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In October 2020, the chemistry Nobel Prize was awarded to Emmanuelle Charpentier and Jennifer A. Doudna for the discovery of a new promising genome-editing tool: the genetic scissors of CRISPR-Cas9. The identification of CRISPR arrays and the subsequent identification of cas genes, which together represent an adaptive immunological system that exists not only in bacteria but also in archaea, led to the development of diverse strategies used for precise DNA editing, providing new insights in basic research and in clinical practice. Due to their advantageous features, the CRISPR-Cas systems are already employed in several biological and medical research fields as the most suitable technique for genome engineering. In this review, we aim to describe the CRISPR-Cas systems that have been identified among prokaryotic organisms and engineered for genome manipulation studies. Furthermore, a comprehensive comparison between the innovative CRISPR-Cas methodology and the previously utilized ZFN and TALEN editing nucleases is also discussed. Ultimately, we highlight the contribution of CRISPR-Cas methodology in modern biomedicine and the current plethora of available applications for gene KO, repression and/or overexpression, as well as their potential implementation in therapeutical strategies that aim to improve patients’ quality of life.

Background Despite significant advancements in multiple myeloma (MM) therapy, the highly heterogenous treatment response hinders reliable prognosis and tailored therapeutics. Herein, we have studied the clinical utility of miRNAs in ameliorating patients’ management. Methods miRNA-seq was performed in bone marrow CD138+ plasma cells (PCs) of 24 MM and smoldering MM (sMM) patients to analyze miRNAs profile. CD138+ and circulating miR-25 levels were quantified using in house RT-qPCR assays in our screening MM/sMM cohort (CD138+ plasma cells n = 167; subcohort of MM peripheral plasma samples n = 69). Two external datasets (Kryukov et al . cohort n = 149; MMRF CoMMpass study n = 760) served as institutional-independent validation cohorts. Patients’ mortality and disease progression were assessed as clinical endpoints. Internal validation was performed by bootstrap analysis. Clinical benefit was estimated by decision curve analysis. Results miRNA-seq highlighted miR-25 of CD138+ plasma cells to be upregulated in MM vs. sMM, R-ISS II/III vs. R-ISS I, and in progressed compared to progression-free patients. The analysis of our screening cohort highlighted that CD138+ miR-25 levels were correlated with short-term progression (HR = 2.729; p = 0.009) and poor survival (HR = 4.581; p = 0.004) of the patients; which was confirmed by Kryukov et al . cohort (HR = 1.878; p = 0.005) and MMRF CoMMpass study (HR = 1.414; p = 0.039) validation cohorts. Moreover, multivariate miR-25-fitted models contributed to superior risk-stratification and clinical benefit in MM prognostication. Finally, elevated miR-25 circulating levels were correlated with poor survival of MM patients (HR = 5.435; p = 0.021), serving as a potent non-invasive molecular prognostic tool. Conclusions Our study identified miR-25 overexpression as a powerful independent predictor of poor treatment outcome and post-treatment progression, aiding towards modern non-invasive disease prognosis and personalized treatment decisions.

A bidirectional negative relationship between Hepatitis C virus (HCV) replication and gene expression of the catecholamine biosynthetic enzyme L-Dopa decarboxylase (DDC) was previously shown in the liver and attributed at least to an association of DDC with phosphatidylinositol 3-kinase (PI3K). Here, we report that the biosynthesis and uptake of catecholamines restrict HCV replication in hepatocytes, while HCV has developed ways to reduce catecholamine production. By employing gene silencing, chemical inhibition or induction of the catecholamine biosynthetic and metabolic enzymes and transporters, and by applying the substrates or the products of the respective enzymes, we unravel the role of the different steps of the pathway in viral infection. We also provide evidence that the effect of catecholamines on HCV is strongly related with oxidative stress that is generated by their autoxidation in the cytosol, while antioxidants or treatments that lower cytosolic catecholamine levels positively affect the virus. To counteract the effect of catecholamines, HCV, apart from the already reported effects on DDC, causes the down-regulation of tyrosine hydroxylase that encodes the rate-limiting enzyme of catecholamine biosynthesis and suppresses dopamine beta-hydroxylase mRNA and protein amounts, while increasing the catecholamine degradation enzyme monoamine oxidase. Moreover, the NS4B viral protein is implicated in the effect of HCV on the ratio of the ~50 kDa DDC monomer and a ~120 kDa DDC complex, while the NS5A protein has a negative effect on total DDC protein levels.

Colorectal cancer (CRC), one of the most prevalent types of cancer, requires the discovery of new tumor biomarkers for accurate patient prognosis. In this work, the prognostic value of the tRNA fragment i-tRF-GlyGCC in CRC was examined. Total RNA extraction from 211 CRC patient cancer tissue specimens and 83 adjacent normal tissues was conducted. Each RNA extract was subjected to in vitro polyadenylation and reverse transcription. A real-time quantitative PCR assay was used to quantify i-tRF-GlyGCC in all samples. Extensive biostatics analysis showed that i-tRF-GlyGCC levels in CRC tissues were significantly lower than in matched normal colorectal tissues. Additionally, the disease-free survival (DFS) and overall survival (OS) time intervals were considerably shorter in CRC patients with high i-tRF-GlyGCC expression. i-tRF-GlyGCC expression maintained its prognostic value independently of other established prognostic factors, as shown by the multivariate Cox regression analysis. Additionally, survival analysis after TNM stage stratification revealed that higher i-tRF-GlyGCC levels were linked to shorter DFS time intervals in patients with TNM stage II tumors, as well as an increased probability of having a worse OS for patients in TNM stage II. In conclusion, i-tRF-GlyGCC has the potential to be a useful molecular tissue biomarker in CRC, independent of other clinicopathological variables.

Previously, the association between the catecholamine biosynthetic enzyme L-Dopa decarboxylase (DDC) and Dengue virus (DV) replication was demonstrated in liver cells and was found to be mediated at least by the interaction between DDC and phosphoinositide 3-kinase (PI3K). Here, we show that biogenic amines production and uptake impede DV replication in hepatocytes and monocytes, while the virus reduces catecholamine biosynthesis, metabolism, and transport. To examine how catecholamine biosynthesis/metabolism influences DV, first, we verified the role of DDC by altering DDC expression. DDC silencing enhanced virus replication, but not translation, attenuated the negative effect of DDC substrates on the virus and reduced the infection related cell death. Then, the role of the downstream steps of the catecholamine biosynthesis/metabolism was analyzed by chemical inhibition of the respective enzymes, application of their substrates and/or their products; moreover, reserpine, the inhibitor of the vesicular monoamine transporter 2 (VMAT2), was used to examine the role of uptake/storage of catecholamines on DV. Apart from the role of each enzyme/transporter, these studies revealed that the dopamine uptake, and not the dopamine-signaling, is responsible for the negative effect on DV. Accordingly, all treatments expected to enhance the accumulation of catecholamines in the cell cytosol suppressed DV replication. This was verified by the use of chemical inducers of catecholamine biosynthesis. Last, the cellular redox alterations due to catecholamine oxidation were not related with the inhibition of DV replication. In turn, DV apart from its negative impact on DDC, inhibits tyrosine hydroxylase, dopamine beta-hydroxylase, monoamine oxidase, and VMAT2 expression.

The discovery of therapeutic miRNAs is one of the most exciting challenges for pharmaceutical companies. Since the first miRNA was discovered in 1993, our knowledge of miRNA biology has grown considerably. Many studies have demonstrated that miRNA expression is dysregulated in many diseases, making them appealing tools for novel therapeutic approaches. This review aims to discuss miRNA biogenesis and function, as well as highlight strategies for delivering miRNA agents, presenting viral, non-viral, and exosomic delivery as therapeutic approaches for different cancer types. We also consider the therapeutic role of microRNA-mediated drug repurposing in cancer therapy.

CircRNAs have become a novel scientific research hotspot, and an increasing number of studies have shed light on their involvement in malignant progression. Prompted by the apparent scientific gap in circRNAs from apoptosis-related genes, such as BOK, we focused on the identification of novel BOK circRNAs in human ovarian and prostate cancer cells. Total RNA was extracted from ovarian and prostate cancer cell lines and reversely transcribed using random hexamer primers. A series of PCR assays utilizing gene-specific divergent primers were carried out. Next, third-generation sequencing based on nanopore technology followed by extensive bioinformatics analysis led to the discovery of 23 novel circRNAs. These novel circRNAs consist of both exonic and intronic regions of the BOK gene. Interestingly, the exons that form the back-splice junction were truncated in most circRNAs, and multiple back-splice sites were found for each BOK exon. Moreover, several BOK circRNAs are predicted to sponge microRNAs with a key role in reproductive cancers, while the presence of putative open reading frames indicates their translational potential. Overall, this study suggests that distinct alternative splicing events lead to the production of novel BOK circRNAs, which could come into play in the molecular landscape and clinical investigation of ovarian and prostate cancer.

Circular RNAs (circRNAs), a novel RNA type generated by back‐splicing, are key regulators of gene expression, with deregulated expression and established involvement in leukemia. The products of BCL2 and its homologs, including BAX and BCL2L12, are implicated in chronic lymphocytic leukemia (CLL). However, to the best of our knowledge, nothing is known about circRNAs produced by these two genes and their role in CLL. We sought to further elucidate the contribution of BAX and BCL2L12 in CLL by unraveling the identity, localization, and potential role of their circRNAs. Therefore, total RNA from the EHEB cell line and peripheral blood mononuclear cells (PBMCs) of CLL patients and non‐leukemic blood donors was extracted and reverse‐transcribed using random hexamers. Next, nested PCRs with divergent primers were performed and the purified PCR products were subjected to 3rd generation nanopore sequencing. Nested PCRs were also applied to first‐strand cDNAs synthesized from total RNA extracts of PBMCs from CLL patients and non‐leukemic blood donors. Lastly, a single‐molecule resolution fluorescent in situ hybridization method called circFISH was used to visualize the circRNA distribution in EHEB cells. We discovered several novel circRNAs produced by BAX and BCL2L12, which were characterized by great exon structure diversity. In addition, intriguing findings regarding their formation emerged. Interestingly, visualization of the most abundant circRNAs showed distinct intracellular localization. Moreover, a complex BAX and BCL2L12 circRNA expression pattern was revealed in CLL patients and non‐leukemic blood donors. Our data suggest a multifaceted role of BAX and BCL2L12 circRNAs in B‐cell CLL. Novel circular RNAs (circRNAs) deriving from BAX and BCL2L12 were discovered using nanopore sequencing. These circRNAs exhibit diverse exon structures, distinct intracellular localization, and may interact with microRNAs with a proven role in chronic lymphocytic leukemia (CLL). A complex circRNA expression pattern in CLL patients and non‐leukemic blood donors was found as well. Our findings suggest a multifaceted role of these circRNAs in B‐cell CLL.

Circular RNAs (circRNAs) are associated with the pathobiology of multiple myeloma (MM). Recent findings regarding circCCT3 support its involvement in the development and progression of MM, through microRNA sponging. Thus, we aimed to examine the expression of circCCT3 in smoldering and symptomatic MM and to assess its clinical importance. Three cell lines from plasma cell neoplasms were cultured and bone marrow aspirate (BMA) samples were collected from 145 patients with MM or smoldering MM. Next, CD138+ enrichment was performed in BMA samples, followed by total RNA extraction and reverse transcription. Preamplification of circCCT3 and GAPDH cDNA was performed. Finally, a sensitive assay for the relative quantification of circCCT3 using nested real‐time quantitative polymerase chain reaction was developed, optimized, and implemented in the patients' samples and cell lines. MM patients exhibited significantly higher intracellular circCCT3 expression in their CD138+ plasma cells, compared to those from SMM patients. In addition, MM patients overexpressing circCCT3 had longer progression‐free and overall survival intervals. The favorable prognostic significance of high circCCT3 expression in MM was independent of disease stage (either International Staging System [ISS] or revised ISS [R‐ISS]) and age of MM patients. Interestingly, circCCT3 expression could serve as a surrogate molecular biomarker of prognosis in MM patients, especially those of R‐ISS stage II. In conclusion, our study sheds new light on the significance of circCCT3 as a promising molecular marker for predicting MM patients' prognosis.

Subtle alternative splicing leads to the formation of RNA variants lacking or including a small number of nucleotides. To date, the impact of subtle alternative splicing phenomena on protein biosynthesis has been studied in frame-preserving incidents. On the contrary, mRNA isoforms derived from frame-shifting events were poorly studied and generally characterized as non-coding. This work provides evidence for a frame-shifting subtle alternative splicing event which results in the production of a novel protein isoform. We applied a combined molecular approach for the cloning and expression analysis of a human RNase κ transcript (RNase κ-02) which lacks four consecutive bases compared to the previously isolated RNase κ isoform. RNase κ-02 mRNA is expressed in all human cell lines tested end encodes the synthesis of a 134-amino-acid protein by utilizing an alternative initiation codon. The expression of RNase κ-02 in the cytoplasm of human cells was verified by Western blot and immunofluorescence analysis using a specific polyclonal antibody developed on the basis of the amino-acid sequence difference between the two protein isoforms. The results presented here show that subtle changes during mRNA splicing can lead to the expression of significantly altered protein isoforms.