistance. CMV also cannot be prevented or eliminated with a vaccine. Fortunately, letermovir which targets the human CMV (HCMV) terminase complex has been recently approved to treat CMV infections in humans. The growing point is developing antiviral agents against both lytically and latently infected cells. The nucleic acid-based therapeutic approaches including the external guide sequences (EGSs)-RNase, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and transcription activator-like effector nucleases (TALENs) are being explored to remove acute and/or latent CMV infections. HCMV vaccine is being developed for prophylaxis. Additionally, adoptive T cell therapy (ACT) has been experimentally used to combate drug-resistant and recurrent CMV in patients after cell and/or organ transplantation. Developing antiviral agents is promising in this area to obtain fruitful outcomes and to have a great impact on humans for the therapy of CMV infections.

show abstract

“…Herpesviruses are nuclear-replicating DNA viruses with large dsDNA genomes (100-200 kb) that encode 100 to 200 genes (8). They frequently undergo homologous recombination during their replication cycle and can be efficiently edited by CRISPR-Cas9 (9)(10)(11)(12). These properties enabled the design of a new gene drive strategy that doesn't involve sexual reproduction, but relies on coinfection of a given cell by a wildtype and an engineered virus ( Fig.…”

Section: Main Textmentioning

confidence: 99%

Viral gene drive in herpesviruses

Walter

Verdin

2019

Preprint

View full text Add to dashboard Cite

Herpesviruses are ubiquitous pathogens in need of novel therapeutic solutions. Current engineered gene drive strategies rely on sexual reproduction, and are thought to be restricted to sexual organisms. Here, we report on the design of a novel gene drive system that allows the spread of an engineered trait in populations of DNA viruses and, in particular, herpesviruses. We describe the successful transmission of a gene drive sequence between distinct strains of human cytomegalovirus (human herpesvirus 5) and show that gene drive viruses can efficiently target and replace wildtype populations in cell culture experiments. Our results indicate that viral gene drives can be used to suppress a viral infection and may represent a novel therapeutic strategy against herpesviruses.

show abstract

Potential Application of the CRISPR/Cas9 System against Herpesvirus Infections

Cited by 63 publications

References 40 publications

CRISPR/Cas9-Mediated Knockout and In Situ Inversion of the ORF57 Gene from All Copies of the Kaposi's Sarcoma-Associated Herpesvirus Genome in BCBL-1 Cells

CRISPR/Cas9-Mediated Knockout and In Situ Inversion of the ORF57 Gene from All Copies of the Kaposi's Sarcoma-Associated Herpesvirus Genome in BCBL-1 Cells

Antiviral Agents as Therapeutic Strategies Against Cytomegalovirus Infections

Viral gene drive in herpesviruses

Contact Info

Product

Resources

About