[Construction and characterization of a stable Cas9-expressing monoclonal WSL cell line].

[CRISPR-Cas9 activation screening identifies candidate chemokine regulators of ter-tiary lymphoid structure formation in bladder cancer].

Engineering Stable Lentiviral Vector Producer Cells Improves LVV Production.

Stable lentiviral vector producer cell lines represent a promising platform for scalable and cost-efficient vector manufacturing, yet their productivity is often limited by intrinsic host-cell constraints. In this study, we aimed to identify cellular factors restricting LVV production in stable doxycycline-inducible GPRTG producer cell lines and to evaluate whether targeted host-cell engineering can improve vector yield. Comparative transcriptomic analysis of high- and low-producing clones revealed distinct differences in cellular pathways related to transcription, translation, energy metabolism, lipid homeostasis, stress response, and chromatin regulation. Based on differential gene expression, candidate genes were modified by CRISPR/Cas9-mediated knockout or overexpression and functionally analyzed in a low-producing clone. Single-gene KO screening identified H1-2, ADAMTS1, INSIG1, GADD45B, and HSPA1B to increase cell-specific LVV productivity up to 2.6-fold, with H1-2 showing the strongest effect. In addition, combinatorial disruption further improved productivity. In contrast, overexpression of selected candidates did not enhance LVV production. Overall, our results demonstrate that transcriptomics-guided host-cell engineering is an effective strategy to identify and relieve intrinsic bottlenecks in stable LVV producer cell lines.

Establishment of a scalable engineered cell-line platform for direct, GMP-grade production of eVLP vectors enabling streamlined generation of gene-edited CAR-T/NK cells

An industrial-grade platform based on monoclonal producer cell lines that enables the continuous and scalable generation of engineered virus-like particles (eVLPs) co-packaging Cas9–gRNA ribonucleoproteins (RNPs) and provides a GMP-compliant and broadly adaptable strategy for the streamlined manufacturing of next-generation autologous and allogeneic gene-edited CAR-T/NK therapies.

Isolation, Characterization and Reverse Genetic System Establishment of a Highly Virulent PEDV Strain

Porcine epidemic diarrhea virus (PEDV) G2c variants have recently emerged, posing significant challenges to swine health management. As a major coronavirus affecting the swine industry, PEDV exhibits extensive genetic variability, which has greatly complicated disease control. Current vaccines provide suboptimal protection under field conditions. Therefore, the isolation of recently circulating strains and the establishment of a robust reverse genetics system are critical for advancing the study of emerging variants and facilitating rational vaccine development. In this study, a PEDV field strain designated PEDV-BJ-2023 was isolated from diarrheic piglets in Guizhou, China. Phylogenetic analysis based on the complete genome and spike gene classified PEDV-BJ-2023 within the emerging G2c lineage. To facilitate functional studies, a full-length infectious cDNA clone was constructed using transformation-associated recombination cloning in yeast. Furthermore, an enhanced green fluorescent protein reporter virus was generated via CRISPR/Cas9-assisted homologous recombination by inserting an EGFP-2A cassette upstream of the nucleocapsid gene. The recombinant viruses displayed virion morphology and plaque characteristics similar to those of the parental wild-type PEDV-BJ-2023 strain, although the parental virus exhibited faster replication during the early stage of infection in vitro. In 5-day-old piglets, all three viruses caused severe diarrhea, weight loss, and intestinal lesions; however, recombinant viruses exhibited slightly reduced viral shedding and pathogenicity, with rPEDV-EGFP being the most attenuated. Notably, rPEDV-EGFP maintained stable EGFP expression over eight serial passages. This study establishes a reverse genetics platform for an emerging G2c PEDV strain and provides a stable fluorescent reporter virus, offering valuable tools for visualizing viral infection and investigating virus–host interactions.

Targeting Cas9 to Perform Rescue Experiments in Stably Transduced Knockout HEK293 Cells.

Cas9 ablation enabled restoration of CUL4B expression and function following introduction of a CUL4B expression plasmid, providing proof of concept for a broadly applicable approach to studying protein function through rescue experiments.

SLC25A38 gene modification mediated by CRISPR/Cas9 in HEK293T cell line.

It is reported that the choice of sgRNA target site affects its targeted cleavage activity and show that the combination of guideRNA with SpCas9 can effectively delete the exonic fragment of the SLC25A38 gene in HEK293T cells.