Although next-generation sequencing (NGS) technology revolutionized sequencing, offering a tremendous sequencing capacity with groundbreaking depth and accuracy, it continues to demonstrate serious limitations. In the early 2010s, the introduction of a novel set of sequencing methodologies, presented by two platforms, Pacific Biosciences (PacBio) and Oxford Nanopore Sequencing (ONT), gave birth to third-generation sequencing (TGS). The innovative long-read technologies turn genome sequencing into an ease-of-handle procedure by greatly reducing the average time of library construction workflows and simplifying the process of de novo genome assembly due to the generation of long reads. Long sequencing reads produced by both TGS methodologies have already facilitated the decipherment of transcriptional profiling since they enable the identification of full-length transcripts without the need for assembly or the use of sophisticated bioinformatics tools. Long-read technologies have also provided new insights into the field of epitranscriptomics, by allowing the direct detection of RNA modifications on native RNA molecules. This review highlights the advantageous features of the newly introduced TGS technologies, discusses their limitations and provides an in-depth comparison regarding their scientific background and available protocols as well as their potential utility in research and clinical applications.

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.

RNA-targeting techniques have emerged as powerful tools in cancer research and therapeutics, offering precise and programmable control over gene expression at the post-transcriptional level. Once viewed as passive intermediates in the central dogma, RNA molecules are now recognized as dynamic regulators of cellular function, capable of influencing transcription, translation, and epigenetic regulation. Advances in high-throughput sequencing technologies, transcriptomics, and structural RNA biology have uncovered a diverse landscape of coding and non-coding RNAs involved in oncogenesis, drug resistance, and tumor progression. In response, several RNA-targeting strategies have been developed to modulate these transcripts, including antisense oligonucleotides (ASOs), RNA interference (RNAi), CRISPR-Cas13 systems, small molecules, and aptamers. This review provides a comparative analysis of these technologies, highlighting their molecular mechanisms, therapeutic potential, and current limitations. Emphasis is placed on the translational progress of RNA-targeting agents, including recent FDA approvals and ongoing clinical trials for cancer indications. Through a critical comparison of these strategies, this review underscores the growing significance of RNA-targeting technologies as a foundation for next-generation cancer therapeutics and precision oncology.

Non-coding RNAs (ncRNAs) are critical regulators of gene expression, taking part in the modulation of multiple biological functions across a range of cell types. Initially dismissed as transcriptional noise, ncRNAs are now recognized for their significant roles in key cellular mechanisms, including differentiation, apoptosis, and proliferation, as well as their profound implications for the pathogenesis of numerous human diseases. Due to their remarkable stability, tissue-specific expression patterns, and abundance in body fluids, ncRNAs hold significant promise as non-invasive biomarkers for diagnosis, prognosis, and therapeutic monitoring. Furthermore, advances in RNA-targeted therapeutics have introduced novel strategies to modulate ncRNA activity, although challenges related to delivery efficiency, specificity, and clinical validation remain. This review comprehensively summarizes the classification, biogenesis, and molecular functions of ncRNAs, elucidates their involvement in health and disease, and evaluates their potential as clinical biomarkers and therapeutic targets. Additionally, it discusses the emerging technologies for RNA manipulation, including CRISPR-based RNA editing, that can advance ncRNA research and revolutionize ncRNA-based therapeutics.

Colorectal cancer (CRC) is a highly heterogenous malignancy with an increased mortality rate. Aberrant splicing is a typical characteristic of CRC, and several studies support the prognostic value of particular transcripts in this malignancy. l-DOPA decarboxylase (DDC) and its derivative neurotransmitters play a multifaceted role in physiological and pathological states. Our recent data support the existence of 6 DDC novel exons. In this study, we investigated the existence of additional DDC novel exons and transcripts, and their potential value as biomarkers in CRC. Next-generation sequencing (NGS) in 55 human cell lines coupled with Sanger sequencing uncovered 3 additional DDC novel exons and 20 splice variants, 7 of which likely encode new protein isoforms. Eight of these transcripts were detected in CRC. An in-house qPCR assay was developed and performed in TNM II and III CRC samples for the quantification of transcripts bearing novel exons. Extensive biostatistical analysis uncovered the prognostic value of specific DDC novel exons for patients’ disease-free and overall survival. The revised DDC exon structure, the putative protein isoforms with distinct functions, and the prognostic value of novel exons highlight the pivotal role of DDC in CRC progression, indicating its potential utility as a molecular biomarker in CRC.

The human E74-like ETS transcription factor 3 (Elf-3) is an epithelium-specific member of the ETS family, all members of which are characterized by a highly conserved DNA-binding domain. Elf-3 plays a crucial role in epithelial cell differentiation by participating in morphogenesis and terminal differentiation of the murine small intestinal epithelium, and also acts as an indispensable regulator of mesenchymal to epithelial transition, underlying its significant involvement in development and in pathological states, such as cancer. Although previous research works have deciphered the functional role of Elf-3 in normal physiology as well as in tumorigenesis, the present study highlights for the first time the wide spectrum of ELF3 mRNAs that are transcribed, providing an in-depth analysis of splicing events and exon/intron boundaries in a broad panel of human cell lines. The implementation of a versatile targeted nanopore sequencing approach led to the identification of 25 novel ELF3 mRNA transcript variants (ELF3 v.3–v.27) with new alternative splicing events, as well as two novel exons. Although the current study provides a qualitative transcriptional profile regarding ELF3, further studies must be conducted, so the biological function of all novel alternative transcript variants as well as the putative protein isoforms are elucidated.