🧬 PubMed RNA Editing Daily Digest

Advancements in synthetic genetic circuits have enabled programmable and condition-dependent control of microbial cell growth. CRISPR-Cas9-based kill switches, genetic systems that program cells to lose viability in response to specific conditions, have recently been demonstrated for bacterial cell factories but not yet in yeast. In this study, we present a foundational demonstration for a CRISPR-based This work highlights the potential of harnessing a CRISPR-based kill switch in The online version contains supplementary material available at 10.1186/s12934-026-02959-2.

Alpha-herpesviruses have evolved strategies to break through immune defenses and cause severe host damage. Here, we demonstrate that the tegument protein UL48 in pseudorabies virus (PRV) inhibits type I interferon signaling by triggering STING1 degradation via a selective macroautophagy/autophagy pathway. Mechanistically, UL48 recruits the E3 ligase TRIM21 (tripartite motif containing 21), which catalyzes the ubiquitination of STING1 to form a K33/K63 linkage and is captured by the cargo receptor CALCOCO2/NDP52 for lysosomal degradation. In addition, multiple α-herpesvirus tegument protein UL48 homologs also target STING1 for degradation. Importantly, this phenotype was also observed in other herpesviruses driven by PRV UL48 homologs (herpes simplex virus-1 [HSV-1] and cercopithecine alphaherpesvirus 2 [CHV-2]). In addition, UL48-deficient PRV and HSV-1 mutant viruses attenuated pathogenicity in mice. In conclusion, this study describes a novel mechanism by which α-herpesviruses utilize UL48 proteins to promote viral escape from the host immune response.

The degradation of seagrass beds, which serve as pivotal indicators of coastal ecosystem health, has emerged as a critical reflection of global nearshore environmental stress. In this study, we employed two closely related seagrass species, Halophila beccarii and H. ovalis, as case studies to investigate the structural and evolutionary dynamics of their mitochondrial genomes through comparative genomics and evolutionary analyses. Our findings reveal striking structural disparities in the mitochondrial genomes of the two species: H. beccarii comprises 28 free circular elements totaling 1.98 Mb, whereas H. ovalis harbors 12 circular units spanning 0.58 Mb. Codon usage analysis demonstrated a significant A/U preference at the third position of high-frequency codons. Analyses of relative substitution rates (Ka/Ks) showed that the nad3 gene consistently exhibited ratios greater than 1 in both marine-freshwater pairwise comparisons and across the phylogeny, distinguishing it from other mitochondrial genes. Halophila species showed a reduced complement of RNA editing sites. Specifically, genes nad3 and nad6 in H. beccarii, and cox1, nad3, and nad6 in H. ovalis completely lacked RNA editing under the experimental conditions employed. Collectively, this work characterizes the diverse mitogenomic architecture in seagrasses, offering a genomic foundation for future studies on seagrass evolution and environmental response.

The CRISPR/Cas9 technology has improved the ability to introduce targeted modifications in cells and embryos in diverse species. The use of this technology enables the establishment of genetically modified livestock models to study human diseases or improve food production. However, one of the main concerns with employing this technology is the possibility of introducing unintended genome modifications induced by the Streptococcus pyogenes Cas9 (SpCas9), a commonly used Cas9 protein. Recent advancements in CRISPR/Cas9 technology offer Cas9 variants that are designed to improve gene editing specificity. Here, three high-fidelity SpCas9 variants (eSpCas9, HiFi Cas9 and LZ3 Cas9) were employed to examine their efficacy and specificity in pig embryos. To introduce targeted modifications, mRNA coding for each Cas9 variant was mixed with IGH single guide RNA (sgRNA) and were injected into fertilized pig zygotes. The frequency of on- and off-targeting was calculated by amplifying IGH, AR, and RBFOX1 regions from genomic DNA derived from the injected embryos at the blastocyst stage and sent for Sanger sequencing. The sgRNA targeting IGH locus resulted in a 100% on-target editing rate using SpCas9. However, SpCas9 introduced off-targeting events in AR and RBFOX1 at a high frequency (> 60%) in embryos. Injecting each Cas9 variant at 20 ng/µl could modify the target gene (IGH) at 100% efficiency except for LZ3 Cas9 (59.1%). Importantly, off-target events on AR and RBFOX1 were not detected in any Cas9 variant groups. Gradually reducing the concentration of Cas9 mRNAs lowered the efficacy of on-targeting in all groups; however, the reduction was more dramatic in HiFi Cas9 and LZ3 Cas9 injected embryos. No embryonic toxicity was identified in embryo injected with Cas9 variants and more embryos reached blastocyst stage when injected with either eSpCas9 or HiFiCas9 mRNA. In vivo competency of embryos receiving eSpCas9 was examined by embryo transfer and fetuses recovered from a pregnant sow presented 100% on-target editing efficiency without any detectable off-target events. In summary, among the Cas9 variants examined, eSpCas9 presented the highest specificity with no detectable off-target events and supported the development of gene-edited fetuses. Our findings indicate that the use of Cas9 variants can advance the field of gene editing in livestock models. Pigs are an important food animal species and a translational model for human disease. The development of gene editing systems such as the CRISPR/Cas9 system has greatly improved the efficiency of introducing precise genetic modifications in pigs. Although efficient, the system can introduce random DNA break in the genome and cause toxicity. To minimize the toxicity, diverse CRISPR/Cas9 systems have been developed. In this study, the efficiency and specificity of selected Cas9 variants (eSpCas9, HiFi Cas9, and LZ3 Cas9) were evaluated using swine embryos as a model. The efficiency of eSpCas9 was comparable to conventional Cas9 and could effectively suppress DNA mutations at non-target locations. In summary, the fidelity of different Cas9 variants was investigated to reduce toxicity of gene editing events without compromising on-target success in swine embryos. The findings indicate that the use of Cas9 variants can advance the field of gene editing in the livestock.

Phosphorothioate (PS) modifications are important chemical modifications across oligonucleotide therapeutics. However, the introduction of PS linkages significantly increases analytical complexity due to the generation of diastereomers. Here, we extend beyond the limited studies of PS diastereomer ion mobility mass spectrometry (IM-MS) through the comprehensive and systematic analysis of model poly-dT PS oligonucleotides using structures for lossless ion manipulation (SLIM) high-resolution IM (HRIM) coupled to high-resolution mass spectrometry (HRMS). For the first time, we thoroughly investigate the impact of charge state, adduct state, oligonucleotide length, sequence composition/symmetry, and the number and location of PS modifications on HRIM-MS separation. Our data showcase that increased sequence asymmetry and lower oligonucleotide