As strange as it sounds, most adults actually carry more than one of the many known herpes viruses. After the initial acute infection, these viruses establish life-long infections within their hosts and cause things such as cold sores, keratitis, genital herpes, shingles, and infectious mononucleosis along with a wide range of other diseases. Some strains of the herpes virus can lead to cancer.
During the latent phase of the infection, the virus remains dormant for long periods of time but is still able to cause occasional reactivations which can lead to disease. A study which was published on June 30th in PLOS Pathogens journal suggests that attacking the herpes virus DNA with CRISPR/Cas9 genome editing technology can suppress virus replication and in some cases lead to complete elimination of the virus.
The CRISPR/Cas9 works by targeting specific DNA sequences and inducing clean cuts across both strands of the DNA. When it comes to mammalian cells, these cuts are flagged and abruptly repaired by an emergency repair system known as the NHEJ (non-homologous end-joining). NHEJ is efficient but not very accurate and oftentimes results in the insertion or complete deletion of a few DNA bases at the repair site. Because DNA is read in codons of three bases at a single time, small changes such as these in critical positions often destroy the function of the respective gene and its protein product.
Robert Jan Lebbink from the University Medical Center in Utrecht, The Netherlands along with colleagues say that CRISPR/Cas9 was able to target and mutate latent herpes virus DNA in infected human cells and may potentially prevent diseases associated with the virus. To test the theory, researchers devised specific guide (g)RNAs, which are sequences that are complementary to vital parts of the viral genome and function as “molecular addresses”. These gRNAs, when combined with the molecular scissors part of the CRISPR/Cas9 system should lead to specific cuts and subsequent mutations in the herpes virus DNA, crippling the virus.
In this systematic approach, researchers looked at three different members of the herpes virus group: herpes simplex virus type 1 (HSV-1) which cause cold sores and herpes keratitis, human cytomegalovirus (HCMV), which is the most common viral cause of birth defects. This is when the virus is transmitted from mother directly into the fetus. Lastly, they looked at Epstein-Barr virus (EBV) which causes infectious mononucleosis and multiple types of cancer.
Researchers worked with lymphoma cells that were latently infected with EBV and were able to show that the introduction of gRNAs that target specific EBV DNA sequences can introduce mutations at the targeted sites. These types of mutations can get rid of essential functions of the virus as well as destabilize the viral DNA molecules. The team reported that by using two different gRNAs targeting an essential EBC gene they are able to induce loss of over 95% of EBV genomes from the host cells.
During the latent infection, HCMV genomes exist as circular DNA molecules in the nucleus of host cells. When the virus was reactivated, HCMV replication proceeded very slowly. When appropriate gRNAs were used, the researchers discovered that CRISPR/Cas9 editing is able to effectively impair HCMC replication. They also found that emergence of escape variants that bypass CRISPR/Cas9 editing, suggesting that simultaneous editing at multiple critical sites in the HCMV genome is required in order to avoid the development of resistant genomes.
In comparison to HCMV, HSV-1 multiples far quicker. When the researchers tested various gRNAs targeting different essential HSV-1 genes in conjunction with CRISPR/Cas9, they found that many of them were able to reduce the replication of the virus. When they combined two of the gRNAs, simultaneously targeting two essential genes, they were able to completely suppress the replication of HSV-1. At the same time, this allowed them to induce editing during the latent phase when the viral DNA was not actively multiplying.
The researchers say they observed highly efficient and specific clearance of EBV from latently infected tumor cells and impairment of HSV-1 and HCMV replication in human cells. Although CRISPR/Cas9 was inefficient at directing genome engineering of quiescent HSV-1, virus replication upon reactivation of quiescent HSV-1 was efficiently abrogated using anti-HSV-1 gRNAs. They hope the results may allow the design of effective therapeutic strategies that will be able to target human herpes viruses during both latent and productive infections.
