🧬 PubMed RNA Editing Daily Digest

CRISPR/Cas12-based nucleic acid detection has revolutionized molecular diagnostics but shows limited single-nucleotide specificity, limited high-fidelity subtype discrimination, and limited compatibility with one-pot assays, restricting its broader clinical application. Here, we report a transposon-associated transposase B (TnpB) ortholog, SfaTnpB, with high trans-cleavage activity, robust single-base mismatch discrimination, and broad temperature tolerance. By stepwise engineering of its guide RNA (ωRNA), we developed an enhanced SfaTnpB (enSfaTnpB) system with markedly improved trans-cleavage efficiency. In combination with a TAM-independent split-activator strategy, this system enables precise detection of single-nucleotide polymorphisms. We further developed TOPIC (TnpB-based One-Pot nucleIC acid detection), a one-pot detection platform coupling enSfaTnpB with recombinase-aided amplification (RAA) or loop-mediated isothermal amplification that enables ultrasensitive detection of human papillomavirus (HPV) subtypes 16 and 18 (∼4 copies/μl) and African swine fever virus DNA (∼3 copies/μl). Finally, RAA-TOPIC accurately detected and genotyped 14 high-risk HPV subtypes with high-fidelity subtype discrimination, showing complete concordance with quantitative real-time PCR-based clinical diagnostics. These findings establish TOPIC as a compact, programmable, and scalable molecular detection tool with broad potential for precision diagnostics and point-of-care testing, particularly in resource-limited settings.

Circular RNAs (circRNAs) are RNA molecules formed through the backsplicing of linear exons. Several thousand have been identified, yet relatively few are functionally characterized due to challenges in distinguishing effects of circular from linear RNA targets. Recently, CRISPR-Cas13 systems have been utilized to directly target unique junctions formed through backsplicing, potentially allowing for selective degradation of circular isoforms. Applying this approach in pooled screens has indeed identified circRNAs proposed to affect viability in several cancer cell lines. However, the design limitations of applying Cas13d to study circRNAs are not fully characterized. Here, we assessed the limitations of Cas13d-mediated circRNA knockdowns by performing essentiality screens on 900 highly expressed circRNAs in K562, an ENCODE tier 1 cell line. We observed consistent off-target knockdown of linear isoforms by certain circRNA-targeting single-guide RNAs (sgRNAs). Re-analysis of existing Cas13d screens in other cell types revealed similar off-target effects. Using machine learning models that predict Cas13d sgRNA efficacy, we further found that most circRNA-targeting sgRNAs are unlikely to induce strong knockdown. After accounting for these design constraints, 0 of 346 circRNAs testable in our screens had detectable effects on proliferation. Our findings highlight key limitations of junction-targeting strategies, with implications for future circRNA perturbation studies.

Posterior capsular opacification (PCO) is the most common complication following cataract surgery and a significant cause of vision impairment. PCO arises from the proliferation, migration, and epithelial-mesenchymal transition (EMT) of residual lens epithelial cells (LECs), driven by an activated mTOR signalling pathway. Previous research has demonstrated that inhibiting mTOR activity effectively reduces LEC proliferation and EMT in rabbit models. However, achieving sustained mTOR inhibition remains a challenge. In this study, we encapsulated the CRISPR/Cas9 system targeting mTOR into chitosan nanoparticles (Chi-gRNA) with an average size of 135 nm. These nanoparticles exhibited resistance to DNase I digestion. To prolong release duration, we incorporated these Chi-gRNA nanoparticles onto the surface of intraocular lenses (IOLs) via layer-by-layer (LbL) assembly. The LbL coatings consisted of alternating layers of positively charged polyethyleneimine (PEI) and negatively charged heparin, interspersed with Chi-gRNA nanoparticles over five consecutive cycles. Spectral analysis confirmed the successful integration and coating of nanoparticles, with characteristic peaks validating the electrostatic assembly of the layers. In vitro assays demonstrated that Chi-gRNA-coated IOLs significantly inhibited the proliferation, migration, and adhesion of human lens epithelial cells (hLECs). These findings highlight the potential of LbL-coated IOLs to deliver CRISPR/Cas9 system-targeting mTOR nanoparticles as a novel and effective strategy to prevent PCO in patients undergoing cataract surgery. This approach offers a promising avenue for the long-term management of this prevalent postoperative complication.

A major advantage of producing therapeutic proteins in mammalian cells is their ability to tailor proteins with human-like posttranslational modifications such as glycosylation, which ultimately defines aspects like stability, protein folding or immunogenicity. However, producing therapeutic proteins with a consistent and reproducible glycoprofile remains a major challenge for the biopharmaceutical industry, especially with biosimilar production. While the enzymes responsible for glycosylation of proteins have been the subject of various cell engineering approaches, tuning their gene expression to precise levels is still difficult to achieve. While CRISPR/Cas9 enabled the genetic engineering of cells to drastically overexpress or remove a target gene, CRISPR/dCas9-based epigenetic editing by targeted DNA methylation promises to stably change the expression pattern of target genes after transient transfection of the CRISPR-tool. Application of targeted DNA methylation so far has mostly been used to completely silence gene expression by fully methylating the corresponding promoter regions. Here, we aim to tune expression of the associated gene by DNA methylation of confined promoter regions and to apply this technique as a new glycoengineering approach. By coupling CRISPR-based targeted DNA methylation with lectin-FACS assisted sorting we obtained CHO cell lines with a fine-tuned phenotype. First, dCas9-DNMT3A3L in combination with one single gRNA is targeted to the FUT8 promoter to induce confined DNA methylation, resulting in a phenotypically diversified population. Next, a window sorting strategy based on lectin-stained cells using five different sorting gates spanning from low to high FUT8 expression was applied to isolate single clones with a defined phenotype. Isolated clones were phenotypically assessed and re-sorted to obtain a homogenous expression profile. The resulting clonal cell lines showed either tuned or knock-down phenotypes with varying gene expression levels. Two out of seven clones that showed tuned FUT8 gene expression were phenotypically stable over 60 days. Gene expression levels, on the other hand, showed a steady decline over time that in part, however, can be explained by the general variation of FUT8 expression in different growth phases. Importantly, glycan analysis of recombinant EpoFc produced in the tuned clonal cell lines showed ranges of 35-70 % fucosylation, demonstrating that isolated clones can produce recombinant proteins with a distinct glycosylation profile. To understand why some clones showed tuned FUT8 gene expression levels while others were completely knocked-down, we analyzed the DNA methylation status of their respective FUT8 promoter. Critical areas within the FUT8 promoter were identified, with some associated with general repression and others with the tuning of FUT8 gene expression when affected by DNA methylation. Additionally, a combination of histone marks associated with active and repressed promoters was found to potentially define clones with a fine-tuned expression. Combined, the data demonstrates that using targeted DNA methylation in a manner confined to specific promoter regions opens new engineering strategies to fine-tune gene expression in mammalian cells.

Chromosomal inversion is a key structural variation impacting cellular fitness and genomic integrity. Here we developed prime-editing-based inversion with enhanced performance (PIE) to efficiently induce large-scale inversions in mammalian cells. PIEv1 uses a prime-editing guide RNA (pegRNA) pair but yields one imprecise junction. PIEv2 and PIEv3 add a second pegRNA pair for precise inversion, with PIEv3b further enhancing coupling precise inversion through improved plasmid design. PIEv3b achieves inversion efficiencies up to 61.7% for 1 Mb and 14.2% for 50 Mb segments and shows 4-20-fold higher efficiency compared to twin prime editing with integrase, across ranges of 100 kb to 30 Mb. Additionally, PIEv3b outperforms nuclease-based approaches in both inversion efficiency and precision. Using PIE, we convert human chromosomes from metacentric to telocentric configurations by inverting 30-Mb and 100-Mb chromosomal segments. Our work represents a powerful tool for engineering chromosomal structural variations, with broad implications for medicine and biotechnology.