🧬 PubMed RNA Editing Daily Digest

The repair of pathological gene variants is an ultimate goal in treating genetic diseases; however, developing distinct therapeutic reagents for each of the numerous variants within a gene may not be scalable. Here, we investigated whether base editing to introduce a gain-of-function variant in blood coagulation factor IX (FIX) can increase FIX activity as a targeted therapeutic approach for hemophilia B. We engineered a G:C to A:T substitution at c.1151 of F9 by cytosine base editing to generate R338Q (the Shanghai F9 variant), which markedly increases coagulation factor activity. An adeno-associated virus vector harboring the base editor converted >60% of the target G:C to A:T and increased FIX activity in HEK293 cells harboring patient-derived F9 variants as well as in knock-in mice carrying a human F9 complementary DNA. Furthermore, administration of lipid nanoparticles containing the base-editor mRNA and guide RNA increased FIX activity in mice. These data indicate that cytosine base editing to generate R338Q in FIX is a broadly applicable genome-editing strategy for hemophilia B with residual FIX activity.

CRISPR-Cas13 exclusively targets RNA. In prokaryotes, Cas13 cleaves both target and non-target RNA indiscriminately upon activation by a specific target RNA, but in eukaryotic cells collateral cleavage activity has been limited. Here we report that LbuCas13a exhibits strong collateral RNA cleavage activity in human cells when delivered as ribonucleoprotein, independent of cell line and targeting both exogenous and endogenous transcripts. Collateral RNA cleavage starts within 50 minutes of ribonucleoprotein delivery resulting in major alterations to the total RNA profile. In response to the collateral RNA cleavage, cells upregulate genes associated with the stress and innate immune response, ultimately leading to apoptotic cell death. This enables us to use LbuCas13a as a flexible and repeatable target-RNA-specific cell elimination tool. Finally, using both total RNA sequencing and Nanopore sequencing, we find that LbuCas13a activation leads to rapid and near-global depletion of cytoplasmic RNAs, and that cleavage occurs at specific nucleotide positions.

Eukaryotic Fanzor proteins are compact, programmable RNA-guided nucleases with substantial potential for genome editing, although their efficiency in mammalian cells remains suboptimal. Here, we present a combinatorial engineering strategy to optimize a representative Fanzor system, MmeFz2-ωRNA. AlphaFold3-powered rational redesign produced a minimized ωRNA scaffold that is 30% smaller while maintaining up to 82.2% efficiency. Synergistic structure-guided and AI-augmented protein engineering generated two variants, enMmeFz2 and evoMmeFz2, which exhibited an average ~32-fold increase in activity across 38 genomic loci. Moreover, fusion of the non-specific DNA-binding domain HMG-D further enhanced editing performance (enMmeFz2-HMG-D and evoMmeFz2-HMG-D). Notably, evoMmeFz2-HMG-D demonstrated robust in vivo genome editing activity, enabling dystrophin restoration in humanized male Duchenne muscular dystrophy mouse models via single adeno-associated virus (AAV) delivery. This study establishes Fanzor2 as a gene editing platform for genome engineering and therapeutic applications, and underscores the power of AI-guided engineering to accelerate genome editor development while reducing experimental burden.

CRISPR-Cas13a biosensing enables rapid, amplification-free RNA diagnostics, yet assay sensitivity varies widely because guide RNAs (gRNAs) differ in their ability to activate the enzyme. Two factors, including the gRNA-target binding affinity and the structural accessibility of the target site, have been proposed to govern activation efficiency, but their relative importance remains unclear. In this study, we systematically disentangle these contributions by measuring binding affinities for gRNAs that span a spectrum of site accessibilities and by comparing their Michaelis-Menten kinetic parameters. Three ciRS-7-specific gRNAs were designed with high, intermediate, and low spacer accessibility. Isothermal titration calorimetry (ITC) quantified site accessibility through entropy changes (ΔS = -862, -813, and -615 cal/mol/K), confirming greater structural exposure for less structured spacers, and also determined binding affinity for each gRNA-target pair. Michaelis-Menten analysis showed k Our data establish site accessibility as a critical determinant of Cas13a activation for amplification-free RNA sensing. Prioritizing unstructured spacer regions enables improved enzyme activation efficiency, providing a clear design rule for next-generation CRISPR diagnostics. This accessibility-driven strategy will facilitate the development of faster, simpler, and more sensitive point-of-care assays for diverse RNA biomarkers.

Single-cell RNA sequencing (scRNA-seq) has identified intermediate epithelial states in pulmonary fibrosis, including KRT5-/KRT17+ aberrant basaloid cells in humans and Krt8+ alveolar differentiation intermediates (ADIs) in mice. Their functional contributions to fibrogenesis, however, remain unclear. Here, we introduce an RNA-sensing-dependent protein translation technology that enables selective targeting of Krt8+ ADI cells in vitro and in vivo. Transcriptomic analysis revealed Small Proline-Rich Protein 1 A (SPRR1A) mRNA as a shared marker of murine Krt8+ ADIs and human KRT5-/KRT17+ basaloid cells, distinguishing them from other lung cell populations. Using programmable RNA sensors, we demonstrated selective EGFP-labeling of Krt8+ ADI cells in vivo, which faithfully recapitulated their transcriptomic and phenotypic features. To test function, we developed an RNA-sensing-driven diphtheria toxin receptor (DTR) system for conditional ablation of Sprr1a+ cells. Targeted depletion markedly reduced fibrosis in bleomycin-injured mice, establishing transitional epithelial cells as pathogenic drivers and highlighting their potential as therapeutic targets in pulmonary fibrosis.