In recent years, the interplay between N6-methyladenosine (m6A) modifications and non-coding RNAs (ncRNAs) has emerged as a pivotal research area, owing to their crucial involvement in the pathophysiological mechanisms underlying various diseases. A significant hurdle in cancer therapy is therapeutic resistance, which frequently contributes to adverse patient outcomes. Recent investigations have underscored the vital role that interactions between m6A modifications and ncRNAs play in mediating cancer therapeutic resistance via the MAPK, PI3K/Akt/mTOR, Wnt/β-catenin, HIPPO, and NF-κB pathways. This review elucidates how these interactions drive tumor therapeutic resistance by modulating these pathways. By dissecting the regulatory dynamics between m6A and ncRNAs in the context of cancer therapeutic resistance, this review aims to deepen the understanding of m6A-ncRNA interaction in cancer therapeutic resistance and identify potential therapeutic targets to improve cancer treatment efficacy.

Spatial RNA organization has a pivotal role in diverse cellular processes and diseases1-4. However, functional implications of the spatial transcriptome remain largely unexplored due to limited technologies for perturbing endogenous RNA within specific subcellular regions1,5. Here we present CRISPR-mediated transcriptome organization (CRISPR-TO), a system that harnesses RNA-guided, nuclease-dead dCas13 for programmable control of endogenous RNA localization in live cells. CRISPR-TO enables targeted localization of endogenous RNAs to diverse subcellular compartments, including the outer mitochondrial membrane, p-bodies, stress granules, telomeres and nuclear stress bodies, across various cell types. It allows for inducible and reversible bidirectional RNA transport along microtubules via motor proteins, facilitating real-time manipulation and monitoring of RNA localization dynamics in living cells. In primary cortical neurons, we demonstrate that repositioned mRNAs undergo local translation along neurites and at neurite tips, and co-transport with ribosomes, with β-actin mRNA localization enhancing the formation of dynamic filopodial protrusions and inhibiting axonal regeneration. CRISPR-TO-enabled screening in primary neurons identifies Stmn2 mRNA localization as a driver of neurite outgrowth. By enabling large-scale perturbation of the spatial transcriptome, CRISPR-TO bridges a critical gap left by sequencing and imaging technologies, offering a versatile platform for high-throughput functional interrogation of RNA localization in living cells and organisms.

Gene therapy stands at the forefront of medical innovation, offering unique potential to treat the underlying causes of genetic disorders and broadly enable regenerative medicine. However, unregulated production of therapeutic genes can lead to decreased clinical utility due to various complications. Thus, many technologies for controlled gene expression are under development, including regulated transgenes, modulation of endogenous genes to leverage native biological regulation, mapping and repurposing of transcriptional regulatory networks, and engineered systems that dynamically react to cell state changes. Transformative therapies enabled by advances in tissue-specific promoters, inducible systems, and targeted delivery have already entered clinical testing and demonstrated significantly improved specificity and efficacy. This review highlights next-generation technologies under development to expand the reach of gene therapies by enabling precise modulation of gene expression. These technologies, including epigenome editing, antisense oligonucleotides, RNA editing, transcription factor-mediated reprogramming, and synthetic genetic circuits, have the potential to provide powerful control over cellular functions. Despite these remarkable achievements, challenges remain in optimizing delivery, minimizing off-target effects, and addressing regulatory hurdles. However, the ongoing integration of biological insights with engineering innovations promises to expand the potential for gene therapy, offering hope for treating not only rare genetic disorders but also complex multifactorial diseases.

Many existing methods for post-transcriptional RNA modification rely on a single-step approach, limiting the ability to reversibly control m6A methylation at specific sites. Here, we address this challenge by developing a multi-step system that builds on the concept of sequential RNA bioorthogonal chemistry. Our strategy uses an azide-based reagent (NAI-N3) capable of both cleavage and ligation reactions, thereby allowing iterative and reversible modifications of RNA in living cells. By applying this approach in CRISPR (clustered regularly interspaced short palindromic repeats)-based frameworks, we demonstrate tailored editing of m6A marks at targeted RNA sites, overcoming the one-way restriction of conventional bioorthogonal methods. This sequential protocol not only broadens the scope for fine-tuned RNA regulation but also provides a versatile platform for exploring dynamic m6A function in genetic and epigenetic research.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) technologies have evolved rapidly over the past decade with the continuous discovery of new Cas systems. In particular, RNA-targeting CRISPR-Cas13 proteins are promising single-effector systems to regulate target mRNAs without altering genomic DNA, yet the current Cas13 systems are restrained by suboptimal efficiencies. Here, we show that U1 promoter-driven CRISPR RNAs (crRNAs) increase the efficiency of various applications, including RNA knockdown and editing, without modifying the Cas13 protein effector. We confirm that U1-driven crRNAs are exported into the cytoplasm, while conventional U6 promoter-driven crRNAs are mostly confined to the nucleus. Furthermore, we reveal that the end positions of crRNAs expressed by the U1 promoter are consistent regardless of guide sequences and lengths. We also demonstrate that U1-driven crRNAs, but not U6-driven crRNAs, can efficiently repress the translation of target genes in combination with catalytically inactive Cas13 proteins. Finally, we show that U1-driven crRNAs can counteract the inhibitory effect of miRNAs. Our simple and effective engineering enables unprecedented cytosolic RNA-targeting applications.

Plant hormones are essential signalling molecules that control and coordinate diverse physiological processes in plant development and adaptation to ever-fluctuating environments. This hormonal regulation of plant development and environmental responses has recently been shown to extensively involve the most widespread RNA modification, N6-methyladenosine (m6A). Here we discuss the current understanding of the crosstalk between m6A and plant hormones, focusing on their reciprocal regulation, where hormonal signals induce m6A reprogramming and m6A affects hormone biosynthesis and signalling cascades. We also highlight new insights into how m6A contributes to the hormonal control of plant development and stress responses. Furthermore, we discuss future prospects for unveiling the regulatory networks that orchestrate epitranscriptome-hormone interactions and harnessing the related knowledge accrued to enhance crop productivity and resilience in changing environments.

N6-methyladenosine (m6A) is the most prevalent internal modification on messenger RNA. Although recent studies have shown m6A effects on determining the fate of mRNA through modulating various aspects of plant mRNA metabolism, whether and how m6A affects gene transcription in plants remains elusive. Here we show that NEEDED FOR RDR2-INDEPENDENT DNA METHYLATION (NERD), a plant-specific protein, is an essential component of the m6A methyltransferase complex required for regulating the transcription of a central floral repressor FLOWERING LOCUS C (FLC) in Arabidopsis. NERD interacts with and stabilizes the two core methyltransferases, mRNA adenosine methylases A and B, to promote m6A modification of nascent RNA, conferring an overall negative effect on gene transcription. At the FLC locus, NERD-mediated m6A modification on the nascent transcript negatively affects H3K36me3 deposition and FLC transcription through NERD interaction with the H3K36me3 methyltransferase SET DOMAIN GROUP 8. Collectively, our findings reveal that NERD mediates the crosstalk between epitranscriptomic and epigenetic regulation of FLC to modulate flowering in Arabidopsis.

The RNA N6-methyladenosine (m6A) modification is a critical regulator of various biological processes, but precise and dynamic control of m6A remains a challenge. In this work, we present a red/far-red light-inducible m6A editing system that enables efficient and reversible modulation of m6A levels with minimal off-target effects. By engineering the CRISPR dCas13 protein and sgRNA with two pairs of light-inducible heterodimerizing proteins, ΔphyA/FHY1 and Bphp1/PspR2, we achieved targeted recruitment of m6A effectors. This system significantly enhances m6A writing efficiency and allows dynamic regulation of m6A deposition and removal on specific transcripts, such as SOX2 and ACTB. Notably, reversible m6A editing was achieved through cyclic modulation at a single target site, demonstrating the ability to influence mRNA expression and modulate the differentiation state of human embryonic stem cells. This optogenetic platform offers a precise, versatile tool for cyclic and reversible m6A regulation, with broad implications for understanding RNA biology and its potential applications in research and medicine.

Ribonucleic acid (RNA) plays a pivotal role in regulating biological processes within living systems, with modified nucleosides serving as critical modulators of various aspects of biological functions. Therefore, the development of efficient methodologies for late-stage, site-selective RNA modification is of considerable interest, as it facilitates the functional exploration of RNA chemical modifications and their implications for therapeutic applications. Precise RNA modification holds significant promise for the treatment of genetic diseases by enabling the correction of mutated nucleobases to their wild-type forms. Additionally, the site-selective incorporation of synthetic labeling groups into RNA provides invaluable tools for structural and functional studies, thereby uncovering previously hidden dimensions of RNA's role in biological systems. In this review, we provide a comprehensive overview of three principal approaches to site-selective, late-stage RNA modifications: enzyme-mediated strategies, catalytic nucleic acid-based techniques, and chemical methodologies. These approaches predominantly target the nucleobase or the 2'-hydroxyl (2'-OH) group of RNA nucleosides. We evaluate the advantages and limitations of each strategy and discuss future directions for advancing this field of research.

The significance of m6A modifications in several biological processes has been increasingly recognized, particularly in the context of cancer. For instance, m6A modifications in gastric cancer (GC) have been significantly implicated in tumor progression, metastasis, and treatment resistance. GC is characterized by the differential expression of m6A regulators. High expression writers such as METTL3 and WTAP are associated with poor prognosis and aggressive clinical features. Conversely, low expression of METTL14 is linked to worse clinical outcomes, whereas elevated levels of demethylases, such as FTO and ALKBH5, correlate with better survival rates. These m6A regulators influence several cellular biological functions, including proliferation, invasion, migration, glycolysis, and chemotherapy resistance, thereby affecting tumor growth and therapeutic outcomes. The assessment of m6A modification patterns and the expression profiles of m6A-related genes hold substantial potential for improving the clinical diagnosis and treatment of GC. In this review, we provide an updated and comprehensive summary of the role of m6A modifications in GC, emphasizing their molecular mechanisms, clinical significance, and translational applications in developing novel diagnostic and therapeutic strategies.

The chemical modifications of DNA and proteins are powerful mechanisms for regulating molecular and biological functions, influencing a wide array of signaling pathways in eukaryotes. Recent advancements in epitranscriptomics have shown that RNA modifications play crucial roles in diverse biological processes. Since their discovery in the 1970s, scientists have sought to decipher, identify, and elucidate the functions of these modifications across biological systems. Over the past decade, mounting evidence has demonstrated the importance of RNA modification pathways in plants, prompting significant efforts to decipher their physiological relevance. With the advent of high-resolution mapping techniques for RNA modifications and the gradual uncovering of their biological roles, our understanding of this additional layer of regulation is beginning to take shape. In this review, we summarize recent findings on the major RNA modifications identified in plants, with an emphasis on N6-methyladenosine (m6A), the most extensively studied modification. We discuss the functional significance of the effector components involved in m6A modification and its diverse roles in plant biotic interactions, including plant-virus, plant-bacterium, plant-fungus, and plant-insect relationships. Furthermore, we highlight new technological developments driving research progress in this field and outline key challenges that remain to be addressed.

Epitranscriptomic chemical modifications of RNAs have emerged as potent regulatory mechanisms in the process of plant stress adaptation. Currently, over 170 distinct chemical modifications have been identified in mRNAs, tRNAs, rRNAs, microRNAs (miRNAs), and long noncoding RNAs (lncRNAs). Genetic and molecular studies have identified the genes responsible for addition and removal of chemical modifications from RNA molecules, which are known as "writers" and "erasers," respectively. N6-methyladenosine (m6A) is the most prevalent chemical modification identified in eukaryotic mRNAs. Recent studies have identified m6A writers and erasers across different plant species, including Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), cotton (Gossypium hirsutum), and tomato (Solanum lycopersicum). Accumulating discoveries have improved our understanding of the functions of RNA modifications in plant stress responses. This review highlights the latest research on RNA modification, emphasizing the biological and cellular roles of diverse chemical modifications of mRNAs, tRNAs, rRNAs, miRNAs, and lncRNAs in plant responses to environmental and hormonal signals. We also propose and discuss critical questions and future challenges for enhancing our understanding of the cellular and mechanistic roles of RNA modifications in plant stress responses. Integrating molecular insights into the regulatory roles of RNA modifications in stress responses with novel genome- and RNA-editing technologies will facilitate the breeding of stress-tolerant crops through precise engineering of RNA modifications.

The challenges faced by today's agronomists, plant breeders, and their managers encompass adapting sustainably to climate variability while working with limited budgets. Besides, managers are dealing with a multitude of issues with different organizations working on similar initiatives and projects, leading to a lack of a sustainable impact on smallholder farmers. To transform the current food systems as a more sustainable and resilient model efficient solutions are needed to deliver and convey results. Challenges such as logistics, labour, infrastructure, and equity, must be addressed alongside adapting to increasingly unstable climate conditions which affect the life cycle of transboundary pathogens and pests. In this context, transforming food systems go far beyond just farmers and plant breeders and it requires substantial contributions from industry, global finances, transportation, energy, education, and country developmental sectors including legislators. As a result, a holistic approach is essential for achieving sustainable and resilient food systems to sustain a global population anticipated to reach 9.7 billion by 2050 and 11.2 billion by 2100. As of 2021, nearly 193 million individuals were affected by food insecurity, 40 million more than in 2020. Meanwhile, the digital world is rapidly advancing with the digital economy estimated at about 20% of the global gross domestic product, suggesting that digital technologies are increasingly accessible even in areas affected by food insecurity. Leveraging these technologies can facilitate the development of climate-smart cultivars that adapt effectively to climate variation, meet consumer preferences, and address human and livestock nutritional needs. Most economically important traits in crops are controlled by multiple loci often with recessive alleles. Considering particularly Africa, this continent has several agro-climatic zones, hence crops need to be adapted to these. Therefore, targeting specific loci using modern tools offers a precise and efficient approach. This review article aims to address how these new technologies can provide a better support to smallholder farmers.

The orchestration of dynamic epigenetic and epitranscriptomic modifications is pivotal for the fine-tuning of gene expression. However, these modifications are traditionally examined independently. Recent compelling studies have disclosed an interesting communication and interplay between m6A RNA methylation (m6A epitranscriptome) and epigenetic modifications, enabling the formation of feedback circuits and cooperative networks. Intriguingly, the interaction between m6A and DNA methylation machinery, coupled with the crosstalk between m6A RNA and histone modifications shape the transcriptional profile and translational efficiency. Moreover, m6A modifications interact also with non-coding RNAs, modulating their stability, abundance, and regulatory functions. In the light of these findings, m6A imprinting acts as a versatile checkpoint, linking epigenetic and epitranscriptomic layers toward a multilayer and time-dependent control of gene expression and cellular homeostasis. The scope of the present review is to decipher the m6A-coordinated circuits with DNA imprinting, chromatin architecture, and non-coding RNAs networks in normal physiology and carcinogenesis. Ultimately, we summarize the development of innovative CRISPR-dCas engineering platforms fused with m6A catalytic components (m6A writers or erasers) to achieve transcript-specific editing of m6A epitranscriptomes that can create new insights in modern RNA therapeutics.

Clustered regularly interspaced short palindromic repeats-Cas13 effectors are used for RNA editing but the adeno-associated virus (AAV) packaging limitations because of their big sizes hinder their therapeutic application. Here we report the identification of the Cas13j family, with LepCas13j (529 aa) and ChiCas13j (424 aa) being the smallest and most highly efficient variants for RNA interference. The miniaturized Cas13j proteins enable the development of compact RNA base editors. Chi-RESCUE-S, by fusing dChiCas13j with hADAR2dd, demonstrates high efficiency and specificity in A-to-G and C-to-U conversions. Importantly, this system is compatible with single-AAV packaging without the need for protein sequence truncation. It successfully corrected pathogenic mutations, such as APOC3D65N and SCN9AR896Q, to the wild-type forms. In addition, we developed an optimized system, Chi-RESCUE-S-mini3, which pioneered efficient in vivo C-to-U RNA editing of PCSK9 in mice through single-AAV delivery, resulting in reduced total cholesterol levels. These results highlight the potential of Cas13j to treat human diseases.

Post-transcriptional chemical modifications to RNA, or the epitranscriptome, play important roles in RNA metabolism, gene regulation, and human disease, including viral pathogenesis. Modifications to the RNA viral genome and transcripts of human immunodeficiency virus 1 (HIV-1) have been reported, including methylation of adenosine (m6A) and cytosine (m5C), acetylation of cytosine, pseudouridylation (psi), and conversion of adenosine to inosine, and their effects on virus and host biology have been investigated. However, diverse experimental approaches have been used, making clear correlations across studies difficult to assess. To address this need, we propose the establishment of a reference HIV-1 epitranscriptome. We sequenced the model NL4-3 HIV-1 genome from infected Jurkat CD4+ T cells cells using the latest nanopore chemistry, custom RNA preparation methods, and commercial base-calling algorithms. This resulted in a reproducible sense and preliminary antisense HIV-1 epitranscriptome where m6A, m5C, psi, ands inosine could be identified by multiplexed base-calling. Multiplexed base-calling miscalled modifications due to sequence and neighboring modification contexts, which we demonstrate can be corrected with synthetic HIV-1 RNA fragments. We validate m6A modification sites with a small molecule inhibitor of methyltransferase-like 3 (METTL3), STM2457. We conclude that modifications do not change substantially under combination antiretroviral therapy (cART) treatment or in primary CD4+ T cells. Samples from patients living with HIV reveal conservation of certain modifications, such as m6A. Our approach and reference data offer a straightforward benchmark that can be adopted to help advance rigor, reproducibility, and uniformity across future HIV-1 epitranscriptomics studies.

Recent advances in gene editing and precise regulation of gene expression based on CRISPR technologies have provided powerful tools for the understanding and manipulation of gene functions. Fusing RNA aptamers to the sgRNA of CRISPR can recruit cognate RNA-binding protein (RBP) effectors to target genomic sites, and the expression of sgRNA containing different RNA aptamers permit simultaneous multiplexed and multifunctional gene regulations. Here, we report an intracellular directed evolution platform for RNA aptamers against intracellularly expressed RBPs. We optimize a bacterial CRISPR-hybrid system coupled with FACS, and identified high affinity RNA aptamers orthogonal to existing aptamer-RBP pairs. Application of orthogonal aptamer-RBP pairs in multiplexed CRISPR allows effective simultaneous transcriptional activation and repression of endogenous genes in mammalian cells.

Objective: Despite N6-methyladenosine (m6A) being closely involved in various pathophysiological processes, its potential role in liver injury is largely unknown. We designed the current research to study the potential role of fat mass and obesity-associated protein (FTO), an m6A demethylase, on hepatic ischemia-reperfusion injury (IRI). Methods: Wild-type mice injected with an adeno-associated virus carrying fat mass and obesity-associated protein (AAV-FTO) or adeno-associated virus carrying green fluorescent protein (GFP) (AAV-GFP) were subjected to a hepatic IRI model in vivo. Hematoxylin-eosin staining was performed to observe IRI. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to observe the cell apoptosis. Reverse transcription polymerase chain reaction (RT-PCR) was used to observe the expression of FTO. The protein levels of FTO, apoptosis, or autophagy-associated signaling proteins were detected by western blot. Reactive oxygen species (ROS) levels were determined by flow cytometry, and immunohistochemistry was used to detect the FTO and LC3-II expression. For in vitro experiments, cultured hepatocytes were subjected to hypoxia/reoxygenation (H/R) stimulation. Monodansylcadaverine (MDC) staining was used to visualize autophagic vesicles. Results: In the present study, we showed that FTO was involved in hepatic IRI, apoptosis, and autophagy. Specifically, the expression level of FTO was significantly reduced in the hepatic IRI. Besides, increasing FTO expression (AAV-FTO) ameliorated the hepatic IRI in animal models, accompanied by decreased apoptosis and autophagy. Furthermore, the FTO inhibitor (FB23-2) aggravated autophagy in hepatocytes upon H/R-induced damage. Conclusion: FTO could act as a protective effector during hepatic IRI, associated with decreased apoptosis and autophagy. FTO-mediated m6A demethylation modification may be an important therapeutic target for hepatic IRI.

Epitranscriptomic regulation has emerged as a crucial layer of gene control where RNA modifications, particularly N6-methyladenosine (m6A), introduce complexity and versatility to gene regulation. Increasing evidence suggests that epitranscriptomic regulation through phase separation plays critical roles in mediating RNA metabolism during plant development and stress responses. m6A-associated biomolecular condensates formed via phase separation act as dynamic cellular hotspots where m6A effectors, RNAs, and other regulatory proteins coalesce to facilitate RNA regulation. Moreover, m6A modulates condensate assembly. Herein, I summarize the current understanding of how m6A- and m6A effector-mediated formation of biomolecular condensates mediates plant development and stress adaptation. I also discuss several working models for m6A-associated biomolecular condensates and highlight the prospects for future research on epitranscriptomic regulation through phase separation.

Recent advancements in tools using programmable RNA binding proteins and m6A-erasers enable sequence-selective and timing-controllable m6A demethylation. However, off-target effects are still a concern. This study addresses the problem by reducing the RNA-binding ability of m6A-erasers. The modulated m6A-erasers achieved sequence-specific and timing-controllable m6A demethylation with minimal off-target activity.

Significant advances in the development of new cancer therapies have given rise to multiple novel therapeutic options in chemotherapy, radiotherapy, immunotherapy, and targeted therapies. Although the development of resistance is often reported along with temporary disease remission, there is often tumor recurrence of an even more aggressive nature. Resistance to currently available anticancer drugs results in poor overall and disease-free survival rates for cancer patients. There are multiple mechanisms through which tumor cells develop resistance to therapeutic agents. To date, efforts to overcome resistance have only achieved limited success. Epitranscriptomics, especially related to m6A RNA modification dysregulation in cancer, is an emerging mechanism for cancer therapy resistance. Here, recent studies regarding the contributions of m6A modification and its regulatory proteins to the development of resistance to different cancer therapies are comprehensively reviewed. The promise and potential limitations of targeting these entities to overcome resistance to various anticancer therapies are also discussed.

Abscisic acid (ABA)-based chemically induced proximity (CIP) is primarily mediated by the interaction of the ABA receptor pyrabactin resistance 1-like 1 (PYL1) and the 2C-type protein phosphatase ABI1, which confers ABA-induced proximity to their fusion proteins, and offers precise temporal control of a wide array of biological processes. However, broad application of ABA-based CIP has been limited by ABA response intensity. In this study, we demonstrated that ABA-induced interaction between another ABA receptor pyrabactin resistance 1 (PYR1) and ABI1 exhibited higher ABA response intensity than that between PYL1 and ABI1 in HEK293T cells. We engineered PYR1-ABI1 and PYL1-ABI1 into ABA-induced transcriptional activation tools in mammalian cells by integration with CRISPR/dCas9 and found that the tool based on PYR1-ABI1 demonstrated better ABA response intensity than that based on PYL1-ABI1 for both exogenous and endogenous genes in mammalian cells. We further achieved ABA-induced RNA m6A modification installation and erasure by combining ABA-induced PYR1-ABI1 interaction with CRISPR/dCas13, successfully inhibiting tumor cell proliferation. We subsequently improved the interaction of PYR1-ABI1 through phage-assisted continuous evolution (PACE), successfully generating a PYR1 mutant (PYR1m) whose interaction with ABI1 exhibited a higher ABA response intensity than that of the wild-type. In addition, we tested the transcriptional activation tool based on PYRm-ABI1 and found that it also showed a higher ABA response intensity than that of the wild type. These results demonstrate that we have developed a novel ABA-based CIP and further improved upon it using PACE, providing a new approach for the modification of other CIP systems.

Current research on long non-coding RNA (lncRNA) has predominantly focused on identifying their protein partners and genomic binding sites, leaving their RNA partners largely unknown. To address this gap, the study has developed a method called sarID (sgRNA scaffold assisted RNA-RNA interaction detection), which integrates Cas13-based RNA targeting, sgRNA engineering, and proximity RNA editing to investigate lncRNA-RNA interactomes. By applying sarID to the lncRNA NEAT1, over one thousand previously unidentified binding transcripts are discovered. sarID is further expanded to investigate binders of XIST, MALAT1, NBR2, and DANCR, demonstrating its broad applicability in identifying lncRNA-RNA interactions. The findings suggest that lncRNAs may regulate gene expression by interacting with mRNAs, expanding their roles beyond known functions as protein scaffolds, miRNA sponges, or guides for epigenetic modulators. sarID has the potential to be adapted for studying other specific RNAs, providing a novel immunoprecipitation-free method for uncovering RNA partners and facilitating the exploration of the RNA-RNA interactome.

The intricate world of RNA modifications, collectively termed the epitranscriptome, covers over 170 identified modifications and impacts RNA metabolism and, consequently, almost all biological processes. In this review, we focus on the regulatory roles and biological functions of a panel of dominant RNA modifications (including m 6A, m 5C, Ψ, ac 4C, m 1A, and m 7G) on three RNA types-mRNA, tRNA, and rRNA-in mammalian development, particularly in the context of reproduction as well as embryonic development. We discuss in detail how those modifications, along with their regulatory proteins, affect RNA processing, structure, localization, stability, and translation efficiency. We also highlight the associations among dysfunctions in RNA modification-related proteins, abnormal modification deposition and various diseases, emphasizing the roles of RNA modifications in critical developmental processes such as stem cell self-renewal and cell fate transition. Elucidating the molecular mechanisms by which RNA modifications influence diverse developmental processes holds promise for developing innovative strategies to manage developmental disorders. Finally, we outline several unexplored areas in the field of RNA modification that warrant further investigation.

Manipulating RNA species in mammalian cells has emerged as an important strategy for precise gene expression control. Here we review recent advances in precision transcriptome editing with a focus on tools that engineer specific transcripts for abundance, translation, base editing, alternative isoforms, and chemical modifications. While some of these methods have demonstrated efficiency in therapeutically relevant cellular or in vivo models, most require further study on their clinical safety and efficacy. Precision transcriptome engineering holds great potential for both mechanistic study of RNA biology and future gene and cell-based therapeutic applications.

RNA modification is the critical mechanism for regulating post-transcriptional processes. There are more than 150 RNA modifications reported so far, among which N6-Methyladenosine is the most prevalent one. M6A RNA modification complex consists of 'writers', 'readers' and 'erasers' which together in a group catalyze, recognize and regulate the methylation process of RNA and thereby regulate the stability and translation of mRNA. The discovery of erasers also known as demethylases, revolutionized the research on RNA modifications as it revealed that this modification is reversible. Since then, various studies have focused on discovering the role of m6A modification in various diseases especially cancers. Aberrant expression of these 'readers', 'writers', and 'erasers' is found to be altered in various cancers resulting in disturbance of cellular homeostasis. Acute leukemias are the most common cancer found in pediatric patients and account for 20% of adult cases. Dysregulation of the RNA modifying complex have been reported in development and progression of hematopoietic malignancies. Further, targeting m6A modification is the new approach for cancer immunotherapy and is being explored extensively. This review provides detailed information about current information on the role of m6A RNA modification in acute leukemia and their therapeutic potential.

N 6-Methyladenine (m6A) RNA methylation plays a key role in RNA processing and translational regulation, influencing both normal physiological and pathological processes. Yet, current techniques for studying RNA methylation struggle to isolate the effects of individual m6A modifications. Engineering of RNA methyltransferases (RNA MTases) could enable development of improved synthetic biology tools to manipulate RNA methylation, but it is challenging due to limited understanding of structure-function relationships in RNA MTases. Herein, using ancestral sequence reconstruction, we explore the sequence space of the bacterial DNA methyltransferase EcoGII (M.EcoGII), a promising target for protein engineering due to its lack of sequence specificity and its residual activity on RNA. We thereby created an efficient non-specific RNA MTase termed SUPer RNA EcoGII Methyltransferase (SUPREM), which exhibits 8-fold higher expression levels, 7°C higher thermostability and 12-fold greater m6A RNA methylation activity compared with M.EcoGII. Immunofluorescent staining and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis confirmed SUPREM's higher RNA methylation activity compared with M.EcoGII in mammalian cells. Additionally, Nanopore direct RNA sequencing highlighted that SUPREM is capable of methylating a larger number of RNA methylation sites than M.EcoGII. Through phylogenetic and mutational analysis, we identified a critical residue for the enhanced RNA methylation activity of SUPREM. Collectively, our findings indicate that SUPREM holds promise as a versatile tool for in vivo RNA methylation and labeling.

Mitochondria have a complete and independent genetic system with necessary biological energy for cancer occurrence and persistence. Mitochondrial RNA (mt-RNA) methylation, as a frontier in epigenetics, has linked to cancer progression with growing evidences. This review has comprehensively summarized detailed mechanisms of mt-RNA methylation in regulating cancer proliferation, metastasis, and immune infiltration from the mt-RNA methylation sites, biological significance, and its methyltransferases. The mt-RNA methylation also plays a very significant role via epigenetic crosstalk between nucleus and mitochondria. Importantly, the unique structures and functional characteristics of mt-RNA methyltransferases and the potential targeting treatment drugs for cancer are also analyzed. Revealing human mt-RNA methylation regulatory system and the relationship with cancer will contribute to identifying potential biomarkers and therapeutic targets for precise prevention, detection, intervention and treatment in the future.

Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV), a novel bunyavirus primarily transmitted by Haemaphysalis longicornis, induces severe disease with a high mortality rate. N6-methyladenosine (m6A) is a prevalent internal chemical modification in eukaryotic mRNA that has been reported to regulate viral infection. However, the role of m6A modification during SFTSV infection remains elusive. We here reported that SFTSV RNAs bear m6A modification during infection. Manipulating the expressions or activities of host m6A regulators significantly impacted SFTSV infection. Mechanistically, SFTSV recruited m6A regulators through the nucleoprotein to modulate the m6A modification of viral RNA, eventually resulting in enhanced infection by promoting viral mRNA translation efficiency and/or genome RNA stability. m6A mutations in the S genome diminished virus particle production, while m6A mutations in the G transcript impaired the replication of recombinant vesicular stomatitis virus (rVSV) expressing G protein in vitro and in vivo. Interestingly, m6A modification was evolutionarily conserved and facilitated SFTSV infection in primary tick cells. These findings may open an avenue for the development of m6A-targeted anti-SFTSV vaccines, drugs, and innovative strategies for the prevention and control of tick-borne disease.

The development of precise RNA-editing tools is essential for the advancement of RNA therapeutics. CRISPR (clustered regularly interspaced short palindromic repeats) PspCas13b is a programmable RNA nuclease predicted to offer superior specificity because of its 30-nucleotide spacer sequence. However, its design principles and its on-target, off-target and collateral activities remain poorly characterized. Here, we present single-base tiled screening and computational analyses that identify key design principles for potent and highly selective RNA recognition and cleavage in human cells. We show that the de novo design of spacers containing guanosine bases at precise positions can greatly enhance the catalytic activity of inefficient CRISPR RNAs (crRNAs). These validated design principles (integrated into an online tool, https://cas13target.azurewebsites.net/ ) can predict highly effective crRNAs with ~90% accuracy. Furthermore, the comprehensive spacer-target mutagenesis revealed that PspCas13b can tolerate only up to four mismatches and requires ~26-nucleotide base pairing with the target to activate its nuclease domains, highlighting its superior specificity compared to other RNA or DNA interference tools. On the basis of this targeting resolution, we predict an extremely low probability of PspCas13b having off-target effects on other cellular transcripts. Proteomic analysis validated this prediction and showed that, unlike other Cas13 orthologs, PspCas13b exhibits potent on-target activity and lacks collateral effects.

In Huntington's disease (HD), aberrant processing of huntingtin (HTT) mRNA produces HTT1a transcripts that encode the pathogenic HTT exon 1 protein. The mechanisms behind HTT1a production are not fully understood. Considering the role of m6A in RNA processing and splicing, we investigated its involvement in HTT1a generation. Here, we show that m6A methylation is increased before the cryptic poly(A) sites (IpA1 and IpA2) within the huntingtin RNA in the striatum of Hdh+/Q111 mice and human HD samples. We further assessed m6A's role in mutant Htt mRNA processing by pharmacological inhibition and knockdown of METTL3, as well as targeted demethylation of Htt intron 1 using a dCas13-ALKBH5 system in HD mouse cells. Our data reveal that Htt1a transcript levels are regulated by both METTL3 and the methylation status of Htt intron 1. They also show that m6A methylation in intron 1 depends on expanded CAG repeats. Our findings highlight a potential role for m6A in aberrant splicing of Htt mRNA.

Alternative splicing allows multiple transcripts to be generated from the same gene to diversify the protein repertoire and gain new functions despite a limited coding genome. It can impact a wide spectrum of biological processes, including disease. However, its significance has long been underestimated due to limitations in dissecting the precise role of each splicing isoform in a physiological context. Furthermore, identifying key regulatory elements to correct deleterious splicing isoforms has proven equally challenging, increasing the difficulty of tackling the role of alternative splicing in cell biology. In this work, we take advantage of dCasRx, a catalytically inactive RNA targeting CRISPR-dCas13 ortholog, to efficiently switch alternative splicing patterns of endogenous transcripts without affecting overall gene expression levels cost-effectively. Additionally, we demonstrate a new application for the dCasRx splice-editing system to identify key regulatory RNA elements of specific splicing events. With this approach, we are expanding the RNA toolkit to better understand the regulatory mechanisms underlying alternative splicing and its physiological impact in various biological processes, including pathological conditions.

Inactive Cas13 orthologs have been fused to a mutant human ADAR2 deaminase domain at the C terminus to enable programmable adenosine-to-inosine (A-to-I) RNA editing in selected transcripts. Although promising, existing RNA-editing tools generally suffer from a trade-off between efficacy and specificity, and off-target editing remains an unsolved problem. Here we describe the development of an optimized RNA-editing platform by rational protein engineering, CasRx-based Programmable Editing of RNA Technology (xPERT). We demonstrate that the topological rearrangement of a CasRx K940L mutant by circular permutation results in a robust scaffold for the tethering of a deaminase domain. We benchmark our tool against the REPAIR system and show that xPERT exhibits strong on-target activity like REPAIRv1 but low off-target editing like REPAIRv2. Our xPERT platform can be used to alter RNA sequence information without risking genome damage, effect temporary cellular changes and customize protein function.

N6-methyladenosine (m6A) is the most prevalent internal modification of mRNA and plays an important role in regulating plant growth. However, there is still a lack of effective tools to precisely modify m6A sites of individual transcripts in plants. Here, programmable m6A editing tools are developed by combining CRISPR/dCas13(Rx) with the methyltransferase GhMTA (Targeted RNA Methylation Editor, TME) or the demethyltransferase GhALKBH10 (Targeted RNA Demethylation Editor, TDE). These editors enable efficient deposition or removal of m6A modifications at targeted sites of endo-transcripts GhECA1 and GhDi19 within a broad editing window ranging from 0 to 46 nt. TDE editor significantly decreases m6A levels by 24%-76%, while the TME editor increases m6A enrichment, ranging from 1.37- to 2.51-fold. Furthermore, installation and removal of m6A modifications play opposing roles in regulating GhECA1 and GhDi19 mRNA transcripts, which may be attributed to the fact that their m6A sites are located in different regions of the genes. Most importantly, targeting the GhDi19 transcript with TME editor plants results in a significant increase in root length and enhanced drought resistance. Collectively, these m6A editors can be applied to study the function of specific m6A modifications and have the potential for future applications in crop improvement.

The application of rapidly growing CRISPR toolboxes and methods has great potential to transform biomedical research. Here, we provide a snapshot of up-to-date CRISPR toolboxes, then critically discuss the promises and hurdles associated with CRISPR-based nuclear genome editing, epigenome editing, and mitochondrial editing. The technical challenges and key solutions to realize epigenome editing in vivo, in vivo base editing and prime editing, mitochondrial editing in complex tissues and animals, and CRISPR-associated transposases and integrases in targeted genomic integration of very large DNA payloads are discussed. Lastly, we discuss the latest situation of the CRISPR/Cas9 clinical trials and provide perspectives on CRISPR-based gene therapy. Apart from technical shortcomings, ethical and societal considerations for CRISPR applications in human therapeutics and research are extensively highlighted.

Among over 170 different types of chemical modifications on RNA nucleobases identified so far, RNA methylation is the major type of epitranscriptomic modifications existing on almost all types of RNAs, and has been demonstrated to participate in the entire process of RNA metabolism, including transcription, pre-mRNA alternative splicing and maturation, mRNA nucleus export, mRNA degradation and stabilization, mRNA translation. Attributing to the development of high-throughput detection technologies and the identification of both dynamic regulators and recognition proteins, mechanisms of RNA methylation modification in regulating the normal development of the organism as well as various disease occurrence and developmental abnormalities upon RNA methylation dysregulation have become increasingly clear. Here, we particularly focus on three types of RNA methylations: N6-methylcytosine (m6A), 5-methylcytosine (m5C), and N7-methyladenosine (m7G). We summarize the elements related to their dynamic installment and removal, specific binding proteins, and the development of high-throughput detection technologies. Then, for a comprehensive understanding of their biological significance, we also overview the latest knowledge on the underlying mechanisms and key roles of these three mRNA methylation modifications in gametogenesis, embryonic development, immune system development, as well as disease and tumor progression.

N6-methyladenosine (m6A) plays a crucial regulatory role in the control of cellular functions and gene expression. Recent advances in sequencing techniques for transcriptome-wide m6A mapping have accelerated the accumulation of m6A site information at a single-nucleotide level, providing more high-confidence training data to develop computational approaches for m6A site prediction. However, it is still a major challenge to precisely predict m6A sites using in silico approaches. To advance the computational support for m6A site identification, here, we curated 13 up-to-date benchmark datasets from nine different species (i.e., H. sapiens, M. musculus, Rat, S. cerevisiae, Zebrafish, A. thaliana, Pig, Rhesus, and Chimpanzee). This will assist the research community in conducting an unbiased evaluation of alternative approaches and support future research on m6A modification. We revisited 52 computational approaches published since 2015 for m6A site identification, including 30 traditional machine learning-based, 14 deep learning-based, and 8 ensemble learning-based methods. We comprehensively reviewed these computational approaches in terms of their training datasets, calculated features, computational methodologies, performance evaluation strategy, and webserver/software usability. Using these benchmark datasets, we benchmarked nine predictors with available online websites or stand-alone software and assessed their prediction performance. We found that deep learning and traditional machine learning approaches generally outperformed scoring function-based approaches. In summary, the curated benchmark dataset repository and the systematic assessment in this study serve to inform the design and implementation of state-of-the-art computational approaches for m6A identification and facilitate more rigorous comparisons of new methods in the future.

The CRISPR-Cas13 system has emerged as a revolutionary tool for RNA editing, offering new opportunities for the development of nucleic acid therapeutics. Unlike DNA-targeting CRISPR-Cas9, Cas13 targets and cleaves RNA, enabling gene silencing and preventing genomic instability. Its applications include suppressing disease-causing genes, correcting splicing errors, and modulating immune responses. Despite these advances, challenges persist, such as the need to refine specificity, mitigate off-target impacts, and ensure effective delivery. This review provides an overview of the CRISPR-Cas13 mechanism, elucidating its role in RNA-targeted therapies and its transformative potential for disease treatment. Furthermore, it addresses the ongoing challenges that the scientific community is striving to overcome.