🧬 PubMed RNA Editing Daily Digest

Frameshift mutations, responsible for >20% of Mendelian inherited diseases, pose substantial therapeutic challenges. Here we developed Template-Independent Genome Editing for Restoration (TIGER), a platform for the efficient and precise correction of frameshift mutations across various models. By identifying reproducible nucleotide-level factors that influence therapeutic efficacy across cells and tissues, we developed a scoring system for guide RNA (gRNA)-Cas9 outcomes. Approximately 75% of deletion and 50% of insertion mutations produced ≥30% in-frame products, sufficient for phenotypic restoration, with 38% and 65% achieving wild-type correction, respectively. To expand the applicability of TIGER across species and genome wide, we retrained the inDelphi algorithm to predict therapeutic gRNAs for single-nucleotide frameshifts. In a mouse model of deafness, delivery of SpCas9 and optimal gRNA via dual adeno-associated virus restored hearing thresholds to wild-type levels, with ~90% of in-frame edits being wild type. TIGER provides a robust and broadly applicable strategy for in vivo correction of inherited frameshift diseases.

Double-stranded RNA (dsRNA) is a universal indicator of viral replication and dysregulated RNA metabolism. Detection of dsRNA triggers some of the most powerful innate immune responses in human cells. Although these molecules differ in origin and structure, viral dsRNAs share the defining geometric and electrostatic features of the A-form helix, enabling their sequence-independent recognition by multiple sensor systems. Cytosolic receptors, like retinoic acid-inducible gene I (RIG-I), melanoma differentiation associated gene 5 (MDA5), and protein kinase R (PKR), as well as the oligoadenylate synthase (OAS)/RNase L pathway, convert dsRNA binding into interferon induction, translational arrest, and widespread RNA decay, while endosomal Toll-like receptor 3 (TLR3) and the inflammasome sensor NLR family pyrin domain containing 1 (NLRP1) expand surveillance to internalised or structurally disruptive RNAs. Counterbalancing these pathways, the RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) marks endogenous dsRNA through A-to-I conversion, preventing inadvertent activation of innate immune response and maintaining self versus non-self discrimination. Although all of these sensors recognise the A-form helix, each extracts distinct structural and chemical information from dsRNA and converts it into a specific response: RIG-I detects short duplexes with 5'-triphosphorylated ends; MDA5 assembles cooperatively along long uninterrupted helices; PKR integrates duplex length with translational control; OAS proteins act as strict reporters of helix regularity; and TLR3 as well as NLRP1 respond to dsRNA in compartment- and context-dependent ways. Epitranscriptomic marks and chemical modifications-including 2'-O-methylation, N6-methyladenosine, pseudouridine, and ADAR1-mediated inosine-further refine sensing by modulating helical stability and end structure, establishing a biochemical 'self-code' that shapes RNA immunogenicity. Together, these pathways form an integrated network that distinguishes between viral and endogenous dsRNA and coordinates antiviral defence with immune tolerance.

The persistence of integrated human immunodeficiency virus (HIV) proviral DNA poses a major barrier to viral eradication, as the viral reservoir enables rapid rebound upon treatment interruption, despite effective virus inhibition. CRISPR-Cas-based editing strategies, especially those using double-site cleavage, show promise in excising proviral DNA, yet the rate and determinants of excision efficiency remain poorly understood. In this study, we systematically evaluated both single- and dual-SaCas9/gRNA approaches for HIV-1 inactivation. Sequence analysis revealed that SaCas9 can eliminate all wild-type HIV-1 genomes with a single gRNA, unlike other CRISPR-Cas systems. Dual-gRNA strategies improved antiviral efficacy, with the Gag3 + Pol5 combination achieving 97% excision efficiency. Kinetic analysis showed that excision efficiency correlates with the kinetic compatibility of paired gRNAs. Pairs of gRNAs with fast and similar kinetics achieved the highest excision efficiency. In contrast, the Gag3 + Env4 pair exhibited discordant kinetic characteristics (fast and slow), resulting in the failure to induce excision as the cut DNA will be repaired before the second cut is realized. Consequently, no excision but regular editing occurred at the two target sites. These findings provide a mechanistic framework for optimizing CRISPR-Cas-mediated excision, highlighting the critical role of both antiviral activity and kinetic synergy in guiding gRNA selection.

In response to the loss of microbial efficiency caused by environmental stress in biomanufacturing, CRISPR-Cas gene editing technology has become a core tool for enhancing stress tolerance by accurately targeting genomic loci. This article systematically reviews the progress of its application. By optimizing engineered nucleases, gRNA design, and innovative delivery strategies, this technology successfully regulates key pathways in oxidative stress responses. It integrates functional genome screening with dynamic regulation to examine the networks of multi-gene collaborative tolerance. In the construction of high-stress-tolerant industrial chassis cells, the stress survival rate (>90% in Bacillus subtilis under thermal stress) and product synthesis ability (such as cellulose producing ethanol up to 4.5 g/L) of strains such as Escherichia coli and Corynebacterium glutamicum were significantly improved. Current challenges focus on delivery efficiency, off-target risks, and complex regulatory bottlenecks. In the future, the development of new editing tools and intelligent circuits will promote their industrial application in sustainable bio-manufacturing.