🎙️ 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/