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Proteins capable of switching between distinct active states in response to biochemical cues are ideal for sensing and controlling biological processes. Activatable CRISPR‐Cas systems are significant in precise genetic manipulation and sensitive molecular diagnostics, yet directly controlling Cas protein function remains challenging. Herein, we explore anti‐CRISPR (Acr) proteins as modules to create synthetic Cas protein switches (CasPSs) based on computational chemistry‐directed rational protein interface engineering. Guided by molecular fingerprint analysis, electrostatic potential mapping, and binding free energy calculations, we rationally engineer the molecular interaction interface between Cas12a and its cognate Acr proteins (AcrVA4 and AcrVA5) to generate a series of orthogonal protease‐responsive CasPSs. These CasPSs enable the conversion of specific proteolytic events into activation of Cas12a function with high switching ratios (up to 34.3‐fold). These advancements enable specific proteolysis‐inducible genome editing in mammalian cells and sensitive detection of viral protease activities during virus infection. This work provides a promising strategy for developing CRISPR‐Cas tools for controllable gene manipulation and regulation and clinical diagnostics. [ABSTRACT FROM AUTHOR]

Proteins capable of switching between distinct active states in response to biochemical cues are ideal for sensing and controlling biological processes. Activatable CRISPR‐Cas systems are significant in precise genetic manipulation and sensitive molecular diagnostics, yet directly controlling Cas protein function remains challenging. Herein, we explore anti‐CRISPR (Acr) proteins as modules to create synthetic Cas protein switches (CasPSs) based on computational chemistry‐directed rational protein interface engineering. Guided by molecular fingerprint analysis, electrostatic potential mapping, and binding free energy calculations, we rationally engineer the molecular interaction interface between Cas12a and its cognate Acr proteins (AcrVA4 and AcrVA5) to generate a series of orthogonal protease‐responsive CasPSs. These CasPSs enable the conversion of specific proteolytic events into activation of Cas12a function with high switching ratios (up to 34.3‐fold). These advancements enable specific proteolysis‐inducible genome editing in mammalian cells and sensitive detection of viral protease activities during virus infection. This work provides a promising strategy for developing CRISPR‐Cas tools for controllable gene manipulation and regulation and clinical diagnostics. [ABSTRACT FROM AUTHOR]

The efficient and robust signal reporting ability of CRISPR-Cas system exhibits huge value in biosensing, but its applicability for non-nucleic acid analyte detection relies on the coupling of additional recognition modules. To address this limitation, we described a switchable Cas12a and exploited it for CRISPR-based direct analysis of histone deacetylase (HDAC) activity. Starting from the acetylation-mediated inactivation of Cas12a by anti-CRISPR protein AcrVA5, we demonstrated that the acetyl-inactivated Cas12a could be reversibly activated by HDAC-mediated deacetylation based on computational simulations (e.g., deep learning and protein-protein docking analysis) and experimental verifications. By leveraging this switchable Cas12a for both target sensing and signal amplification, we established a sensitive one-pot assay capable of detecting deacetylase sirtuin-1 with sub-nanomolar sensitivity, which is 50 times lower than the standard two-step peptide-based assay. The versability of this assay was validated by the sensitive assessment of cellular HDAC activities in different cell lines with good accuracy, making it a valuable tool for biochemical studies and clinical diagnostics. [ABSTRACT FROM AUTHOR]

Selective modulation of ligand–receptor interaction is essential in targeted therapy. In this study, we design an intelligent "scan and unlock" DNA automaton (SUDA) system to equip a native protein‐ligand with cell‐identity recognition and receptor‐mediated signaling in a cell‐type‐specific manner. Using embedded DNA‐based chemical reaction networks (CRNs) on the cell surface, SUDA scans and evaluates molecular profiles of cell‐surface proteins via Boolean logic circuits. Therefore, it achieves cell‐specific signal modulation by quickly unlocking the protein‐ligand in proximity to the target cell‐surface to activate its cognate receptor. As a proof of concept, we non‐genetically engineered hepatic growth factor (HGF) with distinct logic SUDAs to elicit target cell‐specific HGF signaling and wound healing behaviors in multiple heterogeneous cell types. Furthermore, the versatility of the SUDA strategy was shown by engineering tumor necrotic factor‐α (TNFα) to induce programmed cell death of target cell subpopulations through cell‐specific modulation of TNFR1 signaling. [ABSTRACT FROM AUTHOR]

Selective modulation of ligand–receptor interaction is essential in targeted therapy. In this study, we design an intelligent "scan and unlock" DNA automaton (SUDA) system to equip a native protein‐ligand with cell‐identity recognition and receptor‐mediated signaling in a cell‐type‐specific manner. Using embedded DNA‐based chemical reaction networks (CRNs) on the cell surface, SUDA scans and evaluates molecular profiles of cell‐surface proteins via Boolean logic circuits. Therefore, it achieves cell‐specific signal modulation by quickly unlocking the protein‐ligand in proximity to the target cell‐surface to activate its cognate receptor. As a proof of concept, we non‐genetically engineered hepatic growth factor (HGF) with distinct logic SUDAs to elicit target cell‐specific HGF signaling and wound healing behaviors in multiple heterogeneous cell types. Furthermore, the versatility of the SUDA strategy was shown by engineering tumor necrotic factor‐α (TNFα) to induce programmed cell death of target cell subpopulations through cell‐specific modulation of TNFR1 signaling. [ABSTRACT FROM AUTHOR]

The comprehensive understanding of the mechanisms underlying the interaction of cells with their membrane microenvironment is of great value for fundamental biological research; however, tracking biomolecules on cell surfaces with high temporal and spatial resolution remains a challenge. Herein, a modular strategy is presented for the construction of cell surface DNA‐based sensors by engineering DNA motifs and synthetic cofactors. In this strategy, a stimuli‐reactive organic molecule is employed as the cofactor for the DNA motif, and the self‐assembly of them forms a FRET‐based holo DNA‐based sensor. With the use of the DNA‐based sensors, the versatility of this modular strategy has been demonstrated in the ratiometric imaging of the cellular extrusion process of endogenous signaling molecules, including sulfur dioxide derivatives and nitric oxide. [ABSTRACT FROM AUTHOR]

The comprehensive understanding of the mechanisms underlying the interaction of cells with their membrane microenvironment is of great value for fundamental biological research; however, tracking biomolecules on cell surfaces with high temporal and spatial resolution remains a challenge. Herein, a modular strategy is presented for the construction of cell surface DNA‐based sensors by engineering DNA motifs and synthetic cofactors. In this strategy, a stimuli‐reactive organic molecule is employed as the cofactor for the DNA motif, and the self‐assembly of them forms a FRET‐based holo DNA‐based sensor. With the use of the DNA‐based sensors, the versatility of this modular strategy has been demonstrated in the ratiometric imaging of the cellular extrusion process of endogenous signaling molecules, including sulfur dioxide derivatives and nitric oxide. [ABSTRACT FROM AUTHOR]

A charge designable and tunable green fluorescent protein (GFP)-based protein delivery strategy was proposed. The acquired His29GFP selectively permeates the cell membrane at a target pH of 6.5 and escapes from the endosome efficiently. The delivered RNase A caused substantial mRNA degradation in HeLa cells, and proliferation inhibition in different cell lines and a 3D tumor model at pH 6.5. [ABSTRACT FROM AUTHOR]

Macrophages are key responders of inflammation and are closely related with oxidative stress. Activated macrophages can enhance oxygen depletion, which causes an overproduction of reactive oxygen species (ROS) and leads to further excessive inflammatory response and tissue damage. Agmatine, an endogenous metabolite of L-arginine, has recently been shown to have neuroprotective effects based on its antioxidant properties. However, the antioxidant effects of agmatine in peripheral tissues and cells, especially macrophages, remain unclear. In this study we explored the role of agmatine in mediating antioxidant effects in RAW 264.7 cells and studied its antioxidant mechanism. Our data demonstrate that agmatine is an activator of Nrf2 signaling that markedly enhances Nrf2 nuclear translocation, increases nuclear Nrf2 protein level, up-regulates the expression of the Nrf2 downstream effector HO-1, and attenuates ROS generation induced by Lipopolysaccharide (LPS). We further demonstrated that the agmatine-induced activation of Nrf2 is likely through the PI3K/Akt pathway. LY294002, a specific PI3K/Akt inhibitor, abolished agmatine-induced HO-1 up-regulation and ROS suppression significantly. Inhibiting HO-1 pathway significantly attenuated the antioxidant effect of agmatine which the products of HO-1 enzymatic activity contributed to. Furthermore, the common membrane receptors of agmatine were evaluated, revealing that α2-adrenoceptor, I1-imidazoline receptor or I2-imidazoline receptor are not required by the antioxidant properties of agmatine. Taken together, our findings revealed that agmatine has antioxidant activity against LPS-induced ROS accumulation in RAW 264.7 cells involving HO-1 expression induced by Nrf2 via PI3K/Akt pathway activation. [ABSTRACT FROM AUTHOR]