The rapid development of CRISPR genome editing technologies has established a transformative paradigm within biomedical research, drug discovery, and gene therapy. Despite the robust nuclease activity and programmable targeting exhibited by these systems, the clinical translation of CRISPR-mediated therapeutics remains substantially impeded by systemic and cellular delivery constraints. Specifically, the secure, highly efficient, and spatiotemporally controlled administration of CRISPR machinery to designated tissues or distinct cell populations constitutes the primary bottleneck in the field. Physiological and biological impediments, including rapid clearance, limited tissue penetration, endosomal entrapment, and potential adaptive immune responses, necessitate the development of highly specialized delivery vectors to ensure therapeutic viability and mitigate off-target effects. To circumvent these biological barriers, contemporary research has extensively evaluated a spectrum of delivery vehicles engineered to package and protect diverse CRISPR cargoes, including DNA, RNA, and pre-assembled ribonucleoprotein complexes. These diverse cargos are actively investigated in conjunction with established viral vectors, and an expanding array of non-viral vectors encompassing physical methods, nanoparticle‑based systems, and peptide/protein‑mediated platforms. This review delineates the recent advancements in discovery of CRISPR‑based genome editing tools, with a particular emphasis on delivery strategies across in vitro modeling, ex vivo cellular engineering, and in vivo clinical interventions.
Li Du, Xiao-Feng Hua, Qianquan Ma et al.· Molecular Medicine· 0 citations
Human noroviruses (HuNoVs) are one of the main causative agents of non-bacterial gastroenteritis. The viral capsid protein VP1 is the main target for diagnostics. Although eukaryotic expression systems can produce correctly assembled VP1, they are associated with high costs and complexity. The Escherichia coli prokaryotic expression system is attractive for large-scale expression; however, VP1 often aggregates into insoluble inclusion bodies in conventional strains (BL21 (DE3)). The ArcticExpress (DE3) strain co-expresses psychrophilic chaperonins to improve protein folding at low temperatures enhancing the soluble yield of difficult-to-express targets. In this study, we demonstrated that E. coli ArcticExpress (DE3) under low-temperature induction enabled the high-yield production of soluble full-length VP1 for HuNoVs GII.4 and GII.17. In BL21 (DE3), the mentioned antigens were aggregated into insoluble inclusion bodies. Using ArcticExpress(DE3), VP1 yield was identified as approximately 18-93 mg/L across the tested genotypes. In BL21 (DE3), the protein outcome was genotype-dependent: GII.4 could be recovered only via insoluble-fraction processing, GII.17 remained undetectable or present only at trace levels under the conditions tested. The ArcticExpress-derived viral antigen retained antigenic properties and was recognized by cross-reactive anti-GII antibodies. To our knowledge, this study provides evidence that ArcticExpress (DE3) enable the soluble production of full-length HuNoVs VP1 suitable for downstream immunochemical analysis.
I. Kazlouski, N. Paklonskaya, I. Belskaya et al.· FEMS Microbiology Letters· 0 citations
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