Aug 2026· Molecular Biotechnology· 0 citations· 34 references
Medicine
TL;DR
This study demonstrated that UCOE-mediated transcriptional stabilization establishes a robust expression framework while EGFP-based FACS screening enables efficient enrichment of high-producing cells and enhances protein yield with potential for further optimization in larger-scale applications.
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.
Jona Röscheise, Lena-Marie Eberle, Holger Laux et al.· Biotechnology and Bioenginee...· 0 citations
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.
Wei Lin, Jiaru Shi, Hanyi Chen et al.· Frontiers in Immunology· 0 citations
Mosaic animals are highly valuable for investigating complex biological processes and cell lineages in vivo. Traditional mosaic techniques in Drosophila, such as the FRT/Flp system, rely on exogenous site-specific recombination sequences, preventing their application to unmodified mutant chromosomes or wild-derived strains. Mosaic analysis by gRNA-induced crossing-over (MAGIC) overcomes this limitation by utilizing the CRISPR/Cas9 system to generate targeted double-strand breaks (DSBs) that induce somatic homologous recombination in precursor cells. Here, we describe a comprehensive protocol for applying MAGIC with a newly developed, genome-wide MAGIC kit. This protocol utilizes optimized gRNA-markers with the Qtg2.1 scaffold for high-efficiency clone induction, alongside improved fluorescent labeling strategies for both positive MAGIC (pMAGIC) and negative MAGIC (nMAGIC). The procedure details the genetic crossing schemes, temporal induction of clones, and tissue processing for diverse Drosophila cell types. This method enables convenient mosaic analysis across all chromosomes and allows for the study of pericentromeric genes, deficiency chromosomes, and species-specific alleles in interspecific hybrids. Key features • Recombinase-independent: Generates somatic mosaic clones using CRISPR/Cas9 without requiring pre-inserted FRT sequences on the test chromosome. • Genome-wide application: Includes a complete toolkit of pMAGIC and nMAGIC gRNA-markers optimized for all Drosophila chromosomal arms (X, 2L, 2R, 3L, 3R, and 4). • Optimized labeling: Employs destabilized Gal80 for brighter pMAGIC clones and tub-3xHA-BFP/IFP for unambiguous visualization of nMAGIC clones. • Broad compatibility: Applicable to a wide variety of tissues (e.g., neurons, glia, imaginal discs, polyploid tissues) and complex genetic backgrounds, including pericentromeric mutations and deficiencies.
Self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-spe-cific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each target locus, substantially lowers the bar on mouse and human transgenesis.
Mandana Arbab, Sharanya Srinivasan, Tatsunori Hashimoto et al.· 0 citations
A virus-like particle (VLP)-based toolkit that delivers diverse CRISPR editing modalities to human monocytes, macrophages and dendritic cells with high efficiency while preserving viability and innate immune responsiveness is presented.
Hyuncheol Jung, Pascal Devant, Carter Ching et al.· Nature Biotechnology· 0 citations
The CRISPR-condensate system merges the dramatically enhanced transcriptional efficacy with the reduced complexity of components, providing a modular system for fine-tuned gene expression regulation and makes biomolecular condensation a general principle for enhancing CRISPR gene regulation.
Aolin Li, Congcong Cao, Chunyan Yang et al.· Theranostics· 0 citations