Some embodiments of the systems, methods and compositions provided herein relate to a compound protease. In some embodiments, the compound protease includes a protease domain and a cut site for another enzyme. In some embodiments, the compound protease includes an association domain. In some embodiments, the compound protease is part of a protein circuit.

Provided are fusion proteins and related molecules useful for conducting base editing with reduced or no off-target mutations. The fusion protein may include a first fragment comprising a nucleobase deaminase or a catalytic domain thereof, a second fragment comprising a nucleobase deaminase inhibitor, and a protease cleavage site between the first fragment and the second fragment. Also provided are improved prime editing systems, including prime editing guide RNA with improved stability.

Disclosed herein include methods, compositions, and kits suitable for robust and tunable control of payload gene expression. Some embodiments provide rationally designed circuits, including miRNA-level and/or protein-level incoherent feed-forward loop circuits, that maintain the expression of a payload at an efficacious level. The circuit can comprise a promoter operably linked to a polynucleotide encoding a fusion protein comprising a payload protein, a protease, and one or more self-cleaving peptide sequences. The payload protein can comprise a degron and a cut site the protease is capable of cutting to expose the degron. The circuit can comprise a promoter operably linked to a polynucleotide comprising a payload gene, a silencer effector cassette, and one or more silencer effector binding sequences.

Polynucleotide constructs that express an engineered foot-and-mouth disease (FMDV) P1 precursor protein and a non-FMDV TEV protease and methods for safe and efficient recombinant production of FMDV antigens and immunogens. Recombinant production of FMDV antigens avoids the need to culture highly-infectious FMDV, while conventional culture methods for producing FMDV antigens rely on the native FMDV 3C protease which exerts toxic effects on host cells. The inventors have developed a new system that efficiently and safely processes FMDV P1 precursor without the FMDV 3C protease, thus avoiding the toxic effects associated with use of the 3C protease. The invention is also directed to the FMDV antigens and virus-like particles produced by this system as well as to FMDV vaccines, diagnostics and other biologics.

Provided are methods and compositions relating to conjugation of nucleic acids and proteins of interest under conditions that maintain protein activity. The nucleic acid-protein conjugates may be used in nucleic acid nanostructures such as those generated using DNA origami methods.

Disclosed herein include methods, compositions, and kits suitable for use in winner-take-all neural network computation in mammalian cells. In some embodiments, de novo designed protein heterodimers and engineered viral proteases are combined to implement a synthetic protein circuit that performs winner-take-all neural network computation. The synthetic protein circuit can include modules that compute weighted sums of input protein concentrations through reversible binding interactions, and allow for self-activation and mutual inhibition of protein components using irreversible proteolytic cleavage reactions.

Provided are fusion proteins and related molecules useful for conducting base editing with reduced or no off-target mutations. The fusion protein may include a first fragment comprising a nucleobase deaminase or a catalytic domain thereof, a second fragment comprising a nucleobase deaminase inhibitor, and a protease cleavage site between the first fragment and the second fragment. Also provided are improved prime editing systems, including prime editing guide RNA with improved stability.

Disclosed herein include methods, compositions, and kits suitable for use in detecting the activation level of a signal transducer. In some embodiments, there are provided synthetic protein circuits wherein recruitment of synthetic protein circuit components to an association location upon activation of a signal transducer generates an active effector protein. The effector protein can be configured to carry out a variety of functions when in an active state, such as, for example, inducing cell death. Methods of treating a disease or disorder characterized by aberrant signaling are provided in some embodiments.

The present disclosure provides methods in which adherent cells are treated with small molecules, cultured, lysed, and then analyzed by mass spectrometry to measure the activities of endogenous enzymes. The implementation of this method relies on the use of surfaces that are nanopatterned with cell adhesion ligands to mediate cell attachment and a peptide that is a substrate for the desired enzyme activity in the lysate.

The present invention relates to a nucleic acid molecule encoding a fusion protein, wherein the nucleic acid molecule comprises: (a) a first nucleic acid sequence encoding a first biosensor, wherein said first biosensor is a first molecule capable of interacting with a second molecule; (b) a second nucleic acid sequence encoding an effector-activating module, wherein the effector-activating module comprises a nucleic acid sequence encoding a first part of a protease, wherein said first part of the protease is capable of interacting with a second part of said protease to form an active form of said protease; (c) a third nucleic acid sequence encoding a third biosensor comprising a protease cleavage site, wherein the protease cleavage site is sterically occluded in the absence of a stimulus for said third biosensor and wherein the protease cleavage site becomes accessible in the presence of said stimulus.