CRISPR-Cas9 Targeted Enrichment and Next-Generation Sequencing for Mutation Detection.

Despite the rapid application of next-generation sequencing (NGS) technologies, target sequencing in regions of the genome is often required to diagnose many genetic diseases. Target enrichment can be an effective factor in reducing the cost of sequencing and the duration of sequencing. Recently, several clustered system regularly interspaced short palindromic repeats (CRISPR)-based methods (amplification-free sequencing) have been developed to target enrichment in combination with one of the NGS platforms. CRISPR-based target enrichment strategies act as an auxiliary tool to improve NGS analytical performance, thereby indirectly facilitating nucleic acid detection. The direct DNA cleavage approach by CRISPR-CRISPR-associated (Cas) at genome-specific sites enhances the possibility of separating native large fragments from disease-related genomic regions. The CRISPR-Cas can isolate the target region without any amplification; subsequently, long-read sequencing technologies were also implemented. These methods, as promising tools, have the ability to assess genetic and epigenetic composition for clinical application and treatment responses in cancer precision medicine. By modifying CRISPR-based enrichment protocols, it is possible to identify different types of mutations, including structural variants, short tandem repeats, fusion genes, and mobile elements. The Cas9 can specifically eliminate wild-type sequences, and it also enables the enrichment and detection of small amounts of tumor DNA fragments among the highly heterogeneous fragments of wild-type DNA.

Preclinical and Clinical Aspects of using Tazemetostate in Human Cancers.

Epigenetic drugs are novel drug categories with promising effects in different cancers. Tazemetostate is among the drugs that were recently used in clinical settings, especially in the treatment of specific tumors and lymphomas. There are a growing number of ongoing clinical trials evaluating the safety and efficacy of tazemetostate in different cancers. The present review addressed the available preclinical studies evaluating the combination of tazemetostate and other chemotherapy agents in treating different cancers and summarized the limited clinical evidence available regarding the efficacy of this novel Enhancer of Zeste Homolog 2 (EZH2) inhibitor in cancer. Based on the available clinical studies, tazemetostate could be considered a safe epigenetic agent with limited adverse events for treating specific types of lymphomas and solid tumors. However, the superiority of using tazemetostate over other chemotherapy agents in patients with cancer as well as using the drug for other clinical conditions, including non-alcoholic steatohepatitis, needs further investigation. Moreover, the effect of tazemetostate on human germline cells is clearly evaluated as some animal studies demonstrated that the drug can affect germline epigenome suggesting further studies on this issue.

Circulating miR-455-3p, miR-5787, and miR-548a-3p as potential noninvasive biomarkers in the diagnosis of acute graft-versus-host disease: a validation study.

Currently, acute graft-versus-host disease (aGVHD) diagnosis is based on clinical features and pathological findings. Until now, there is no non-invasive diagnostic test for aGVHD. MicroRNAs may act as promising predictive, diagnostic, or prognostic biomarkers for aGVHD. The purpose of the current study was to validate circulating microRNAs as diagnostic biomarkers to assist clinicians in promptly diagnosing aGVHD, so that treatment can be initiated earlier. In the present study, we evaluated six microRNAs (miR-455-3p, miR-5787, miR-6729-5p, miR-6776-5p, miR-548a-3p, and miR-6732-5p) selected from miRNA array data in 40 aGVHD patients compared to 40 non-GVHD patients with RT-qPCR. Target genes of differentially expressed microRNAs (DEMs) were predicted using Targetscan, miRanda, miRDB, miRWalk, PICTAR5, miRmap, DIANA, and miRTarBase algorithms, and their functions were analyzed using EnrichNet, Metascape, and DIANA-miRPath databases. The expressions of plasma miR-455-3p and miR-5787 were significantly downregulated, whereas miR-548a-3p was significantly upregulated in aGVHD patients compared to non-GVHD patients. Moreover, DEMs showed potentially high diagnostic accuracy for aGVHD. In silico analysis of DEMs provided valuable information on the role of DEMs in GVHD, immune regulation, and inflammatory response. Our study suggested that miR-455-3p, miR-5787, and miR-548a-3p could be used as potential noninvasive biomarkers in the diagnosis of aGVHD in addition to possible therapeutic targets in aGVHD.

Molecular Genetics of Cleidocranial Dysplasia.

BACKGROUND: Cleidocranial dysplasia (CCD) is a genetic disorder with an autosomal dominant inheritance pattern. CCD characterized by abnormal clavicles, patent sutures and fontenelles, supernumerary teeth and short stature. Approximately 60-70% of CCD patients have mutations in the RUNX2 gene. The RUNX2 gene is an essential transcription factor for chondrocyte maturation, osteoblast differentiation and bone formation. Runx2 regulates mesenchymal cell proliferation in sutures and suture closure by inducing the signaling pathways of the genes of Fgf, Pthlh, hedgehog and Wnt. Material and Methods: We summarized molecular genetics aspects of CCD. Result: Approximately 94% of CCD patients have dental anomalies, the most common of which are supernumerary tooth. Dental anomalies are not determined solely by gene mutations of RUNX2, but are also affected by modifier genes, environmental factors, epigenetic factors and copy number variations. Conclusion: a definite diagnosis of CCD should include the patient's clinical history, symptoms and signs, as well as genetic analyses.

CRISPR-based biosensing systems: a way to rapidly diagnose COVID-19.

The outbreak of the emerging SARS-CoV-2 virus has highlighted the challenges of detecting viral infections, especially in resource-limited settings. The SARS-CoV-2 virus transmission chain is interrupted when screening and diagnosis can be performed on a large scale by identifying asymptomatic or moderately symptomatic patients. Diagnosis of COVID-19 with reverse transcription polymerase chain reaction (RT-PCR) has been limited due to inadequate access to complex, expensive equipment and reagents, which has impeded efforts to reduce the spread of virus transmission. Recently, the development of several diagnostic platforms based on the CRISPR-Cas system has reduced the dependence on RT-PCR. The first CRISPR-based diagnostic test for SARS-CoV-2 was recently approved by the U.S. Food and Drug Administration. The biosensing systems have several important features that make them suitable for point-of-care tests, including the speed of design and synthesis of each platform in less than a few days, an assay time of 1-2 h, and the cost of materials and reagents less than one dollar per test. The HUDSON-SHERLOCK and STOPCovid biosensing systems, as field-deployable and rapid diagnostic tests, can detect low-copy viruses in body fluids without nucleic acid extraction and with minimal equipment. In addition, Cas13-based treatment strategies could potentially be an effective antiviral strategy for the prevention and treatment of emerging pandemic viruses such as SARS-CoV-2. In this review, we describe recent advances in CRISPR-based diagnostic platforms with an emphasis on their use in the rapid diagnosis and potential treatment of COVID-19.

MicroRNAs as Potential Diagnostic, Prognostic, and Predictive Biomarkers for Acute Graft-versus-Host Disease.

Successful treatment of various hematologic diseases with allogeneic hematopoietic stem cell transplantation is often limited due to the occurrence of acute graft-versus-host disease (aGVHD). So far, there are no approved molecular biomarkers for the diagnosis and prediction of aGVHD at the clinical level due to our incomplete understanding of the molecular biology of the disease. Various studies have been conducted on animal models and humans to investigate the role of microRNAs in aGVHD pathogenesis to implicate them as biomarkers and therapeutic targets. Because of their high stability, tissue specificity, ease of measurement, low cost, and simplicity, they are excellent targets for biomarkers. In this review, we focused on microRNA expression profiling studies that were performed recently in both animal models and human cases of aGVHD to identify diagnostic and predictive biomarkers for this disease. The expression pattern of microRNAs can be specific to cells and tissues. Because aGVHD affects several organs, microRNA signatures in target tissues may help to understand the molecular pathology of the disease. Identification of organ-specific microRNAs in aGVHD can be promising to categorize patients for organ-specific therapies. Thus, microRNAs can be used as noninvasive diagnostic tests in clinic to improve prophylaxis, predict incidence and severity, and reduce morbidity.