Abstract: Disclosed herein include methods, compositions, and kits suitable for use in tuned dosage-invariant expression of a payload protein. Compositions (e.g., nucleic acid compositions, one or more cells) provided herein can comprise a first promoter sequence operably linked to a first polynucleotide comprising one or more miRNA cassettes, and a second promoter sequence operably linked to a second polynucleotide comprising a payload gene. In some embodiments, the first promoter sequence is capable of inducing transcription of the first polynucleotide to generate a first transcript, and the first transcript is capable of being processed to generate said miRNA. The payload gene can comprise a miRNA target region comprising one or more miRNA target sequences.

Abstract: Disclosed herein include methods, compositions, and kits suitable for use in capturing lineage relationships in dividing cell populations. Disclosed herein include compact phylogenetic recording systems for high resolution lineage reconstruction over long time scales. In some embodiments, the system comprises one or more hypercascade array(s) each comprising p hypercascade units, n layer guide RNAs, and an editor. In some embodiments, the system comprises one or more target array(s) comprising n editable target sites, n guide RNAs gRNAs, and a base editor capable of adenine (A)-to-guanine (G) base editing.

Abstract: Disclosed herein include methods, compositions, and kits suitable for use in the measurement of the states of living cells across time and for use in the delivery of polyribonucleotides and circuits. There are provided, in some embodiments, RNA exporter proteins comprising an RNA-binding domain, a membrane-binding domain, and an interaction domain capable of nucleating self-assembly. Disclosed herein include polynucleotides encoding reporter RNA molecule(s) or cargo RNA molecule(s). In some embodiments, a plurality of RNA exporter proteins are capable of self-assembling into lipid-enveloped nanoparticles (LNs) secreted from a cell in which the RNA exporter proteins are expressed, thereby generating a population of LNs comprising exported reporter RNA molecule(s) or a fusogen and exported cargo RNA molecule(s). Disclosed herein include export modulator proteins capable of, e.g., enhancing or suppressing LN export.

Abstract: Disclosed herein include circuits, compositions, nucleic acids, populations, systems, and methods enabling cells to sense, control, and/or respond to their own population size. In some embodiments, an orthogonal communication channel allows specific communication between engineered cells. Also described herein, in some embodiments, is an evolutionarily robust ‘paradoxical’ regulatory circuit architecture in which orthogonal signals both stimulate and inhibit net cell growth at different signal concentrations. In some embodiments, engineered cells autonomously reach designed densities and/or activate therapeutic or safety programs at specific density thresholds. Methods of treatment are also provided in some embodiments.

Abstract: Disclosed herein include circuits, compositions, nucleic acids, populations, systems, and methods enabling cells to sense, control, and/or respond to their own population size. In some embodiments, an orthogonal communication channel allows specific communication between engineered cells. Also described herein, in some embodiments, is an evolutionarily robust ‘paradoxical’ regulatory circuit architecture in which orthogonal signals both stimulate and inhibit net cell growth at different signal concentrations. In some embodiments, engineered cells autonomously reach designed densities and/or activate therapeutic or safety programs at specific density thresholds. Methods of treatment are also provided in some embodiments.

Abstract: 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.

Abstract: Disclosed herein include methods, compositions, and kits suitable for use in signal amplification. There are provided, in some embodiments, protease-based signal amplification modules. Disclosed herein include amplifier proteins comprising a first part of a first protease domain, a first dimerization domain, a first cut site a protease in a protease active state is capable of cutting, a second dimerization domain, a second cut site a protease in a protease active state is capable of cutting, and a first caging domain. Disclosed herein include companion amplifier proteins comprising a second part of a first protease domain, a third dimerization domain, a third cut site a protease in a protease active state is capable of cutting, a fourth dimerization domain, a fourth cut site a protease in a protease active state is capable of cutting, and a second caging domain.

Abstract: Disclosed herein include methods, compositions, and systems suitable for use in delivering a polynucleotide to a target cell of a subject in need thereof. In some embodiments, a viral vector comprises a polynucleotide encoding nucleoprotein (N), phosphoprotein (P), matrix protein (M), RNA-dependent RNA polymerase (L), and one or more transgenes. The viral vector can comprise one or more of a conditionally stable fusion protein, a protease fusion protein, a degron fusion protein, and/or a glycoprotein derived of another species than the viral vector polynucleotide to enable control of viral vector transduction and/or replication.

Abstract: Disclosed herein include methods, compositions, and kits suitable for use in thresholding of protein signals. There are provided, in some embodiments, synthetic protein circuits that respond to inputs only above or below a certain threshold concentration. In some embodiments, the threshold value itself is tunable. Methods of treating a disease or disorder characterized by aberrant signaling are provided in some embodiments.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: Disclosed herein include methods, compositions, and systems suitable for use in delivering a polynucleotide to a target cell of a subject in need thereof. In some embodiments, a viral vector comprises a polynucleotide encoding nucleoprotein (N), phosphoprotein (P), matrix protein (M), RNA-dependent RNA polymerase (L), and one or more transgenes. The viral vector can comprise one or more of a conditionally stable fusion protein, a protease fusion protein, a degron fusion protein, and/or a glycoprotein derived of another species than the viral vector polynucleotide to enable control of viral vector transduction and/or replication.

Abstract: Disclosed herein include methods, compositions, and kits suitable for use in thresholding of protein signals. There are provided, in some embodiments, synthetic protein circuits that respond to inputs only above or below a certain threshold concentration. In some embodiments, the threshold value itself is tunable. Methods of treating a disease or disorder characterized by aberrant signaling are provided in some embodiments.

Abstract: Disclosed herein include methods, compositions, and kits suitable for use in the measurement of the states of living cells across time. There are provided, in some embodiments, RNA exporter proteins comprising an RNA-binding domain, a membrane-binding domain, and an interaction domain capable of nucleating self-assembly. Disclosed herein include polynucleotides encoding reporter RNA molecule(s). In some embodiments, a plurality of RNA exporter proteins are capable of self-assembling into lipid-enveloped nanoparticles (LNs) secreted from a reporter cell in which the RNA exporter proteins are expressed, thereby generating a population of LNs comprising exported reporter RNA molecule(s).

Abstract: Disclosed herein include methods, compositions, and kits suitable for use in the delivery of polyribonucleotides and circuits. There are provided, in some embodiments, RNA exporter proteins comprising an RNA-binding domain, a membrane-binding domain, and an interaction domain capable of nucleating self-assembly. Disclosed herein include polynucleotides encoding cargo RNA molecule(s). In some embodiments, a plurality of RNA exporter proteins are capable of self-assembling into lipid-enveloped nanoparticles (LNs) secreted from a sender cell in which the RNA exporter proteins are expressed, thereby generating a population of LNs comprising a fusogen and exported cargo RNA molecule(s).

Abstract: 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.

Abstract: Disclosed herein include systems, methods, compositions, and kits for in situ readout of barcodes, such as DNA barcodes. Barcode constructs containing a promoter (e.g., a phage promoter) that is inactive in live cells can be integrated in the genomes of cells. Cells can be fixed, and phage RNA polymerase can be used for transcription of the barcode to RNA transcripts. The RNA transcripts can be detected using, for example, fluorescent imaging and used to determine barcode sequences.

Abstract: Disclosed herein include methods, compositions, and systems suitable for use in delivering a polynucleotide to a target cell of a subject in need thereof. In some embodiments, a viral vector comprises a polynucleotide encoding nucleoprotein (N), phosphoprotein (P), matrix protein (M), RNA-dependent RNA polymerase (L), and one or more transgenes. The viral vector can comprise one or more of a conditionally stable fusion protein, a protease fusion protein, a degron fusion protein, and/or a glycoprotein derived of another species than the viral vector polynucleotide to enable control of viral vector transduction and/or replication.

Abstract: Disclosed herein include systems, methods, compositions, and kits for in situ readout of barcodes, such as DNA barcodes. Barcode constructs containing a promoter (e.g., a phage promoter) that is inactive in live cells can be integrated in the genomes of cells. Cells can be fixed, and phage RNA polymerase can be used for transcription of the barcode to RNA transcripts. The RNA transcripts can be detected using, for example, fluorescent imaging and used to determine barcode sequences.