Synthetic circuits based on split Cas9 to detect cellular events

Abstract Synthetic biology involves the engineering of logic circuit gates that process different inputs to produce specific outputs, enabling the creation or control of biological functions. While CRISPR has become the tool of choice in molecular biology due to its RNA-guided targetability to other nucleic acids, it has not been frequently applied to logic gates beyond those controlling the guide RNA (gRNA). In this study, we present an adaptation of split Cas9 to generate logic gates capable of sensing biological events, leveraging a Cas9 reporter (EGxxFP) to detect occurrences such as cancer cell origin, epithelial to mesenchymal transition (EMT), and cell–cell fusion. First, we positioned the complementing halves of split Cas9 under different promoters—one specific to cancer cells of epithelial origin ( p hCEA) and the other a universal promoter. The use of self-assembling inteins facilitated the reconstitution of the Cas9 halves. Consequently, only cancer cells with an epithelial origin activated the reporter, exhibiting green fluorescence. Subsequently, we explored whether this system could detect biological processes such as epithelial to mesenchymal transition (EMT). To achieve this, we designed a logic gate where one half of Cas9 is expressed under the p hCEA, while the other is activated by TWIST1. The results showed that cells undergoing EMT effectively activated the reporter. Next, we combined the two inputs (epithelial origin and EMT) to create a new logic gate, where only cancer epithelial cells undergoing EMT activated the reporter. Lastly, we applied the split-Cas9 logic gate as a sensor of cell–cell fusion, both in induced and naturally occurring scenarios. Each cell type expressed one half of split Cas9, and the induction of fusion resulted in the appearance of multinucleated syncytia and the fluorescent reporter. The simplicity of the split Cas9 system presented here allows for its integration into various cellular processes, not only as a sensor but also as an actuator

Genetically Engineered Lung Cancer Cells for Analyzing Epithelial–Mesenchymal Transition

Cell plasticity, defined as the ability to undergo phenotypical transformation in a reversible manner, is a physiological process that also exerts important roles in disease progression. Two forms of cellular plasticity are epithelial–mesenchymal transition (EMT) and its inverse process, mesenchymal–epithelial transition (MET). These processes have been correlated to the poor outcome of different types of neoplasias as well as drug resistance development. Since EMT/MET are transitional processes, we generated and validated a reporter cell line. Specifically, a far-red fluorescent protein was knocked-in in-frame with the mesenchymal gene marker VIMENTIN (VIM) in H2170 lung cancer cells. The vimentin reporter cells (VRCs) are a reliable model for studying EMT and MET showing cellular plasticity upon a series of stimulations. These cells are a robust platform to dissect the molecular mechanisms of these processes, and for drug discovery in vitro and in vivo in the future

Optogenetic control of NOTCH1 signaling

The Notch signaling pathway is a crucial regulator of cell differentiation as well as tissue organization, whose deregulation is linked to the pathogenesis of different diseases. NOTCH1 plays a key role in breast cancer progression by increasing proliferation, maintenance of cancer stem cells, and impairment of cell death. NOTCH1 is a mechanosensitive receptor, where mechanical force is required to activate the proteolytic cleavage and release of the Notch intracellular domain (NICD). We circumvent this limitation by regulating Notch activity by light. To achieve this, we have engineered an optogenetic NOTCH1 receptor (optoNotch) to control the activation of NOTCH1 intracellular domain (N1ICD) and its downstream transcriptional activities. Using optoNotch we confirm that NOTCH1 activation increases cell proliferation in MCF7 and MDA-MB-468 breast cancer cells in 2D and spheroid 3D cultures, although causing distinct cell-type specific migratory phenotypes. Additionally, optoNotch activation induced chemoresistance on the same cell lines. OptoNotch allows the fine-tuning, ligand-independent, regulation of N1ICD activity and thus a better understanding of the spatiotemporal complexity of Notch signaling. Video Abstract

Optogenetic control of NOTCH1 signaling

Abstract The Notch signaling pathway is a crucial regulator of cell differentiation as well as tissue organization, whose deregulation is linked to the pathogenesis of different diseases. NOTCH1 plays a key role in breast cancer progression by increasing proliferation, maintenance of cancer stem cells, and impairment of cell death. NOTCH1 is a mechanosensitive receptor, where mechanical force is required to activate the proteolytic cleavage and release of the Notch intracellular domain (NICD). We circumvent this limitation by regulating Notch activity by light. To achieve this, we have engineered an optogenetic NOTCH1 receptor (optoNotch) to control the activation of NOTCH1 intracellular domain (N1ICD) and its downstream transcriptional activities. Using optoNotch we confirm that NOTCH1 activation increases cell proliferation in MCF7 and MDA-MB-468 breast cancer cells in 2D and spheroid 3D cultures, although causing distinct cell-type specific migratory phenotypes. Additionally, optoNotch activation induced chemoresistance on the same cell lines. OptoNotch allows the fine-tuning, ligand-independent, regulation of N1ICD activity and thus a better understanding of the spatiotemporal complexity of Notch signaling. Video Abstrac