Construction of an Exudative Age-Related Macular Degeneration Diagnostic and Therapeutic Molecular Network Using Multi-Layer Network Analysis, a Fuzzy Logic Model, and Deep Learning Techniques: Are Retinal and Brain Neurodegenerative Disorders Related?

Neovascular age-related macular degeneration (nAMD) is a leading cause of irreversible visual impairment in the elderly. The current management of nAMD is limited and involves regular intravitreal administration of anti-vascular endothelial growth factor (anti-VEGF). However, the effectiveness of these treatments is limited by overlapping and compensatory pathways leading to unresponsiveness to anti-VEGF treatments in a significant portion of nAMD patients. Therefore, a system view of pathways involved in pathophysiology of nAMD will have significant clinical value. The aim of this study was to identify proteins, miRNAs, long non-coding RNAs (lncRNAs), various metabolites, and single-nucleotide polymorphisms (SNPs) with a significant role in the pathogenesis of nAMD. To accomplish this goal, we conducted a multi-layer network analysis, which identified 30 key genes, six miRNAs, and four lncRNAs. We also found three key metabolites that are common with AMD, Alzheimer's disease (AD) and schizophrenia. Moreover, we identified nine key SNPs and their related genes that are common among AMD, AD, schizophrenia, multiple sclerosis (MS), and Parkinson's disease (PD). Thus, our findings suggest that there exists a connection between nAMD and the aforementioned neurodegenerative disorders. In addition, our study also demonstrates the effectiveness of using artificial intelligence, specifically the LSTM network, a fuzzy logic model, and genetic algorithms, to identify important metabolites in complex metabolic pathways to open new avenues for the design and/or repurposing of drugs for nAMD treatment.

Research and Therapeutic Approaches in Stem Cell Genome Editing by CRISPR Toolkit.

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.

Principles and Applications of CRISPR Toolkit in Virus Manipulation, Diagnosis, and Virus-Host Interactions.

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.

Enhanced High Mutation Rate and Natural Selection to Produce Attenuated Viral Vaccine with CRISPR Toolkit in RNA Viruses especially SARS-CoV-2.

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.

A knockdown of the herpes simplex virus type-1 gene in all-in-one CRISPR vectors.

INTRODUCTION: Herpes simplex virus type 1 (HSV-1) is a virus that causes serious human disease and establishes a long-term latent infection. The latent form of this virus has shown to be resistant to antiviral drugs. Clustered Regularly Interspace Short Palindromic Repeats (CRISPR), is an important tool in genome engineering and composed of guide RNA (gRNA) and Cas9 nuclease that makes an RNA-protein complex to digest exclusive target sequences implementation of gRNA. Moreover, CRISPR-Cas9 system effectively suppresses HSV-1 infection by knockout of some viral genes. MATERIALS AND METHODS: To survey the efficacy of Cas9 system on HSV-1 genome destruction, we designed several guide RNAs (gRNAs) that all packaged in one vector. Additionally, we performed a one-step restriction using BamHI and Esp3I enzymes. RESULTS: CRISPR/Cas9 system targeted against the gD gene of HSV-1 was transfected into HEK-AD cells that showed a significant reduction of HSV-1 infection by plaque assay and real-time PCR. CONCLUSION: The pCas-Guide-EF1a-GFP CRISPR vector can create a fast and efficient method for gRNA cloning by restriction enzymes (Esp3I (BsmBI) and BamHI). Therefore, the CRISPR/Cas9 system may be utilized for the screening of genes critical for the HSV-1 infection and developing new strategies for targeted therapy of viral infections caused by HSV-1.

An overview of applications of CRISPR-Cas technologies in biomedical engineering.

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.