🧬 PubMed RNA Editing Daily Digest

CRISPR/Cas9 mediated genome editing is a highly powerful and versatile tool for accelerating crop improvement. The editing efficiency of CRISPR/Cas9 system in planta has been highly variable owing to the variable binding affinity between native CRISPR RNA and Cas9 protein in vivo. In plant systems, systematic, large-scale engineering and benchmarking of guide RNA (gRNA) scaffold variants is still relatively limited compared with work in mammalian systems, despite several important studies demonstrating that scaffold and expression-cassette engineering can substantially improve CRISPR/Cas9 efficacy. Current study addresses the limitations of commonly used gRNA scaffold architecture by incorporating a stabilized stem-loop RAR (tetra loop) extension and a transcription-termination site mutation, resulting in improved RNA folding, increased Cas9 binding affinity, and enhanced in vivo editing outcomes. The synthesized scaffold boosted CRISPR/Cas9 efficiency in monocot or dicot plants across the 19 diverse target sites in Arabidopsis, rice and tomato. Furthermore, the synthetic scaffold is compatible with multiplex genome editing architectures, including polycistronic tRNA-gRNA (PTG) expression systems, enabling efficient simultaneous targeting of multiple genomic loci. The findings of this study have broad applications in precision plant breeding, functional genomics, and agricultural biotechnology, facilitating reliable gene modification across diverse plant species and transformation platforms.

As an important structural protein, porcine epidemic diarrhea virus (PEDV) nucleocapsid (N) plays critical roles in the viral life cycle. Liquid-liquid phase separation (LLPS) is required for multiple biological functions for coronaviridae infection. However, whether PEDV has the ability to cause LLPS and its roles in this process remain unknown. In this study, we carried out bioinformatic analysis to show that PEDV N protein had the strongest potential to induce LLPS among all PEDV proteins. PEDV N protein caused both endoplasmic reticulum stress (ERS) and protein translation stall in vivo, which has been associated with biomolecular condensates mediated by LLPS. At the same time, we purified the N protein and found that either RNA or molecular crowding agent can induce LLPS of N protein in a concentration-dependent manner in vitro. Subsequently, the different truncated domains of N protein were further investigated, of which results showed that the dimerization domain (DD) was responsible for the self-aggregation of N protein, and that the intrinsically disordered region (IDR) played decisive roles in LLPS formation with mRNA. In addition, we showed that the RNA sequence and structural differences in genomic RNA (gRNA) such as 5' end contribute to LLPS triggered by N protein, as revealed by larger, more liquid-like condensates of 5'-UTR combined with N protein. Disruption of LLPS attenuated ERS and interfered with the binding of N protein with RNA, as well as N gene transcription and protein expression. These findings not only provide new insights into the novel functions of PEDV N protein, but also provide a foundation for understanding the mechanism of genome aggregation and its role in the virus life cycle.

The vascular endothelium is a critical regulator of vascular homeostasis and tissue fluid balance, and mouse models are essential for studying these processes in vivo. Here, we present a protocol to generate adult endothelial cell (EC)-specific gene knockout (KO) mouse models. We describe steps for Cas9-active and Cdh5-Cre-positive (Cas9/Cdh5-Cre) mouse line generation, single guide RNA (sgRNA) design for vector construction, plasmid DNA generation, and liposome preparation. We then detail procedures for liposome/plasmid complex injection, lung harvest, homogenization, protein quantification, and verification with western blotting. For complete details on the use and execution of this protocol, please refer to Wang et al.

The Bromeliaceae family contains many horticulturally and economically important species, including Aechmea fasciata and pineapple (Ananas comosus). Compared to their well-studied chloroplast and nuclear genomes, the mitogenomes of bromeliads remain largely unexplored, hindering a full understanding of their organellar evolution and phylogenetic relationships. This study provides the first complete mitogenome assemblies and a comparative analysis for these two high-value bromeliads, revealing their phylogenetic affinities. Our findings suggested that the mitogenomes of Ae. fasciata and An. comosus both exhibit a simple master circle structure. They were 0.93-1.16 Mb in length, containing similar protein-coding genes (PCGs) (42-44), 39 tRNA genes, and three rRNA genes. Notably, the synonymous codons usage analysis revealed a preference for A/U endings, and a total of 542-548 RNA editing sites were detected. Most PCGs were under purifying selection, indicating a strong evolutionary constraint to preserve functional integrity. Additionally, the ATP synthase gene exhibited high nucleotide diversity, suggesting that such genes may need further investigation in the context of future CMS-related studies. Phylogenetically, analyses based on shared PCGs provided robust support for the clustering relationships within Bromeliaceae, confirming that An. comosus and Ae. fasciata form a distinct clade and are separate from Puya raimondii. This study establishes a crucial comparative genomic framework for Bromeliaceae, providing valuable genetic resources for future evolutionary studies and potential marker-assisted breeding applications.