CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells (original) (raw)
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The implication of CRISPR/Cas9 genome editing technology in combating human oncoviruses
It is evidenced that 20% of all tumors in humans are caused by oncoviruses, including human papilloma viruses, Epstein-Barr virus, Kaposi sarcoma virus, human polyoma-viruses, human T-lymphotrophic virus-1, and hepatitis B and C viruses. Human immunodeficiency virus is also involved in carcinogenesis, although not directly, but by facilitating the infection of many oncoviruses through compromising the immune system. Being intracellular parasites with the property of establishing latency and integrating into the host genome, these viruses are a therapeutic challenge for biomedical researchers. Therefore, strategies able to target nucleotide sequences within episomal or integrated viral genomes are of prime importance in antiviral or anticancerous armamentarium. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has emerged as a powerful genome editing tool. Standing out as a precise and efficient oncoviruses method, it has been extensively applied in recent experimental ventures in the field of molecular medicine, particularly in combating infections including tumor inducing viruses. This review is aimed at collating the experimental and clinical advances in CRISPR/Cas9 technology in terms of its applications against oncoviruses. Primarily, it will focus on the application of CRISPR/ Cas9 in combating tumor viruses, types of mechanisms targeted, and the significant outcomes till date. The technical pitfalls of the CRISPR/Cas9 and the comparative approaches in evaluating this technique with respect to other available alternatives are also described briefly. Furthermore, the review also discussed the clinical aspects and the ethical, legal, and social issues associated with the use of CRISPR/Cas9. K E Y W O R D S CRISPR/Cas9, DNA targeting, genome editing, oncoviruses 1 | INTRODUCTION Numerous genetic engineering techniques with promising applications in combating various infections have been described during the last few decades. These methods are based on DNA-protein or RNA-RNA interactions. The premier category includes zinc finger nucleases (ZFN), transcription activator-like effector nucleases, and mega-endonucleases. These genome editing tools use proteins (endonucleases) that interact with DNA and cause double-stranded DNA breaks (DSBs) in the target DNA at specific sites. 1 The second category includes RNA interference (RNAi), which induces posttranscriptional gene silencing through small interfering RNA interaction with messenger RNA (mRNA) of a specific gene. This cleavage is mediated by RNA-induced silencing complex and causes translational repression by degrading RNA. 2 More recently, an J Med Virol. 2018;1-13. wileyonlinelibrary.com/journal/jmv
Journal of World's Poultry Research
Introduction. Clustered-Regularly Interspaced Short Palindromic Repeats (CRISPR), and CRISPR associated (Cas) protein (CRISPR/Cas) structures were first identified in E. coli in 1987 and guard prokaryotic cells from any invading pathogens, harmful events and plasmids by recognizing and cutting foreign nucleic acid sequences that contain short palindromic repeats spacer sequences. Several genome editing approaches have been developed based on these mechanisms; the most recent is known as CRISPR/Cas. Before the CRISPR technique was revealed in 2012, editing the genomes of plants and animals took many years and cost hundreds of thousands of dollars. Thus, CRISPR/Cas has attracted significant interest in the scientific community, especially for disease diagnosis and treatment, as it is quicker, less expensive and more precise than other genome editing approaches. The evidence from gene mutations in specific patients generated using CRISPR/Cas can assist in the prediction of the optimal treatment schedule for individual patients and for innovation purposes in other researches like replication in cell culture of coronaviruses like severe acute respiratory syndrome coronavirus-2 (SARS-CoV2 or COVID-19). However, in numerous situations, the effects of the furthermost significant driver mutations are not yet understood and interpretation of the optimal treatment is impossible. CRISPR/Cas classifications feature highly sensitive and selective tools for the detection of various target genes. When we see the next steps of genomic research, it is obvious that genome-wide association studies are relatively new way to identify the genes involved in human disease. Furthermore, CRISPR/Cas provides a tool to manipulate non-coding regions and will thus accelerate examination of these poorly characterized regions of the genome and play a vital role in the progress of whole genome libraries. Aim. We aimed to review the history of CRISPR/Cas, the mechanisms of CRISPR techniques, its current status as a tool for studying both natural mutations and genomic manipulations, and explore how CRISPR/Cas may improve the treatment of diseases.
An Update on Genome Editing With the Utilization of CRISPR/Cas 9 System for Evaluation and Treatment of Human Diseases - A Systemic Review, 2022
Abstract The CRISPR/Cas9-techique again emerged in 2012 with the generation of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based gene editing that represents a modulation tool that has been obtained from the defense system of the some bacteria, against viruses in addition to plasmids. This is a neconomical, simple approach that has got utilized in a lot of experimental models inclusive of cell lines, various laboratory animals, plants, as well as human Clinical trial. The CRISPR/Cas9 system is constituted of gjuiding the Cas 9 nuclease for generation of site-directed double stranded DNA break with the utilization of small RNA molecules for directing. It is an event that results in permanent manipulation of the genomic target sequence which can heal the injury that occurs in the DNA. Thus here we conducted a systematic review utilizing search engine pubmed, google scholar and other sutilizing the MeSH terms like zinc finger nucleases; TALENs; CRISPR/Cas9 system; DSB; genome editing; trans-encoded small cr RNAs(tracr RNAs); single guide gRNA; Protospacer Adjacent Motif (PAM); endonuclease; HNH domain; Ruv C-like nuclease domain; insertions along with deletions (indels); dead Sp- Cas9 (d Sp- Cas9); n Sp- Cas9 (n Cas9)]. Hirshsprung disease; Megacystis-Microcolon- Intestinal Hyperperistalsis Syndrome (MMIHS); β-haemoglobinopathies, Sickle Cell Disease (SCD); β thallasaemia, is Human Papilloma Virus (HPV); human immunodeficiency virus(HIV),;Hepatitis B virus(HBV); cancers; neurological diseases; from 1990 to 2021 till date. We found 2050 articles out of which we selected 141 articles for this review. No meta-analysis was done. Here we present app approaches as well as manipulations of enzyme Cas9 for removal of target cuts away the various applications of CRISPR/Cas9 system for looking besides activation as well as repression. Furthermore, we outline the therapeutic aspects besides the latest updates in their utilization in various human diseases. Keywords: CRISPR/Cas9 system; Tracr RNAs; PAM; Hirshsprung disease; HPV; HIV; Mitochondrial diseases’ breast cancer
CRISPR/Cas9-Mediated Genome Editing of Herpesviruses Limits Productive and Latent Infections
PLOS Pathogens, 2016
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.
Genome editing: A perspective on the application of CRISPR/Cas9 to study human diseases (Review)
International Journal of Molecular Medicine, 2019
Genome editing reemerged in 2012 with the development of cRISPR/cas9 technology, which is a genetic manipulation tool derived from the defense system of certain bacteria against viruses and plasmids. This method is easy to apply and has been used in a wide variety of experimental models, including cell lines, laboratory animals, plants, and even in human clinical trials. The cRISPR/cas9 system consists of directing the cas9 nuclease to create a site-directed double-strand dNA break using a small RNA molecule as a guide. A process that allows a permanent modification of the genomic target sequence can repair the damage caused to dNA. In the present study, the basic principles of the cRISPR/cas9 system are reviewed, as well as the strategies and modifications of the enzyme cas9 to eliminate the off-target cuts, and the different applications of cRISPR/cas9 as a system for visualization and gene expression activation or suppression. In addition, the review emphasizes on the potential application of this system in the treatment of different diseases, such as pulmonary, gastrointestinal, hematologic, immune system, viral, autoimmune and inflammatory diseases, and cancer.
CRISPR/Cas9 system: a reliable and facile genome editing tool in modern biology
Molecular Biology Reports
Genome engineering has always been a versatile technique in biological research and medicine, with several applications. In the last several years, the discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 technology has swept the scientific community and revolutionised the speed of modern biology, heralding a new era of disease detection and rapid biotechnology discoveries. It enables successful gene editing by producing targeted double-strand breaks in virtually any organism or cell type. So, this review presents a comprehensive knowledge about the mechanism and structure of Cas9-mediated RNA-guided DNA targeting and cleavage. In addition, genome editing via CRISPR-Cas9 technology in various animals which are being used as models in scientific research including Non-Human Primates Pigs, Dogs, Zebra, fish and Drosophila has been discussed in this review. This review also aims to understand the applications, serious concerns and future perspective of CRISPR/Cas9-mediated genome editing.
Advances In Research On Genome Editing Crispr-Cas9 Technology
Journal of Ayub Medical College, Abbottabad : JAMC, 2019
BACKGROUND The current era of genome engineering has been revolutionized by the evolution of a bacterial adaptive immune system, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) into a radical technology that is making an expeditious progress in its mechanism, function and applicability.. METHODS A systematic literature review study was carried out with the help of all available information and online resources.. RESULTS In this review, we intend to elucidate different aspects of CRISPR in the light of current advancements. Utilizing a nonspecific Cas9 nuclease and a sequence specific programmable CRISPR RNA (crRNA), this system cleaves the target DNA with high precision. With a vast potential for profound implications, CRISPR has emerged as a mainstream method for plausible genomic manipulations in a range of organisms owing to its simplicity, accuracy and speed. A modified form of CRISPR system, known as CRISPR/Cpf1 that employs a smaller and simpler endonuclease...
CRISPR–Cas9 in genome editing: Its function and medical applications
Journal of Cellular Physiology, 2018
The targeted genome modification using RNA-guided nucleases is associated with several advantages such as a rapid, easy, and efficient method that not only provides the manipulation and alteration of genes and functional studies for researchers, but also increases their awareness of the molecular basis of the disease and development of new and targeted therapeutic approaches. Different techniques have been emerged so far as the molecular scissors mediating targeted genome editing including zinc finger nuclease, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). CRISPR-Cas9 is a bacterial immune system against viruses in which the single-strand RNA-guided Cas9 nuclease is linked to the targeted complementary sequences to apply changes. The advances made in the transfer, modification, and emergence of specific solutions have led to the creation of different classes of CRISPR-Cas9. Since this robust tool is capable of direct correction of disease-causing mutations, its ability to treat genetic disorders has attracted the tremendous attention of researchers. Considering the reported cases of nonspecific targeting of Cas9 proteins, many studies focused on enhancing the Cas9 features. In this regard, significant advances have been made in choosing guide RNA, new enzymes and methods for identifying misplaced targeting. Here, we highlighted the history and various direct aspects of CRISPR-Cas9, such as precision in genomic targeting, system transfer and its control over correction events with its applications in future biological studies, and modern treatment of diseases.
Targeted gene disruption in Epstein-Barr virus
Virology, 1992
We report the development of a method that should allow the insertion of a selective marker into any region of the Epstein-Barr virus (EBV) genome of strain 895-8 through homologous recombination with plasmids. In this method, EBV recombinants are isolated as G41 a-resistant, immortalized B-cell clones or as G41 a-resistant, latently infected subclones of Burkitt lymphoma cell lines. The presence of the productive replication origin of EBV, oriLyt, on the plasmid was found to increase the number of observed recombinant viruses by approximately 1 OO-fold; this stimulation was observed when oriLytwas separated from the sites of recombination by several kilobases of nonhomologous DNA. Long segments of EBV DNA flanking the marker on the plasmid and/or a large plasmid size were inferred to be important for obtaining a high proportion of recombinant genomes that had recombined on both sides of the selective marker; otherwise, the recombinants that predominated had acquired the entire plasmid by recombining only on one side of the inserted marker. Therefore, to facilitate targeted insertion of genetic markers into the EBV genome, a cosmid vector carrying oriLyt was constructed and tested by using it to generate EBV mutants with the BALF2 openreading frame disrupted. o 1992 Academic PWSS. I~C. 253
CRISPR/Cas9 System: A Breakthrough in Genome Editing
Molecular Biology
Clustered Regularly Interspaced Short Palindromic Repeats is a new and advance gene editing tool using specific nuclease enzyme for specific cleavage. It is the most efficient technique, commonly used for many purposes like gene therapy, production of desired plants and transgenic animals. But it has some limitations like off-target issue and this issue can be minimized by the production of specific sequence of guide RNA. In the future, it can be used for the treatment of many human genetic diseases.