🧬 PubMed RNA Editing Daily Digest

Urinary small extracellular vesicles (sEVs), which can reflect systemic conditions, hold great promise for noninvasive cancer diagnostics, yet the mechanism by which tumor-derived sEVs reach urine remains unclear. Here, we demonstrate that the glomerulus actively transcytoses circulating tumor-derived sEVs into urine. Using CRISPR guide RNA-tagged glioma sEVs and bioluminescent/fluorescent green-enhanced nano-lantern (GeNL)-tagged lung and pancreatic cancer sEVs, we tracked their journey from tumors to urine in multiple mouse models. In vivo and in vitro analyses revealed endocytic uptake and transcytotic release by glomerular cells, accompanied by changes in sEV size and surface composition. GeNL-tagged sEVs consistently showed higher signals in urine than plasma, indicating selective excretion. These findings redefine the glomerulus as a dynamic regulator of sEV processing and establish a mechanistic foundation for urinary liquid biopsy.

Characterizing the protospacer adjacent motif (PAM) requirements of different Cas enzymes is a bottleneck in the discovery of Cas proteins and their engineered variants in mammalian cell contexts. Here, to overcome this challenge and to enable more scalable characterization of PAM preferences, we develop a method named GenomePAM that allows for direct PAM characterization in mammalian cells. GenomePAM leverages genomic repetitive sequences as target sites and does not require protein purification or synthetic oligos. GenomePAM uses a 20-nt protospacer that occurs ~16,942 times in every human diploid cell and is flanked by nearly random sequences. We demonstrate that GenomePAM can accurately characterize the PAM requirement of type II and type V nucleases, including the minimal PAM requirement of the near-PAMless SpRY and extended PAM for CjCas9. Beyond PAM characterization, GenomePAM allows for simultaneous comparison of activities and fidelities among different Cas nucleases on thousands of match and mismatch sites across the genome using a single gRNA and provides insight into the genome-wide chromatin accessibility profiles in different cell types.

The altered life history strategies of heterotrophic organisms often leave a profound genetic footprint on energy metabolism related functions. In parasitic plants, the reliance on host-derived nutrients and loss of photosynthesis in holoparasites have led to highly degraded to absent plastid genomes, but its impact on mitochondrial genome (mitogenome) evolution has remained controversial. By examining mitogenomes from 45 Orobanchaceae species including three independent transitions to holoparasitism and key evolutionary intermediates, we identified measurable and predictable genetic alterations in genomic shuffling, RNA editing, and intracellular (IGT) and horizontal gene transfer (HGT) en route to a nonphotosynthetic lifestyle. In-depth comparative analyses revealed DNA recombination and repair processes, especially conversion of RNA-mediated retroprocessing, as significant drivers for genome structure evolution. In particular, we identified a novel RNA-mediated IGT and HGT mechanism, which has not been demonstrated previously in cross-species and inter-organelle transfers. We propose a dosage effect mechanism to explain the biased transferability of plastid DNA to mitochondria across green plants, especially in heterotrophic lineages like parasites and mycoheterotrophs. Evolutionary rates scaled with these genomic changes, but the direction and strength of selection varied substantially among genes and clades, resulting in high contingency in mitochondrial genome evolution. Finally, we summarize mitochondrial evolutionary trends in Orobanchaceae that are potentially generalizable to other heterotrophic plants: increased recombination and repair activities, rather than relaxed selection alone, lead to differentiated genome structure compared to free-living species.

A fundamental challenge in studying therapy resistance is understanding whether it results from pre-existing cellular states ("priming") or drug-induced changes ("adaptation"). While lineage barcoding enables retrospective analysis of cells before and after treatment, current methods struggle to efficiently capture rare lineages in single-cell RNA sequencing (scRNA-seq) or isolate multiple specific lineages simultaneously for functional study. To overcome these limitations, we developed CloneSweeper, a multiplexed lineage tracking platform that pools enrichment libraries to isolate or enrich multiple rare lineages. CloneSweeper utilizes a dual-function barcode expressed as both a Cas9 gRNA for live-cell sorting and a 3' UTR transcript for high-recovery detection in 10x Genomics scRNA-seq. We applied CloneSweeper to a model of BRAF V600E melanoma, where we identified that resistance to targeted therapy emerges from a polyclonal population of rare, pre-existing lineages. By simultaneously targeting and enriching 21 distinct rare lineages prior to treatment, we defined a heritable, primed state characterized by de-differentiation and elevated mesenchymal markers. We demonstrate that these primed cells are not quiescent but instead exhibit upregulated inflammatory and stress response signaling, specifically via the AP-1 and NF-κB1 pathways. CloneSweeper thus provides a robust framework for dissecting the molecular mechanisms of rare biological phenomena through simultaneous, multiplexed lineage isolation.

Duckweeds (Lemnaceae) have excellent potential for fundamental and applied research due to ease of cultivation, small size, and continuous fast clonal growth. However, their usage as model organisms and platforms for biotechnological applications is often limited by the lack of universal genetic manipulation methods necessary for transgene expression, gene editing, and other methods to modify gene expression. To identify suitable strains for genetic manipulation of the giant duckweed, Spirodela polyrhiza, we screened several genotypes for callus induction and regeneration and established genetic transformation. We identified SP162 to be amenable to Agrobacterium-mediated transformation via tissue culture. The procedure is robust and reproducible across laboratories, allowing stable expression of different reporter genes and selectable markers, enabling CRISPR/Cas9-mediated genome editing. In addition, due to a weak small RNA-based silencing response, S. polyrhiza sustains prolonged periods of transgene activity in transient expression assays. To promote duckweed research and encourage the adoption of S. polyrhiza, we have made SP162 (ID#: 5676) and its genome publicly available and provide here detailed procedures for its cultivation and transformation. Furthermore, we created a web server to explore its genome, retrieve gene sequences, and implement orthologous gene search and a gRNA design function for diverse CRISPR/Cas-based applications (https://agxu.uni-mainz.de/SP162/).