🧬 PubMed RNA Editing Daily Digest

Base editors, composed of engineered deaminases fused with Cas proteins and a guide RNA, enable precise, programmable alteration of single nucleotides within the genome and transcriptome. This innovative technology holds promising therapeutic potential for correcting disease-causing point mutations. However, its clinical translation hinges on both high efficacy and accuracy. Non-specific unintended edits by base editors remain a critical challenge. Efforts to mitigate off-target activity have focused mostly on detecting recurrent RNA deaminations at specific sites. Complementarily, our methodology quantifies the total burden of RNA alterations, which is particularly effective for capturing stochastic off-target edits that evade conventional detection. Here, we applied the RNA editing index algorithm to quantify off-target levels across individual genes and identified 2,844 adenine base editors and 1,253 cytosine base editor hotspot genes susceptible to aberrant editing. Exon-level analysis revealed localized regions within genes that are particularly prone to off-target editing, including regions where edits introduce premature stop codons, a critical risk for therapeutic applications. By uncovering these previously unrecognized off-target landscapes, our study deepens our understanding of base editor specificity and provides a framework for optimizing their precision, accelerating the development of safer next-generation editing tools.

As systematic comparisons of editing efficiency and specificity seldom keep pace with rapid developments in RNA-guided nucleases (RGNs), the current study examined 50 such editing systems and characterized the off-target effects and genomic structural impacts of a subset of high-efficiency RGNs. Among them, AsCas12a-Ultra, LbCpf1, and AsCas12a-Plus demonstrated similar or higher efficiency compared to SpCas9, while the relatively high efficiency and small size of enOsCas12f1 together support its suitability for in vivo delivery. AsCpf1-YH and FnCpf1 exhibited the lowest single-guide RNA-dependent (sgRNA-dependent) off-target risks, whereas DpFNuc showed the highest. Genomic structural analysis revealed that enCas12f-HKRA frequently introduces chromosomal translocations, while Cas12j-SF05 poses a lower risk of such mutations. Notably, the high-efficiency RGNs were associated with translocation hotspots. Additionally, enRhCas12f1 and SpaCas12f1 had the lowest cytotoxicity, while enAsCpf1-HF strongly inhibited cell proliferation. This study establishes the first multidimensional performance evaluation framework for RGNs, providing a data-driven tool to support precise genome editing.

Osteosarcoma is a highly malignant bone tumor which primarily affects the juvenile population and is characterized by high rate of recurrence and metastasis. RNA editing has emerged as a key process in cancer progression. Herein, we investigated the role of RNA editing enzyme ADAR2 (Adenosine Deaminase Acting on RNA 2) in osteosarcoma. We demonstrated that ADAR2 expression increases during osteoblast differentiation and inversely correlates with the aggressiveness of osteosarcoma cells. Interestingly, the overexpression of ADAR2 in osteosarcoma cell lines reduces their tumoral properties and promotes their differentiation in osteoblast-like cells, as shown by gene expression analysis and mineralization assays. These results were also confirmed by in vivo experiments; indeed, intratibial injection of ADAR2-overexpressing osteosarcoma cells in NSG mice resulted in less aggressive tumors compared to mice injected with pEmpty or pInactive ADAR2 E/A vector-transfected cells. To elucidate the mechanisms by which ADAR2 overexpression induces osteogenic terminal differentiation of osteosarcoma cells, we performed RNA-seq analysis of Saos-2 cells and identified IGFBP7 (Insulin-like Growth Factor Binding Protein 7) as the most highly edited transcript in ADAR2-overexpressing cells. We showed that the editing activity of ADAR2 on IGFBP7 abolishes its proliferative effect on osteosarcoma cells and triggers terminal differentiation. Overall, our results indicate that ADAR2 acts as a tumor suppressor in osteosarcoma and may represent a novel therapeutic target for this aggressive pediatric tumor.

Nucleic acid detection methods leveraging Cas9, Cas12, and Cas13 enzymes have recently been widely integrated with isothermal amplification techniques, particularly Loop-Mediated Isothermal Amplification (LAMP), to develop CRISPR-based diagnostic assays for a broad range of pathogens. Coupling these systems with portable result-readout platforms such as lateral flow devices, microfluidics, and smartphones offers a promising pathway for deploying LAMP-CRISPR diagnostics at the point-of-care (PoC), especially in settings where conventional, resource-intensive methods like real-time PCR are not feasible. However, the development of LAMP-CRISPR assays presents unique challenges not typically encountered in real-time PCR workflows. These include the need for a larger number of oligonucleotides, the complexity of integrating multiple biochemical conditions, and a heightened risk of false-positive results. Despite the growing number of bioinformatics tools designed to aid assay development, establishing a robust and reproducible workflow for LAMP-CRISPR remains a significant hurdle. In this review, we critically examine current strategies for designing LAMP-CRISPR assays and offer a detailed, step-by-step guide to achieving high-performance diagnostic tools using this approach. We cover key aspects of target sequence selection, oligonucleotide and CRISPR system design, and the strategic choice of readout methods. We further discuss available tools for LAMP primer and CRISPR guide RNA design, providing practical recommendations for optimizing sequence selection. Various probe formats for Cas-mediated trans-cleavage detection are summarized, and we present best practices for assay standardization and minimizing false-positive signals. Finally, we highlight the current limitations and outline future directions for LAMP-CRISPR diagnostics in decentralized and PoC testing environments.

Toxoplasma gondii is a globally prevalent protozoan parasite capable of establishing lifelong infections in its host. While acute infection is often asymptomatic, reactivation of latent bradyzoites can cause severe disease, particularly in immunocompromised individuals. Current therapies are ineffective against chronic infection, underscoring critical gaps in our understanding of bradyzoite biology and the molecular mechanisms governing stage conversion. Recent studies have identified translational control as a central regulator of T. gondii differentiation. This review highlights the roles of canonical translation initiation factors (eIF2α, eIF1.2, and eIF4E1), RNA-binding proteins (RBPs; BFD2/ROCY1, Alba1, and Alba2), and RNA modifications (with pseudouridylation representing the best-characterized modification currently linked to differentiation), as well as alternative splicing and non-coding RNAs in shaping stage-specific translational programs. This review further discusses underexplored mechanisms, including non-canonical initiation pathways, upstream open reading frames, transcript-level RNA modifications, ribosome heterogeneity and rRNA modifications, elongation and termination control, uncharacterized RBPs, and post-translational modifications of translation factors, that may coordinate proteome remodeling during differentiation. Together, established translational regulators and these emerging pathways highlight translational control as a central driver of parasite persistence and a promising therapeutic target for chronic toxoplasmosis.