The best and most effective way to combat pandemics is to use effective vaccines and live attenuated vaccines are among the most effective vaccines. However, one of the major problems is the length of time it takes to get the attenuated vaccines. Today, the CRISPR toolkit (Clustered Regularly Inerspaced Short Palindromic Repeats) has made it possible to make changes with high efficiency and speed. Using this toolkit to make point mutations on the RNA virus's genome in a coculture of permissive and nonpermissive cells and under controlled conditions can accelerate changes in the genome and accelerate natural selection to obtain live attenuated vaccines.
Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) is one of the major genome editing systems and allows changing DNA levels of an organism. Among several CRISPR categories, the CRISPR-Cas9 system has shown a remarkable progression rate over its lifetime. Recently, other tools including CRISPR-Cas12 and CRISPR-Cas13 have been introduced. CRISPR-Cas9 system has played a key role in the industrial cell factory's production and improved our understanding of genome function. Additionally, this system has been used as one of the major genome editing systems for the diagnosis and treatment of several infectious and non-infectious diseases. In this review, we discuss CRISPR biology, its versatility, and its application in biomedical engineering.
Viruses are one of the most important concerns for human health, and overcoming viral infections is a worldwide challenge. However, researchers have been trying to manipulate viral genomes to overcome various disorders, including cancer, for vaccine development purposes. CRISPR (clustered regularly interspaced short palindromic repeats) is becoming one of the most functional and widely used tools for RNA and DNA manipulation in multiple organisms. This approach has provided an unprecedented opportunity for creating simple, inexpensive, specific, targeted, accurate, and practical manipulations of viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus-1 (HIV-1), and vaccinia virus. Furthermore, this method can be used to make an effective and precise diagnosis of viral infections. Nevertheless, a valid and scientifically designed CRISPR system is critical to make more effective and accurate changes in viruses. In this review, we have focused on the best and the most effective ways to design sgRNA, gene knock-in(s), and gene knock-out(s) for virus-targeted manipulation. Furthermore, we have emphasized the application of CRISPR technology in virus diagnosis and in finding significant genes involved in virus-host interactions.
The most widely used genome editing toolkit is CRISPR (clustered regularly interspaced short palindromic repeats). It provides the possibility of replacing and modifying DNA and RNA nucleotides. Furthermore, with advancements in biological technology, inhibition and activation of the transcription of specific gene(s) has become possible. Bioinformatics tools that target the evolution of CRISPR-associated protein 9 (Cas9) turn this protein into a vehicle that is specific for a DNA or RNA region with single guide RNA (sgRNA). This toolkit could be used by researchers to investigate the function of stem cell gene(s). Here, in this review article, we cover recent developments and applications of this technique in stem cells for research and clinical purposes and discuss different CRISPR/Cas technologies for knock-out, knock-in, activation, or inhibition of gene expression. Additionally, a comparison of several deliveries and off-target detecting strategies is discussed.
Background: Arginine metabolism is an important factor involved in tumorigenesis, progression, and survival of tumor cells. Besides, other metabolites produced in the arginine metabolism process, such as polyamines, nitric oxide, argininosuccinate, and agmatine, play key roles in different stages of tumor development. On the other hand, herbal metabolites are widely used to treat cancer. One of these herbal flavonoids is quercetin. Methods: In this study, according to MTT assay data, two concentrations of quercetin flavonoid were selected (57.5 and 115 µM) to treat human embryonic kidney 293 (HEK293) cells. Then RNA was extracted from the cells and used as a template for cDNA synthesis. Using real-time PCR, the expression of key enzymes involved in arginine metabolism was evaluated, including arginase 2 (Arg2), ornithine carbamoyl transferase (OTC), agmatinase (AGMAT), arginase 1 (Arg1), nitric oxide synthase 1 (nNOS), arginine decarboxylase (ADC), ornithine decarboxylase 1 (ODC), ornithine carbamoyl transferase (OCT), spermidine synthase (SRM), spermine synthase (SMS), argininosuccinate synthase 1 (ASS1), and argininosuccinate lyase (ASL). The Student t-test was used to analyze the data considering a P value of < 0.05 as the significance level. Results: Our results indicated significant changes in the expression of arginine metabolism enzymes after quercetin exposure, confirming a role for quercetin plant flavonoid in regulating arginine metabolism in HEK293 cells. Conclusions: Quercetin could alter the gene expression of the key enzymes involved in arginine metabolism. This was the first study investigating the effects of quercetin on arginine metabolism in HEK293 cells.
Arginine is a semiessential amino acid in human. Metabolism of arginine in human plays a key role in the production of proline, polyamines, urea, creatine, glutamate, ornithine, citrulline, agmatine and nitric oxide. Metabolism of this amino acid changes in neurodegenerative diseases. Association between herpes simplex virus-1 (HSV-1) infection and the probability of developing neurodegenerative diseases in humans has been proven, but the association between the molecular mechanism of HSV-1 infected cells and increased risk of these diseases has not been determined. Human embryonic kidney 293 (HEK 293) cells were cultured and checked for mycoplasma bacterial infection with polymerase chain reaction (PCR) technique, then the cells were divided into two groups. One group of cells became infected with multiplicity of infections 1 and 5 (MOIs 1 and 5) HSV-1 and the other group considered as a control. After 6.5 h, the cells were collected and RNA extracted, then cDNA synthesized, and changes in mRNA expression of key arginine metabolism enzymes genes were investigated with real-time PCR and data were analyzed by using REST 2009 (Corbett Research, United Kingdom) software. The aim of this study was to investigate the expression of the arginine metabolism pathway key enzymes in HEK 293 cells infected with HSV-1 and the interaction probability with neurodegenerative diseases. In this study, it has been shown that most of the key enzymes involved in arginine metabolism in HEK 293-infected cells with HSV-1 has changed in MOIs 1 and 5. These changes are great along with changes in neurodegenerative diseases. Change in arginine metabolism key enzymes mRNA expression after infection with HSV-1 can reflect one of the most important mechanisms of HSV-1 involvement in neurodegenerative diseases.
Background: Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly lethal virus that causes hemorrhagic fever in humans and is endemic in many countries, including Iran. Therefore, fast, accurate, and reliable diagnosis is crucial for patient management and outbreak control. Objectives: This study aims to optimize a TaqMan multiplex real-time RT-PCR for the rapid and specific diagnosis of CCHFV. Methods: In this study, the L (NC_005301.3) and S (NC_005302.1) fragments were used as reference sequences for blast analysis. The L and S sequence segments of CCHFV with more than 90% identity from different areas were downloaded from the Genbank database. Primers and probes were designed based on the best-conserved regions of CCHFV L and S sequence segments. To construct the plasmid, a 1751 bp fragment from the MS2 phage that was previously amplified using cloning primers was inserted into the pET-32a plasmid. The S and L segments of the CCHFV, which were 110 bp and 135 bp, respectively, were inserted downstream of the MS2 phage sequence from HindIII to NotI. The Viral-like particles (VLPs) were produced in Escherichia coli, strain BL-21(DE3), in the presence of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). The stability of VLP particles was confirmed in the presence of the ribonuclease enzyme. The fabrication of VLPs was approved by transmission electron microscopy (TEM) with negative staining (1% phosphotungstic acid). To validate the specificity of the primers and probes sequences, we compared them to the NCBI database and tested them experimentally using extracted DNA and RNA samples from healthy subjects and an infectious panel. Results: The VLPs showed complete resistance in the presence of the ribonuclease enzyme, and the TEM results confirmed that the VLPs were correctly produced. The TaqMan multiplex real-time RT-PCR confirmed that the primers and probes were designed correctly and were completely specific to the CCHFV. The limit of detection (LOD) of the multiplex assay for the L and S genes was one copy of the VLPs per µL. Conclusions: This TaqMan assay is reliable for amplifying CCHFV due to its design on conserved regions of the CCHFV sequences, which have minimal variability and high specificity.
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