🎙️ Episode 27: Nucleocytosolic Vehicles — A CRISPR RNP Delivery Breakthrough

🧬 In this episode of Base by Base, we delve into a novel virus-like particle (VLP) platform, ENVLPE, developed by Geilenkeuser et al. (2025) in Cell. This innovation harnesses nucleocytosolic shuttling and RNA aptamer recruitment to efficiently package and deliver fully assembled CRISPR ribonucleoproteins (RNPs), overcoming spatial and loading limitations of previous VLP approaches.

🔍 Main Highlights:
The ENVLPE system integrates a PP7 coat protein into the HIV-1 Gag polyprotein and appends PP7-tagged guide RNAs to selectively load base editors, prime editors, nucleases, and trans-activators as RNPs rather than mRNA or protein fusions.
Active nuclear export and localization signals shuttle editor RNPs into the cytosol, synchronizing RNP assembly with VLP budding and greatly enhancing packaging efficiency.
Co-expression of the RNA endonuclease Csy4, which remains bound to a 3′ hairpin on pegRNAs, protects guide RNAs from degradation and substantially boosts prime editing performance under low-dose conditions.
A streamlined “miniENVLPE” variant retaining only essential Gag elements and coiled-coil oligomerization domains matches full-length editing efficiency, demonstrating the platform’s modularity and scalability.
In vivo subretinal delivery of ENVLPE in mouse models achieves robust editor uptake by retinal cells and functional gene correction in inherited eye disease—with minimal off-target activity.

🧠 Conclusion:
ENVLPE represents a versatile, high-efficiency vehicle for RNP delivery of CRISPR effectors in both ex vivo and in vivo settings. By combining RNA-mediated recruitment, active transport, and modular VLP engineering, this platform opens new avenues for precise therapeutic genome editing.

📖 Reference:
Geilenkeuser, J., Armbrust, N., Steinmaßl, E., et al. (2025). Engineered nucleocytosolic vehicles for loading of programmable editors. Cell, 188, 2637–2655. https://doi.org/10.1016/j.cell.2025.03.015

📜 License:
This episode is based on an open-access article published under the Creative Commons Attribution 4.0 International (CC BY 4.0) license – https://creativecommons.org/licenses/by/4.0/

🎙️ Episode 26: Reannotation in Focus — Uncovering Functional Non-Coding Mutations in Melanoma

🧬 In this episode of Base por Base, we delve into a breakthrough study by Pepe et al. (2025) in The American Journal of Human Genetics that challenges conventional cancer mutation annotation. The authors present an automated pipeline combining RNA-seq–based transcript quantification and Ensembl VEP reannotation to map somatic variants to the transcripts actually expressed in melanoma tumors. By integrating TCGA and COSMIC datasets, deep-learning predictions, and functional assays, this framework exposes a hidden layer of non-coding promoter mutations driving melanoma pathogenesis.

🔍 Study Highlights:
The reannotation approach revealed that many mutation clusters previously labeled as synonymous or missense events in cancer databases instead reside in promoter regions of expressed genes and adjacent loci, with 22% of 52 hotspots in melanoma belonging to this non-coding category. Functional validation using CRISPR-Cas9–edited melanocyte models and luciferase reporter assays demonstrated that IRF3/BCL2L12 promoter variants downregulate IRF3, BCL2L12, and downstream TP53 signaling, and disrupt ETS and SP/E2F transcription factor binding motifs as predicted by the DeepMEL2 model and PhysBinder/FABIAN-variant analyses. Systematic reannotation of KNSTRN and SLC27A5 clusters further confirmed non-coding promoter activity alterations, underscoring the prevalence and significance of regulatory mutations overlooked by reference-transcript annotation.

🧠 Conclusion:
By anchoring mutation annotation to expressed transcripts, the BayesMRnet reannotation pipeline (Salmon + VEP) ushers in a more precise era of cancer genomics, uncovering functional non-coding drivers in melanoma and offering a scalable tool to refine driver mutation discovery and guide future therapeutic strategies.

📖 Reference:
Pepe, D., Janssens, X., Timcheva, K., Marrón-Liñares, G. M., Verbelen, B., Konstantakos, V., … De Keersmaecker, K. (2025). Reannotation of cancer mutations based on expressed RNA transcripts reveals functional non-coding mutations in melanoma. The American Journal of Human Genetics, 112, 1–21. https://doi.org/10.1016/j.ajhg.2025.04.005

📜 License:
This episode is based on an open-access article published under the Creative Commons Attribution 4.0 International (CC BY 4.0) license – http://creativecommons.org/licenses/by/4.0/

🎙️ Episode 25: Uncovering Mitochondrial DNA Diseases — The Solve-RD Experience with Genotype and Phenotype Integration

🧬 In this episode of Base por Base, we delve into a groundbreaking study by Ratnaike et al. (2025) published in The American Journal of Human Genetics, which presents a semi-automated workflow combining mtDNA variant filtering and MitoPhen-based HPO phenotype similarity scoring applied to exome and genome data from the Solve-RD cohort of undiagnosed rare disease cases .

🔍 Study highlights:
Our workflow applied MToolBox for mtDNA reconstruction and MITOMAP annotations with stringent quality filters (≥50% mtDNA assembly and ≥5× coverage) to prioritize 136 rare variants across 9,923 individuals from 9,483 families without prior suspicion of mitochondrial disease .
Phenotype similarity scoring using the curated MitoPhen database achieved 100% sensitivity at a 0.3 threshold and distinguished confirmed mtDNA disease cases from nuclear genetic diagnoses with an AUC of 0.82 .
A total of 21 confirmed and 16 likely causative mtDNA diagnoses were made, boosting the overall diagnostic yield by 0.4% and uncovering 37 new diagnoses .
The pipeline efficiently handled off-target exome sequencing data, retaining 90% of datasets for analysis and enabling detection of pathogenic variants at heteroplasmy levels as low as 1% .
Structured, phenotype-driven curation underscored the importance of comprehensive HPO annotation and highlighted the value of iterative genotype-phenotype evaluation in improving rare disease diagnostics .

🧠 Conclusion:
This study demonstrates a scalable approach to integrate mtDNA analysis into routine exome and genome reanalysis by leveraging automated bioinformatic filtering and phenotype similarity scoring, offering a powerful tool to improve diagnostic rates for mitochondrial disorders in heterogeneous cohorts .

📖 Reference:
Ratnaike, T., Paramonov, I., Olimpio, C., et al. (2025). Mitochondrial DNA disease discovery through evaluation of genotype and phenotype data: The Solve-RD experience. The American Journal of Human Genetics, 112(1), 1–12. https://doi.org/10.1016/j.ajhg.2025.04.003

📜 License:
This episode is based on an open-access article published under the Creative Commons Attribution 4.0 International (CC BY 4.0) license – http://creativecommons.org/licenses/by/4.0/

🎙️ Episode 24: The Role of the X Chromosome in Complex Trait Genetics — Unraveling Dosage-Compensation Mechanisms
🧬 In this installment of Base por Base, we delve into groundbreaking work by Fu et al. (2025) in The American Journal of Human Genetics, which systematically interrogates how the X chromosome shapes human quantitative traits. Leveraging data from over 350,000 UK Biobank participants with replication in more than 400,000 FinnGen individuals, the study quantifies the X chromosome’s contribution to trait heritability, examines sex-specific genetic variance, and elucidates the interplay between X-inactivation and upregulation processes in balancing dosage between sexes and with the autosomes .

🔍 Key Highlights: In a comprehensive analysis of 48 traits, the authors report that the X chromosome accounts for roughly 3% of the heritability explained by autosomes, demonstrating a proportional yet distinct contribution to complex trait variation; they confirm near-complete X chromosome inactivation in females and hemizygosity in males, leading to approximately double the genetic variance on X in men versus women; compelling evidence emerges for partial escape from inactivation impacting traits such as height; moreover, the study reveals that single active copies of X-linked loci exert allele effects about 1.6-fold greater than those on autosomes, supporting a model of partial transcriptional upregulation on X relative to autosomes .

🧠 Conclusion: By integrating large-scale biobank data with advanced heritability and effect-size modeling, this work inaugurates a new era in understanding X-linked genetic architecture. It highlights how near-complete inactivation, nuanced escape, and dosage compensation via allele-level upregulation collectively maintain balanced contributions of the X chromosome to human phenotypic diversity, offering vital insights for future locus discovery and sex-specific analyses in genomics .

📖 Reference:
Fu, Y., Kenttämies, A., Ruotsalainen, S., Pirinen, M., & Tukiainen, T. (2025). Role of X chromosome and dosage-compensation mechanisms in complex trait genetics. The American Journal of Human Genetics, 112, 1–14. https://doi.org/10.1016/j.ajhg.2025.04.004

📜 License: This episode is based on an open-access article published under the Creative Commons Attribution 4.0 International (CC BY 4.0) license – https://creativecommons.org/licenses/by/4.0/

🎙️ Episode 23: Evaluating the Return of Additional Findings from the 100,000 Genomes Project — Participant Experiences of Secondary Genomic Findings

🧬 In this episode of Base por Base, we explore a mixed-methods study by Stafford-Smith et al. (2025) published in Genetics in Medicine, which examines how participants in England’s 100,000 Genomes Project experienced the disclosure of additional (secondary) genomic findings. By combining a cross-sectional survey of 147 adults who received either cancer- or familial hypercholesterolemia-related findings with in-depth interviews of 35 positive-finding recipients and 29 individuals with no additional findings, this research sheds light on the psychological, behavioural, and communication aspects of returning actionable genetic results through NHS pathways.

🔍 Study highlights:
Participants rated the utility of their results exceptionally high (mean 8.9/10 now; 9.0/10 for future use) and most (82%) reported that knowing their finding would influence health management. Those who learned of a cancer-related variant often felt initial shock and anxiety, whereas individuals with familial hypercholesterolemia findings described relief and readiness to act. Recipients of no additional findings welcomed the reassurance but sometimes struggled to distinguish these results from their primary diagnostic findings. Overall decisional regret was low (median score 5/100), though a minority experienced mild or high regret initially. Thematic analysis revealed that clear, tailored communication and timely clinical support were critical for helping participants adjust, share results with family, and implement risk-reducing strategies.

🧠 Conclusion:
Stafford-Smith et al. demonstrate strong participant support for routinely offering actionable secondary findings alongside genome sequencing, while emphasizing the need for condition-specific pathways, improved consent-to-result timelines, and enhanced informational resources for no-finding recipients. Their work lays the groundwork for more patient-centred genomic medicine practices and underscores the importance of psychosocial support in “genome-first” care models.

📖 Reference:
Stafford-Smith B., Daniel M., Peter M., et al. (2025). Evaluating the return of additional findings from the 100,000 Genomes Project: A mixed methods study exploring participant experiences of receiving secondary findings from genomic sequencing. Genetics in Medicine. https://doi.org/10.1016/j.gim.2025.101446

📜 License:
This episode is based on an open-access article published under the Creative Commons Attribution 4.0 International (CC BY 4.0) license – https://creativecommons.org/licenses/by/4.0/

🎙️ Episode 22: Immune Sensing Unveiled — Ribonuclease Control of Naked Extracellular RNA

🧬 In this episode of Base por Base, we explore a landmark study by Castellano et al. (2025) published in Cell Genomics that uncovers how extracellular ribonucleases regulate the immune detection and functional uptake of naked extracellular RNA (exRNA). Through strategic use of a broad-spectrum ribonuclease inhibitor, the authors demonstrate that, contrary to long-held assumptions, naked exRNAs can be spontaneously internalized by dendritic cells and macrophages, engage both endosomal Toll-like receptors and cytosolic RIG-I-like receptors, and even serve as templates for protein translation, thereby revealing a novel, nonvesicular pathway of intercellular RNA communication .

🔍 Key Highlights: Naked extracellular RNA exhibits intrinsic bioactivity when extracellular RNases are inhibited or absent, triggering pro-inflammatory transcriptional programs via endosomal TLR13 in murine cells and RIG-I/MAVS-dependent pathways in human macrophages; these naked RNAs can escape endosomes to reach the cytosol, where they activate downstream antiviral and inflammatory signaling; in vitro-transcribed mRNAs delivered as naked exRNA are taken up and translated into functional reporter proteins in both primary immune cells and epithelial cell lines in an RNase-sensitive manner; and systemic administration of naked bacterial RNA or synthetic dsRNA with ribonuclease inhibition markedly amplifies immune cell activation in vivo, whereas the high RNase activity of blood normally prevents such systemic inflammation .

🧠 Conclusion: Castellano et al. reveal that extracellular RNase activity serves as a critical barrier to the bioactivity of naked exRNAs, and that in low-RNase environments naked RNA can mediate immune communication and protein expression. These insights fundamentally challenge the prevailing view that vesicular encapsulation is required for functional RNA transfer and position extracellular RNases as key modulators of intercellular RNA signaling and immune homeostasis .

📖 Reference:
Castellano, M., Blanco, V., Li Calzi, M., Costa, B., Witwer, K., Hill, M., Cayota, A., Segovia, M., & Tosar, J.P. (2025). Ribonuclease activity undermines immune sensing of naked extracellular RNA. Cell Genomics, 5, 100874. https://doi.org/10.1016/j.xgen.2025.100874

📜 License: This episode is based on an open-access article published under the Creative Commons Attribution 4.0 International (CC BY 4.0) license – https://creativecommons.org/licenses/by/4.0/

🎙️ Episode 21: High-Throughput Variant Discovery — Pooled Prime Editing in Human Cells

🧬 In this episode of Base by Base, we explore a cutting-edge platform for scalable functional screening of human genetic variants reported by Herger et al. (2025) in Cell Genomics. The study introduces a pooled prime editing workflow in haploid HAP1 cells that optimizes pegRNA scaffold design, employs co-selection via an ATP1A1 resistance mutation, and integrates surrogate targets to enrich and filter highly active edits—enabling interrogation of both coding and non-coding variants in their native genomic context .

🔍 Study highlights:
The platform achieves improved editing efficiencies by incorporating stabilized pegRNA scaffolds, optimized prime editor expression, and co-selection for an ATP1A1 resistance mutation in HAP1 cells .
Deep sequencing of pegRNA–surrogate target cassettes enables negative selection screening in SMARCB1 exons 8–9, accurately identifying loss-of-function single-nucleotide and multinucleotide variants that impair protein function .
Positive selection using 6-thioguanine in MLH1 screens yields precise function scores for SNVs in exon 10, with AUCs reaching 1.00 under stringent surrogate editing thresholds, demonstrating robust discrimination of pathogenic variants .
A comprehensive library covering 874 non-coding ClinVar variants across 60 kb of MLH1 reveals pathogenic splice and regulatory variants at scale, with surrogate target filtering ensuring high data quality .
Validation assays confirm that surrogate target editing correlates strongly with endogenous variant installation, supporting accurate function scoring across large genomic regions .

🧠 Conclusion:
This pooled prime editing platform offers a versatile and scalable tool for functional assessment of genetic variants across both coding and non-coding regions, paving the way for accelerated discovery and classification of disease-associated alleles .

📖 Reference:
Herger, M., Kajba, C. M., Buckley, M., Cunha, A., Strom, M., & Findlay, G. M. (2025). High-throughput screening of human genetic variants by pooled prime editing. Cell Genomics, 5, 100814. https://doi.org/10.1016/j.xgen.2025.100814

📜 License:
This episode is based on an open access article published under the Creative Commons Attribution 4.0 International license (CC BY 4.0) – https://creativecommons.org/licenses/by/4.0/

🎙️ Episode 19: Genotype-Specific Chemoprevention with Sulfadoxine-Pyrimethamine

🧬 In this episode of Base by Base, we examine a comprehensive analysis by Mousa et al. (2025) in Nature Communications that quantifies how mutations in the Plasmodium falciparum dihydropteroate synthase (dhps) gene affect the duration of protection conferred by sulfadoxine-pyrimethamine (SP) chemoprevention across Africa. A Bayesian multi-strain model was applied to reinfection data from seven therapeutic efficacy studies involving 1,639 participants across twelve African sites, enabling simultaneous estimation of genotype-specific protective durations and mapping of SP efficacy in diverse resistance settings .

🔍 Main highlights: Protection against dhps sulfadoxine-susceptible parasites extends beyond 42 days, while protection against the West African dhps GKA genotype lasts a median of 30.3 days (95% Credible Interval: 17.1–45.1). The East African dhps GEA and highly resistant dhps GEG genotypes reduce median protection to 16.5 days (95% CrI: 11.2–37.4) and 11.7 days (95% CrI: 8.0–21.9), respectively; model validation against intermittent preventive treatment in infants (IPTi) trials from Mozambique and Tanzania demonstrated strong agreement between predicted and observed reinfection patterns. Continent-wide predictions based on genotype frequency maps revealed 30-day protective efficacy ranging from 59.3% to 91.5% and median protection durations from 17.2 to 37.2 days, guiding targeted SP deployment strategies. Comparative analyses with SP–amodiaquine (SPAQ) and SP–chloroquine (SPCQ) combinations highlight enhanced protection against resistant genotypes where amodiaquine efficacy remains high .

🧠 Conclusion: By providing robust genotype-specific estimates of SP protective efficacy and developing an interactive prediction tool for local genomic surveillance integration, this study equips malaria control programs with actionable insights to tailor chemoprevention strategies and optimize SP-based interventions across varying transmission and resistance landscapes .

📖 Reference:
Mousa, A., Cuomo-Dannenburg, G., Thompson, H. A., et al. (2025). Impact of dhps mutations on sulfadoxine-pyrimethamine protective efficacy and implications for malaria chemoprevention. Nature Communications, 16, 4268. https://doi.org/10.1038/s41467-025-58326-z

📜 License:
This episode is based on an open-access article published under the Creative Commons Attribution 4.0 International license (CC BY 4.0) – https://creativecommons.org/licenses/by/4.0/