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PubMed2026

A contextual activity score (CAS) for inferring ADAR-associated transcriptional activity across RNA-seq, single-cell, and spatial transcriptomics.

BACKGROUND AND OBJECTIVE: Adenosine-to-inosine RNA editing, catalyzed by Adenosine Deaminases Acting on RNA (ADARs), is a widespread modification involved in neural function, immune regulation, and cancer. The Alu Editing Index (AEI) is the standard metric to estimate ADAR activity but requires raw sequencing reads and is poorly suited for single-cell and spatial transcriptomic data. This study aimed to develop an alternative framework for inferring ADAR-associated transcriptional activity from gene expression data across diverse transcriptomic technologies. METHODS: We developed the Contextual Activity Score (CAS), a framework based on transcriptional signatures from ADAR perturbation experiments. Context-specific signatures were generated for human neurons, mouse neurons, and cancer models to infer ADAR1 and ADAR2 activity. CAS was computed from normalized gene expression matrices using regulon-based enrichment analysis. Performance was evaluated by comparing with the Alu Editing Index across bulk RNA sequencing datasets, simulated sequencing depths, and library preparation protocols. RESULTS: CAS showed strong concordance with the Alu Editing Index across multiple datasets, while remaining robust to reduced sequencing depth and different library protocols. Unlike the Alu Editing Index, CAS can be applied to single-cell and spatial transcriptomic data and enables the independent assessment of ADAR2 activity. In cancer and neuronal contexts, CAS captured biologically meaningful variations in ADAR-associated transcriptional activity at sample, cell-type, and spatial levels. CONCLUSION: CAS provides a scalable approach applicable across multiple RNA-seq protocols for estimating ADAR-associated transcriptional activity using gene expression data. This method, implemented in an open-source R package for broad adoption, expands the ability to study ADAR-associated transcriptional activity across transcriptomic modalities where direct editing quantification is challenging, such as single-cell and spatial transcriptomics.

Adenosine Deaminase↗

PubMed2026

Emerging techniques of CRISPR/Cas system in antiviral therapy and diagnostics: Applications, limitations, and translational perspectives.

The CRISPR/Cas (clustered regularly interspaced short palindromic repeats) system is a versatile technology for developing antiviral medicines and editing viral genomes in both diagnostics and vaccine synthesis. Emerging insights into class 2 effectors, such as Cas9, Cas12, and Cas13, which target viral DNA and RNA, have revolutionized vaccines against viruses such as HIV, HPV, HBV, and EBV. Innovative diagnostic techniques such as SHERLOCK, DETECTR, and FELUDA have demonstrated system's diversity and accuracy in detecting the virus markers, supporting clinical decision-making, indicating adaptability and precision of CRISPR. This review critically evaluates CRISPR's role in RNA editing, emphasizing its importance for functional genomics and development of recombinant vaccines. Translational challenges are critically discussed, including off-target effects, delivery limitations, and ethical issues, for which unique approaches such as high-fidelity Cas variants, non-viral delivery systems, and bioethical frameworks are evaluated to address these limitations. This review also covers other social implications, such as accessibility and biosecurity risks, associated with CRISPR technologies Collectively, these advances underscore the transformative potential of CRISPR technologies in shaping next-generation antiviral diagnostics and therapeutics.

CRISPR-Cas Systems↗

PubMed2024

Proteome Dynamics in iPSC-Derived Human Dopaminergic Neurons.

Dopaminergic neurons participate in fundamental physiological processes and are the cell type primarily affected in Parkinson's disease. Their analysis is challenging due to the intricate nature of their function, involvement in diverse neurological processes, and heterogeneity and localization in deep brain regions. Consequently, most of the research on the protein dynamics of dopaminergic neurons has been performed in animal cells ex vivo. Here we use iPSC-derived human mid-brain-specific dopaminergic neurons to study general features of their proteome biology and provide datasets for protein turnover and dynamics, including a human axonal translatome. We cover the proteome to a depth of 9409 proteins and use dynamic SILAC to measure the half-life of more than 4300 proteins. We report uniform turnover rates of conserved cytosolic protein complexes such as the proteasome and map the variable rates of turnover of the respiratory chain complexes in these cells. We use differential dynamic SILAC labeling in combination with microfluidic devices to analyze local protein synthesis and transport between axons and soma. We report 105 potentially novel axonal markers and detect translocation of 269 proteins between axons and the soma in the time frame of our analysis (120 h). Importantly, we provide evidence for local synthesis of 154 proteins in the axon and their retrograde transport to the soma, among them several proteins involved in RNA editing such as ADAR1 and the RNA helicase DHX30, involved in the assembly of mitochondrial ribosomes. Our study provides a workflow and resource for the future applications of quantitative proteomics in iPSC-derived human neurons.

Humans↗

PubMed2026

Mitogenome assembly and phylogenetic relationships of Phalaris arundinacea.

INTRODUCTION: As a perennial herb of Poaceae, Phalaris arundinacea plays key roles in grazing, production, and soil and water conservation because of its well-developed rhizomes and seed dispersal. We assembled and annotated the first mitogenome of P. arundinacea to support evolutionary and taxonomic research. METHODS: We assembled and annotated the first complete mitochondrial genome of P. arundinacea by integrating Illumina short reads with Nanopore long reads via a hybrid assembly strategy. The genome architecture was comprehensively characterized, encompassing codon usage bias, repetitive sequence organization, and inter-organellar genetic exchange with the chloroplast genome. RESULTS AND DISCUSSION: Assembly of the P. arundinacea mitogenome revealed two circular structures with a combined length of 526,717 bp. The genome comprised a set of 37 protein-coding genes (PCGs), 27 tRNAs, and 8 rRNAs, with the rRNA genes exhibiting full assembly (100% coverage). The mitochondrial genome contained 154 forward and 164 palindromic repeats, along with 25 tandem repeats and 124 simple sequence repeats (SSRs). Notably, 102 SSRs were distributed on contig1, predominantly in tetrameric form. Furthermore, 376 RNA editing sites were predicted. A total of 104 fragments were integrated into the mitochondrial genome from the chloroplast, amounting to 55,866 bp of transferred sequence. Finally, phylogenetic analysis of 28 plant mitogenomes placed P. arundinacea closest to species within the genus Poa (P. chaixii and P. pratensis). Comparative analysis of non-synonymous-to-synonymous substitution rate (Ka/Ks) ratios across divergent species revealed that the mitochondrial genome of P. arundinacea underwent stabilizing evolutionary dynamics, characterized by predominant purifying selection with several lineage-specific variations in selective pressure. Our findings support the close phylogenetic relationship between P. arundinacea and species of the genus Poa and provide a reference mitochondrial genome resource for future comparative studies within Phalaris that incorporate broader taxon sampling. These results support deeper phylogenetic investigations of P. arundinacea and facilitate future work on its germplasm characterization and applied use.

Phalaris arundinacea↗

PubMed2026

Assembly and characterization of the first complete mitochondrial genome of Epimedium sagittatum (Sieb. et Zucc.) Maxim (Berberidaceae):an invaluable traditional Chinese medicine.

BACKGROUND: Epimedium sagittatum (Sieb. et Zucc.) Maxim is an invaluable traditional Chinese medicine plant known for its properties of tonifying kidney yang, strengthening bones and muscles, and dispelling rheumatism. The chloroplast (cp) genome of E. sagittatum have been sequenced, offering critical insights for breeding and phylogenetic research. However, the mitochondrial (mt) genome of E. sagittatum remains uncharacterized, limiting comprehensive insights into its genomic evolution. RESULTS: In this study, we assembled the first complete mt genome of E. sagittatum employing Illumina and Nanopore sequencing technology and subsequently investigated comparative analysis with its closely related species. The mt genome of E. sagittatum was assembled as a multi-branched structure with a length of 339,191 bp, within a GC content of 46.91%. Our annotation results have shown 39 protein-coding genes (PCGs), 22 tRNA genes, three rRNA genes and four pseudogenes in the E. sagittatum mt genome. The analysis of sequence repeats has detected 79 simple sequence repeats (SSRs), 10 tandem repeats and 255 dispersed repeats in the E. sagittatum mt genome. A total of 720 C to U RNA editing sites of the 34 PCGs was predicted in E. sagittatum. The codons exhibited a strong preference for A or U bases in the E. sagittatum mt genome. The analysis of nucleotide diversity (Pi) highlighted differences in genetic variability across the tested genes, with atp9 gene exhibiting the highest genetic variation. Selection pressure analysis showed that most genes were affected by negative selection during evolution, whereas ccmB, rps10, and rps12 underwent positive selection in different plants. Additionally, a Bayesian phylogenetic tree showed that E. sagittatum was closely related to E. wushanense and E. pubescens. In total of 14 homologous fragments totaling 8,954 bp were identified between the cp and mt genomes of E. sagittatum. CONCLUSIONS: This study presents the first assembled and annotated mt genome of E. sagittatum, which provides a valuable genetic resource for the Epimedium genus and lays the foundation for investigating the phylogenetic relationship and genetic variation of this invaluable medicinal plant.

Epimedium↗

PubMed2026

Exploring the Mitochondrial Genomes of Phoebe Species (Lauraceae): Structural Dynamics and Functional Conservation.

Plant mitochondrial genomes (mitogenomes) vary markedly in size and architecture despite generally slow rates of sequence evolution. Phoebe is an ecologically and economically valuable genus of Lauraceae, yet its mitogenome diversity remains poorly characterized. In this study, we newly sequenced, assembled, and annotated the mitogenomes of three nationally protected Class II wild plants (P. bournei, P. chekiangensis, P. zhennan) from China and compared their mitogenomic characteristics. The three assemblies were resolved into representative circular configurations ranging from 808 to 864 kb, with similar GC contents and conserved protein-coding capacity. Each mitogenome contained distinct 41 protein-coding genes, 27-28 transfer RNAs, and three ribosomal RNAs. Synteny analysis revealed extensive changes in homologous-block order and orientation despite substantial sequence homology among the three species. Abundant repeats occurred predominantly in noncoding regions, while plastid-derived fragments documented historical intracellular DNA transfer. The three species exhibited similar codon usage and predicted RNA-editing patterns, whereas low synonymous divergence limited inference from pairwise ratios. Phylogenetic analysis based on mitochondrial protein-coding genes recovered Phoebe as a well-supported monophyletic lineage. These results reveal substantial structural divergence accompanied by conserved nucleotide composition and coding capacity, providing valuable data for further understanding the evolutionary variation of plant mitogenomes of Phoebe and the Lauraceae.

Phoebe↗

PubMed2025

Identification of Potential Therapeutic Agents for Type I Interferonopathy Using iPSC-Based Disease Modeling.

PURPOSE: Type I interferonopathy encompasses disorders marked by systemic inflammation and neurological involvement, arising from genetic mutations that result in the upregulation of type I IFN signaling through various mechanisms. Currently, therapeutic options are limited, and no standard therapy exists. This study aims to develop a strategy for identifying new therapeutic targets for type I interferonopathy using induced pluripotent stem cells (iPSCs). METHODS: The IFIH1 R779H variant was introduced into iPSCs through genome editing. RNA sequencing of iPSC-derived dendritic cells (DCs) was performed, and differentially expressed genes (DEGs) were identified. IFN-α secretion, reactive oxygen species (ROS), and mitochondrial oxygen consumption rate (OCR) were analyzed in iPSC-derived DCs. An in silico prediction of compounds binding to the OAS-like domain was conducted. Candidate compounds were evaluated for their ability to inhibit IFN secretion from IFIH1 R779H-mutated iPSC-derived DCs. RESULTS: Transcriptome analysis indicated upregulation of the IFN-related and metabolic pathways. IFIH1 R779H-mutated iPSC-derived DCs exhibited increased OCR and ROS generation, and blocking mitochondrial metabolism significantly reduced excessive IFN-α secretion. Among the DEGs, PML was upregulated, and targeting this gene with arsenic trioxide (ATO), a PML antagonist, suppressed IFN-α secretion from IFIH1 R779H-mutated iPSC-derived DCs. Additionally, bisantrene, phthalylsulfathiazole and ganaplacide were predicted to bind to the RNA binding groove of OAS-like domain of human OASL in silico, effectively inhibiting IFN-α secretion from IFIH1 R779H-mutated DCs. CONCLUSION: Our iPSC-based disease modeling and drug investigation approach provides a robust platform for validating the efficacy and toxicity of candidate therapeutic agents for rare and intractable human diseases such as type I interferonopathy.

Humans↗

PubMed2026

Viral mimicry escape as a necessary feature of malignant transformation.

Malignant transformation is driven by disruption of pathways regulating proliferation and cell fate, but these same disruptions can create a collateral vulnerability: loss of transcriptional and epigenetic control over transposable elements and other normally silenced genomic regions. Consequently, emerging cancer cells can accumulate transposable element-derived and other endogenous immunogenic nucleic acids capable of triggering antiviral responses, a process termed viral mimicry. Increasing evidence indicates that viral mimicry can eliminate precancerous cells and shape tumour evolution, positioning it as an intrinsic tumour-suppressive mechanism. Here we highlight how cancer-associated changes in DNA methylation, histone modifications, splicing and RNA processing can lead to the presence of immunogenic nucleic acids that can activate viral mimicry pathways. We outline how cancer cells suppress viral mimicry, including compensatory epigenetic repression, RNA editing, nucleic acid decay and dampening of interferon signalling to enable cancer cell growth. Finally, we highlight the evidence suggesting that escaping viral mimicry is a fundamental process for cancer initiation and progression, and suggest that viral mimicry escape is necessary for cancer transformation and a therapeutic target in combination with immunotherapies. By framing viral mimicry escape as a necessary part of cancer transformation, this Review provides a unifying conceptual model for its translational exploitation.

Journal Article↗

PubMed2025

Characterization of Tumor Antigens from Multi-omics Data: Computational Approaches and Resources.

Tumor-specific antigens, also known as neoantigens, have potential utility in anti-cancer immunotherapy, including immune checkpoint blockade (ICB), neoantigen-specific T cell receptor-engineered T (TCR-T), chimeric antigen receptor T (CAR-T), and therapeutic cancer vaccines (TCVs). After recognizing presented neoantigens, the immune system becomes activated and triggers the death of tumor cells. Neoantigens may be derived from multiple origins, including somatic mutations (single nucleotide variants, insertions/deletions, and gene fusions), circular RNAs, alternative splicing, RNA editing, and polymorphic microbiomes. An increasing amount of bioinformatics tools and algorithms are being developed to predict tumor neoantigens derived from different sources, which may require inputs from different multi-omics data. In addition, calculating the peptide-major histocompatibility complex (MHC) affinity can aid in selecting putative neoantigens, as high binding affinities facilitate antigen presentation. Based on these approaches and previous experiments, many resources have been developed to reveal the landscape of tumor neoantigens across multiple cancer types. Herein, we summarize these tools, algorithms, and resources to provide an overview of computational analysis for neoantigen discovery and prioritization, as well as the future development of potential clinical utilities in this field.

Humans↗

PubMed2026

Assembly and comparative analysis of the mitochondrial genome of Pleione yunnanensis: genome structure and evolutionary insights.

BACKGROUND: Pleione yunnanensis a terrestrial or semi-epiphytic herbaceous plant belonging to the Orchidaceae family, is valued for both its medicinal uses and ornamental appeal. Although its chloroplast genomes have been sequenced, its complete mt genome had not previously been resolved, limiting genetic and evolutionary studies of the species. RESULTS: In this work, we assembled and characterized the first complete mt genome of P. yunnanensis, revealing a structurally complex, multibranched system composed of 14 circular-mapping molecules totaling 468,176 bp with a GC content of 44.32%. The genome encodes 44 annotated genes, including 28 protein-coding genes (PCGs), 15 tRNAs, and one rRNA. The multibranched architecture provides new evidence supporting the dynamic and recombinational nature of plant mt genomes. Repeat analysis uncovered 29 simple sequence repeats (SSRs), 19 tandem repeats, and 118 dispersed repeats, indicating a comparatively lower repeat abundance than that found in closely related orchids with similar mt genome sizes. Codon-usage profiling of PCGs showed a marked bias toward A/T-ending codons. Prediction of RNA editing sites identified 4,708 putative edits across mitochondrial PCGs. Most mitochondrial genes displayed Ka/Ks ratios close to 1.0, suggesting relaxed selective constraints or lineage-specific evolutionary patterns rather than strong positive selection. Moreover, we detected 69 chloroplast-derived homologous fragments, including 15 intact genes, suggesting ongoing plastid-mitochondrial DNA transfer. Phylogenetic reconstruction and collinearity comparisons demonstrated that P. yunnanensis clustered closely with Dendrobium species, including D. amplum and D. hancockii, within the Orchidaceae clade. CONCLUSIONS: This study provides the first complete mt genome of P. yunnanensis, providing a foundational genomic resource for the genus Pleione. The results not only improve our understanding of mt genome structure and evolution in Orchidaceae, but also offer valuable molecular evidence for phylogenetic inference, germplasm identification, and conservation of this endangered medicinal species.

Orchidaceae↗

PubMed2025

Enrichment of G-to-U Substitution in SARS-CoV-2 Functional Regions and Its Compensation via Concurrent Mutations.

We surveyed single nucleotide variant (SNV) patterns from 5 903 647 complete SARS-CoV-2 genomes. Among 10 012 SNVs, APOBEC-mediated C-to-U (C > U) deamination was the most prevalent, followed by G > U and other RNA editing-related substitutions including (A > G, U > C, G > A). However, C > U mutations were less frequent in functional regions, for example, S protein, intrinsic disordered regions, and nonsynonymous mutations, where G > U were over-represented. Notably, G-loss substitutions rarely appeared together. Instead, G-gain mutations tended to more frequently co-occur with others, with a marked preference in the S protein, suggesting a compensatory mechanism for G loss in G > U mutations. The temporal patterns revealed C > U frequency declined until late 2021 then resurged in early 2022. Conversely, G > U steadily decreased, with a pronounced drop in January 2022, coinciding with reduced COVID-19 severity. Vaccinated individuals exhibited a slightly but significantly higher C > U frequency and a notably lower G > U frequency compared to the unvaccinated group. Additionally, cancer patients had higher G > U frequency than general patients during the same period. Interestingly, none of the C > U SNVs were uniquely identified in 2724 environmental samples. These findings suggest novel functional roles of G > U in COVID-19 symptoms, potentially linked to oxidative stress and reactive oxygen species, while C > U remains the dominant substitution, likely driven by host immune-mediated RNA editing.

SARS-CoV-2↗

PubMed2026

Synergistic HMGN1 and VP64 Fusions Potentiate High-Precision and PAM-Flexible Base Editing.

RNA-guided CRISPR-derived base editors (BEs) have revolutionized genome editing by enabling targeted base substitutions. However, their application is frequently constrained by the stringent requirement for PAM sequences and low editing precision (bystander editing). Here, we present a robust strategy to overcome these limitations by coupling SpRY, a near-PAM-less Cas9 variant, with truncated CDA1 cytidine deaminases. While this combination enables precise editing of virtually any cytosine in the genome, it initially exhibited suboptimal efficiency. To address this, we systematically screened a diverse panel of candidate DNA-binding proteins and identified that the synergistic fusion of HMGN1 and VP64 substantially enhances editing activity without compromising precision. Importantly, this enhanced editing efficiency was achieved without markedly increasing off-target effects. Our new BEs demonstrated robust performance not only in yeast but also in rice, suggesting broad applicability in gene therapy, precision breeding, and fundamental research.

Gene Editing↗

PubMed2025

A six-repeat PPR protein WPR directly binds target RNAs and coordinates chloroplast RNA processing via dual recruitment of MORF1, MORF8b, and CAF2 proteins in rice.

Pentatricopeptide repeat (PPR) proteins are key regulators of organelle RNA metabolism in plants, yet their precise mechanisms in chloroplast RNA processing remain unclear. Here, we identify WPR, a unique P-type PPR protein in rice (Oryza sativa L.), as a critical factor in chloroplast RNA splicing and editing. A ~112-kb chromosomal inversion upstream of WPR causes an albino panicle rachis phenotype (wpr mutant), while complete loss of WPR function leads to seedling lethality. WPR deficiency disrupts the splicing of multiple group II introns (atpF, ndhA, ndhB, petB, rpl2, and rps12) and impairs RNA editing in transcripts such as ndhA, ndhB, ndhG, rps14, and ycf3. Electrophoretic mobility shift assay (EMSA) data confirm that WPR directly binds to precursor mRNAs of atpF, ndhA, petB, rpl2, and rps12. Strikingly, WPR interacts with both RNA editing factors (MORF1, MORF8b) and the splicing factor CAF2, but not with other PPR proteins targeting the same transcripts. Unlike most PPR proteins, WPR contains only six PPR repeats, which is the fewest among all functionally characterized rice PPR proteins. With few informative repeats, WPR likely possesses a broad, low-specificity RNA-binding activity. Moreover, WPR may act on chloroplast RNA maturation by recruiting MORFs and CAF2 rather than other PPR proteins, highlighting a novel regulatory mode in which P-type PPR protein may act as an RNA-binding scaffold to integrate diverse RNA-processing machineries. This study advances the understanding of PPR protein diversity and provides new insights into the molecular mechanisms of chloroplast RNA processing in rice.

Oryza↗

PubMed2025

A probe-based capture enrichment method for detection of A-to-I editing in low abundance transcripts.

Exactly two decades ago, the ability to use high-throughput RNA sequencing technology to identify sites of editing by ADARs was employed for the first time. Since that time, RNA sequencing has become a standard tool for researchers studying RNA biology and led to the discovery of RNA editing sites present in a multitude of organisms, across tissue types, and in disease. However, transcriptome-wide sequencing is not without limitations. Most notably, RNA sequencing depth of a given transcript is correlated with expression, and sequencing depth impacts the ability to robustly detect RNA editing events. This chapter focuses on a method for enrichment of low-abundance transcripts that can facilitate more efficient sequencing and detection of RNA editing events. An important note is that while we describe aspects of the protocol important for capturing intron-containing transcripts, this probe-based enrichment method could be easily modified to assess editing within any low-abundance transcript. We also provide some perspectives on the current limitations as well as important future directions for expanding this technology to gain more insights into how RNA editing can impact transcript diversity.

RNA Editing↗

PubMed2025

Using Prime Editing Guide Generator (PEGG) for high-throughput generation of prime editing sensor libraries.

Prime editing enables the generation of nearly any small genetic variant. However, the process of prime editing guide RNA (pegRNA) design is challenging and requires automated computational design tools. We developed Prime Editing Guide Generator (PEGG), a fast, flexible, and user-friendly Python package that enables the rapid generation of pegRNA and pegRNA-sensor libraries. Here, we describe the installation and use of PEGG (https://pegg.readthedocs.io) to rapidly generate custom pegRNA-sensor libraries for use in high-throughput prime editing screens.

Gene Editing↗

PubMed2026

dsRNAscan maps human dsRNAome, revealing conservation, intermolecular dsRNA, and correlates of ADAR dependency.

The human transcriptome contains millions of A-to-I editing sites arising from an unclear number of poorly characterized dsRNAs. Editing sites reveal the presence of dsRNA, but this method is limited by transcription levels, read depth, and ADAR expression and cannot identify unedited dsRNA. To address these limitations, we developed dsRNAscan. Applying dsRNAscan to the human genome predicted 5 million dsRNAs, mostly in repetitive and intergenic regions. Machine learning models trained on A-to-I editing and RNA structure-probing data identified ∼2.4 million high-confidence predictions, which were enriched at dsRNA-binding protein binding sites. Additionally, we predicted hundreds of dsRNAs conserved across vertebrates and observed thousands of editing-enriched regions suspected to arise from intermolecular dsRNAs formed with sense-antisense transcripts. Quantifying expression of intramolecular and intermolecular dsRNAs accessible to cytoplasmic immune sensors revealed that their ratio correlated with ADAR dependency across cancer cell lines. The human dsRNAome is available as a resource at https://dsrna.chpc.utah.edu/.

A-to-I RNA editing↗

PubMed2026

DNA-guided CRISPR-Cas12 for cellular RNA targeting.

Here, we present ΨDNA, a DNA-based guide that enables RNA targeting by Cas12 nucleases, overcoming the traditional reliance on RNA-guided systems. We engineer ΨDNA to mimic a CRISPR RNA (crRNA) scaffold in reverse orientation, allowing AsCas12a and Cas12i1 to recognize RNA and trigger strong single-stranded DNA trans-cleavage for sensitive detection of diverse RNA species, including 100% accurate hepatitis C virus RNA detection in clinical samples. ΨDNA also achieves 70-95% multiplex knockdown of endogenous intracellular RNA transcripts through ribosome stalling across multiple human cell lines. Mechanistic studies reveal that activity depends on a stem loop that stabilizes a catalytically competent Cas12-ΨDNA-RNA complex. Lastly, codelivery of crRNA and ΨDNA enables simultaneous DNA editing and RNA knockdown with a single effector and modular fusions of different enzymes to AsCas12a extend ΨDNA to RNase H-mediated RNA degradation and METTL3-based epitranscriptomic editing. Together, ΨDNA guides constitute an adaptable toolkit that extends Cas12 systems beyond genome editing and diagnostics to enable precise, programmable control of cellular transcriptomes and their epitranscriptomic marks.

Journal Article↗

PubMed2026

LDLR Variant Classification Through Activity-Normalized Prime Editing Screening.

BACKGROUND: Inherited variants in the LDL (low-density lipoprotein) receptor (LDLR) gene are the most common cause of familial hypercholesterolemia, significantly increasing coronary artery disease risk. Early identification of pathogenic LDLR variants enables prompt lipid-lowering therapy and cascade testing of at-risk relatives; however, most LDLR variants observed in the population have uncertain or absent clinical classifications, leaving many patients without actionable information. METHODS: We developed the first activity-normalized prime editing screening pipeline to measure the impact of 5184 LDLR coding variants on LDL-cholesterol (LDL-C) uptake. Each prime editing guide RNA is paired with a genotypic outcome reporter to correct for variable editing efficiency, overcoming a key limitation of previous pooled genome editing screens. A statistical framework further improves variant effect estimates by jointly analyzing all missense variants at each amino acid position. RESULTS: We show that prime editing of the reporter construct correlates with endogenous variant installation frequency, validating the activity normalization approach. The resulting scores capture a continuous spectrum of functional effects, robustly separate pathogenic versus benign ClinVar variants, and show concordance with LDL-C levels in UK Biobank participants. We calibrate functional evidence strengths to the ACMG/AMP variant interpretation framework, enabling integration into a clinical variant classification workflow. By combining functional, computational, population, and contextual evidence, 322 of 434 LDLR variants currently classified as variants of uncertain significance, conflicting, or absent from ClinVar appear to meet evidence thresholds for reclassification and can be prioritized for expert review, substantially expanding the pool of actionable variant classifications. The screen also reveals a cluster of gain-of-function variants in LDLR class A repeat 5, at least some of which enhance LDL-C uptake through increased apolipoprotein B interaction, with implications for therapeutic genome editing. Last, prime editing uniquely detects splice-altering coding variants missed by cDNA-based screens and pathogenicity predictors, revealing an advantage of endogenous variant installation. CONCLUSIONS: Altogether, activity-normalized prime editing provides a scalable framework for LDLR variant classification that substantially expands the proportion of variants with evidence for genetic diagnosis and reveals novel biology with therapeutic relevance.

CRISPR screening↗