🧬 PubMed RNA Editing Daily Digest

8-Chloro-adenosine (8-Cl-Ado) is a promising antitumor agent, and ferroptosis plays a critical role in breast cancer progression. Our previous work demonstrated that 8-Cl-Ado inhibits breast cancer cell proliferation by targeting adenosine deaminase acting on RNA 1 (ADAR1), an RNA-editing enzyme. However, whether 8-Cl-Ado exerts its anti-tumor effects through the modulation of ferroptosis remains largely unknown. The effects of 8-Cl-Ado on ferroptosis were assessed in vitro and in vivo. The molecular mechanisms of 8-Cl-Ado were investigated by performing bioinformatics analysis, RNA immunoprecipitation assay (RIP), luciferase reporter assay, fluorescence in situ hybridization (FISH), qRT-PCR, and western blotting. 8-Cl-Ado significantly inhibited the proliferation, migration, and invasion of MCF-7 and MDA-MB-231 breast cancer cells, while promoting ferroptosis, as evidenced by elevated levels of intracellular Fe Our study identifies a novel pathway by which 8-Cl-Ado promotes ferroptosis through the ADAR1/miR-101-3p/SLC7A11 axis to suppress breast cancer progression. These findings highlight the therapeutic potential of 8-Cl-Ado as a ferroptosis-inducing agent by targeting ADAR1.

Promoters and vectors are critical components of gene therapy, enabling the delivery and expression of therapeutic genes to correct both loss- and gain-of-function mutations. Adeno-associated virus (AAV) vectors are the leading platform for in vivo gene delivery; however, the widely used Streptococcus pyogenes Cas9 (SpCas9, 4.1 kb) approaches the AAV packaging limit of 4.7 kb. This constraint often necessitates dual-vector systems, which reduce therapeutic efficiency, or the use of smaller nucleases such as SaCas9 (3.2 kb) and AacCas12b (3.4 kb), which have lower PAM site frequencies. To enhance promoter selection for gene therapy applications, we developed a strategy to identify compact, cell-preferred RNA polymerase II (Pol II) promoters. Analysis of approximately 300 compact Pol II promoters revealed that exogenous expression levels in one cell type correlate more strongly with those in other cell types than with endogenous expression, underscoring the importance of exogenous expression efficiency in promoter selection. Using this approach, we identified a compact Pol II promoter #2 (Pro2, 133 bp) that drives robust transgene expression in human retinal ganglion cells (RGCs). To enable single-AAV delivery of SpCas9, we analyzed three commonly used Pol III promoters (H1, 7SK and U6) and determined their minimal functional lengths using a CRISPR/Cas9 reporter assay. We further engineered three compact hybrid Pol II/III promoters which combined pro2 with minimal H1, 7SK and U6 (276, 294, and 323 bp) capable of co-expressing SpCas9 and gRNA, enabling efficient genome editing in both transfected HEK293 cells (approaching 100%) and human RGCs (up to 55.9%) from human stem cell-derived retinal ganglion cells (RGCs). Together, these findings establish a framework for developing single-AAV CRISPR-based gene therapy strategies. PWZ and DJZ conceived the study, designed the experiments, performed data analysis and interpretation, and were the primary contributors to manuscript writing. STZ played a key role in data collection and correlation analysis. YYC, SL, LF, CJK, YD, CAB, JC, and DW contributed to the execution of essential experiments and subsequent data analysis. All authors have read and approved the final manuscript. The authors declare no conflicts of interest.

Prime editing is a versatile "search-and-replace" genome-editing technology that enables precise and flexible genome correction of genetic sequences by reverse-transcribing an RNA template encoded at the 3' end of a prime editing guide RNA (pegRNA). It supports the introduction of nucleotide substitutions, and insertions or/and deletions (indels) in living cells without requiring double-stranded DNA breaks or exogenous donor templates. Since its introduction in 2019, prime editing has advanced rapidly-from the first-generation prime editor (PE1) to PE7 and other next-generation variants-with editing efficiencies increasing from 0.7 to 5.5 % to more than 50 % in vitro. Optimization strategies including engineering of the Cas9 and reverse transcriptase domains, refinement of pegRNA architecture, recruitment of auxiliary proteins, and modulation of DNA repair pathways have substantially enhanced editing efficiency, product purity, and target scope across diverse cell types and tissues. These developments are particularly relevant to ophthalmology, where many blinding disorders arise from point mutations or small indels ideally suited for prime editing-based correction. Recent work in retinal cells and animal models has demonstrated the growing feasibility of prime editing to treat inherited retinal diseases, modulate pathological angiogenesis, and achieve precise gene repair in post-mitotic photoreceptors and retinal pigment epithelial cells. As delivery vectors and newer PE variants improve, prime editing is a plausible next-generation platform for a wide range of ocular diseases.

Insect cells are one of the uprising expression systems in the biopharmaceutical industry to produce vaccines and gene therapy vectors, but cell line development has been limited by the lack of established genetic engineering tools and genomic characterization. CRISPR-Cas9 has arisen as a powerful tool for gene editing but has seen little application in insect cells. In this work, a gene editing pipeline for the delivery of a ribonucleoprotein (RNP) complex comprised of a guide RNA and the enzyme Cas9 to insect Sf9 cells was implemented and then applied to knockout caspase initiator Sf-Dronc, aiming at alleviating cell apoptosis during an infection process. The resulting engineered cell lines were characterized as per their phenotype and production of three different product modalities. Utilizing the established workflow, a knockout rate of 68% was achieved with the implemented protocol (vs. the 12% presumed efficiency of a previously reported system) when targeting the fdl gene. When applied to Sf-Dronc, mutants containing deletions in several alleles of the host genome were identified and confirmed by next-generation sequencing. Generated clones exhibited higher apoptosis resistance and delayed onset of cell viability drop following infection with baculovirus. While Sf-Dronc deletion was shown to have negligible impact on the production of rAAV and PfRipr5, production of iVLPS showed an > twofold increase over wild-type Sf9. Overall, this study showcases the successful implementation of an efficient CRISPR-Cas9 pipeline, further leveraging the usage of genetic engineering in insect Sf9 cells towards the development of enhanced cell hosts for biopharmaceutical production. KEY POINTS: • Implementation of an efficient CRISPR-Cas9 RNP complex delivery strategy to insect cells. • Establishment of the genome editing pipeline demonstrated through Sf-Dronc knockout, resulting in increased apoptosis resistance and delayed loss of viability upon baculovirus infection. • Sf-Dronc deletion led to over a twofold increase in the production of influenza VLPs compared to wild-type Sf9 cells.

Cas9 can process poly(T) single-stranded DNA molecules upon activation in an RNA-guided manner. Here, we uncover key determinants underlying this function. First, we show that unflanked R-loops in the RNA 5' side favor trans-cleavage activity, which occur when targeting short double-stranded DNA molecules. Second, we show that elongated guide RNA spacers beyond the canonical 20 bases, even by a few bases, severely impair this collateral activity. Third, although trans-cleavage is mediated by the RuvC domain, we show that a catalytically active HNH domain contributes to an efficient process. Analysis of structural models provides tentative mechanistic insights. Together, these findings illustrate that fine modulation of Cas9 function can be achieved.