🧬 PubMed RNA Editing Daily Digest

Eradication of HIV is challenging because of the integration of proviral DNA in reservoir cells. In this study, we evaluated the antiviral effects of synthetic gRNA/Cas12a and gRNA/Cas9 ribonucleoproteins (RNPs) in HIV-infected T cells and demonstrated their specificity and efficacy in disrupting HIV gene replication and expression. Notably, sequential or combinatorial treatments with gRNA4/Cas9 and/or gRNA5/Cas12a RNPs elicited the most effective antiviral effect, with significant reduction in integrated proviral DNA, HIV mRNA, early and late reverse transcripts, and p24 protein levels. DNA sequencing revealed a high rate of insertion and deletion or knockout frequencies at the HIV target genes. Gene alignment analysis showed a high level of conservation with both gRNA4/Cas9 and gRNA5/Cas12a target sequences across diverse regional HIV strains, indicating their potential to target different HIV strains across the world. This study indicates that synthetic gRNA4/Cas9 and gRNA5/Cas12a RNPs can be used for HIV gene disruption and viral eradication.

Since their discovery, CRISPR-Cas systems have been widely applied in areas ranging from genome editing to biosensing, owing to their specific, RNA-guided target recognition. Their performance in complex biological environments has been extensively studied, particularly to optimize guide RNA (gRNA) design and minimize off-target cleavage. Here, we focus on the kinetic inhibition of the interaction between Cas12a-a Class 2, Type V effector-and its target, caused by interference from non-cognate background nucleic acids. This effect is particularly relevant for sensing applications in complex mixtures or cellular contexts, where genome- and transcriptome-derived sequences may impede CRISPR-Cas activity. Using in vitro assays under defined conditions, we systematically examine the influence of background single-stranded RNA and double-stranded DNA (dsDNA) on reaction kinetics. We find that both the purine-to-pyrimidine ratio and the GC content of the gRNA seed region significantly affect kinetic inhibition by background polynucleotides. gRNAs with low GC content and a high purine fraction in the seed region were least affected by background sequences. A gRNA with high uracil content in the seed region exhibited particularly strong inhibition in the presence of a dsDNA background. Experiments with dCas12a-based gene activation in living cells indicate that our in vitro findings may also be relevant for in vivo applications.

The adaptation of CRISPR technologies for molecular detection marks a significant advancement in the field of biosurveillance and infectious disease response. CRISPR-based detection systems offer superior specificity and sensitivity compared to traditional PCR methods by directly binding and cleaving target DNA or RNA sequences, thus signaling the presence of specific pathogens. These advantages include the elimination of non-specific amplification and the reduction of required genetic material, leading to faster time to results without the need for extensive amplification cycling. However, the efficacy of CRISPR technologies heavily depends on the design of specific guide RNA (gRNA) sequences tailored for each genomic target, a process that can be intricate and time-consuming. We present Cas-CRISPR Automated Design and Evaluation (CasCADE), a state-of-the-art gRNA design software platform with a high degree of flexibility and modularity. CasCADE incorporates k-mer set operations to reduce time to answer for large data inputs when compared to computationally costly multiple sequence alignment methodologies and uses an agnostic whole genome approach to maximize gRNA discovery. CasCADE can be scaled efficiently to problems of any input sequence size and can be used for design, candidate evaluation, or both, depending on user need. This work describes our software pipeline Cas-CRISPR Automated Design and Evaluation (CasCADE) that allows for

Polygonatum kingianum, a key species in traditional Chinese medicine, is increasingly valued for its medicinal and nutritional properties. However, wild resources are declining due to over-exploitation, necessitating genomic studies for conservation. Flow cytometry analysis based on genome size revealed the presence of both putative diploid and tetraploid P. kingianum. By further integrating sequencing data from both Illumina and Oxford Nanopore Technologies (ONT), the organelle genomes were assembled. While the chloroplast genomes of different putative ploidy levels of P. kingianum were highly conserved in structure and features, the mitogenome of putative tetraploid P. kingianum consisted of two chromosomes, whereas that of putative diploid P. kingianum contained only one. Mitochondrial gene annotation showed that putative tetraploid P. kingianum possesses one additional copy each of the nad3 and nad5 genes compared to the putative diploid P. kingianum. Analyses of repetitive sequences indicated that, although no obvious correlation was observed between tandem repeats or dispersed repeats and different putative ploidy levels in the chloroplast genomes or between tandem repeats and different putative ploidy levels in the mitogenomes, simple sequence repeats in the organelle genomes correlated with ploidy variation of P. kingianum. Investigation of RNA editing sites revealed ploidy-dependent variations in the cox2 and nad3 genes of the mitogenomes across different ploidy levels of P. kingianum, suggesting that differences in editing sites may be linked to the adaptability of the different ploidies. Phylogenetic analyses based on mitochondrial genes and nucleotide substitution rate analysis showed no significant differences or distinct variations among different ploidy levels of P. kingianum, supporting that the morphologically highly variable P. kingianum comprises only a single species. In this study, we successfully assembled the organelle genomes of P. kingianum with different putative ploidy levels and conducted comparative genomic analyses. We found that the mitogenomes of P. kingianum are structurally complex and variable, playing crucial roles in evolution and adaptation. This study not only enhances our understanding of the organelle genome characteristics of different putative ploidy levels within the same species, but also lays the foundation for the subsequent development and utilization of the medicinal value of Polygonatum.

Astragalus (Fabaceae), the largest genus of flowering plants with its phytogeographic center in Southwest Asia, is ecologically and medicinally vital, as high-quality forage and a source of traditional medicine. However, its ambiguous morphological traits hinder taxonomic clarity, prompting increased reliance on molecular phylogenetic approaches to resolve relationships. In the research, we assembled and annotated mitochondrial genomes of three Astragalus species, integrating them with two published mitochondrial genomes for comparative analysis. Newly sequenced genomes (269,019-364,431 bp) encoded 55-59 genes (31-34 protein-coding, 22-25 tRNAs, 3 rRNAs). Comparative analysis identified four genes (cox2, rps4, atp8, rps12) with high nucleotide variability, establishing them as reliable molecular markers for Astragalus phylogenetics, critical for future taxonomic and evolutionary studies. Mitochondrial plastid sequences were widespread across all five species, with petG and trnW-CCA conserved as chloroplast-derived fragments. Phylogenetic trees based on concatenated mitochondrial genes revealed Astragalus species shared close affinities with A. cicer and A.laxmanii, refining subgeneric classification frameworks. Our study confirms mitochondrial genomes as powerful tools for resolving relationships in morphologically cryptic genera. The novel genomic resources, including repeats and RNA editing sites, and validated markers, lay a foundation for future research into Astragalus biogeography, adaptive evolution, and medicinal resource development-thereby, enhancing the genus' ecological and economic value.