Schmidt, T. J. N. | bioRxiv Science

bioRxiv2025

The extremely low mechanical force generated by nano-pulling induces global changes in the microtubule network, nuclear morphology, and chromatin transcription in neurons

Mechanical force plays a pivotal role in every aspect of axon development. In this paper, we explore the use of nano-pulling, a technology that enables the intracellular generation of extremely low mechanical forces. We demonstrate that force-mediated axon growth also exerts global effects that extend to the nuclear level. Our mechanistic studies support a model in which exogenous forces induce stabilization of microtubules, and a significant remodeling of perinuclear microtubules, which preferentially align perpendicularly to the nuclear envelope. We observed an increase in the lateral tension of the nucleus, leading to substantial remodelling of nuclear morphology, characterized by an increase in nuclear grooves and higher sphericity index (indicating less flattened nuclei). Notably, these changes in nuclear shape are linked to chromatin remodelling, resulting in global transcriptional activation.

neuroscience↗

bioRxiv2024

A Transfection-Free Approach of Gene Editing via a gold-based nanoformulation of the Cas9 protein

In recent years, the CRISPR/Cas9 technology has emerged as a highly efficient tool for cell gene editing. However, the delivery of the CRISPR/Cas9 system into cells remains a significant challenge, drastically limiting in vivo gene therapy applications. In this study, we present a transfection/transduction-free tool for intracellular delivery of the Cas9:gRNA ribonucleoprotein. The Cas9 enzyme is conjugated to a 12 nm gold nanoparticle through affinity binding between the 6x His-tag of the protein and the NTA-Ni{superscript 2}LJ groups on the nanoparticles. This link chemistry allows a fine control of the density of the enzymes decorating the particle surface, the orientation of the bonding and the stability of the interaction. Importantly, the surface chemistry of this nanoformulation has been precisely engineered to modulate the cellular internalization and localization. Thanks to this approach of precision chemistry, this nanoformulation demonstrated the ability to spontaneously enter human melanoma cells as monodispersed particles that localize in cell cytoplasm, endosomes, and nucleus. It also shows effective gene editing efficiency similarly to conventional transfection tools. This gold-based formulation of Cas9 represents a ready-to-use biotech editing tool, and a promising solution for direct in vivo gene editing applications.

molecular biology↗