The present disclosure provides methods for testing whether a target protein regulates viral RNA translation. The methods comprise a) introducing into a test host cell (where the test host cell comprises a catalytically inactive or a catalytically active CRISPR/Cas effector polypeptide): i) a reporter nucleic acid comprising a nucleotide sequence encoding a bicistronic translation monitor; and ii) a regulatory nucleic acid comprising a nucleotide sequence encoding a single guide RNA (sgRNA) that comprises a targeting sequence that specifically binds to a target sequence within a nucleic acid encoding the target protein; and b) detecting expression of the reporter proteins to determine whether a target protein regulates viral RNA translation via a Cap-dependent element or a Cap-independent element based on the expression of the reporter proteins in the test host cell as compared to the expression in the control host cell. Kits for conducting the methods disclosed herein are also provided.

The present invention relates to genetically modified bacteria and methods of optimizing genetically modified bacteria for the production of a metabolite.

Provided herein is a method for manufacturing a microarray system, for example, 3-dimensional lattice microarray system, for DNA sequence detection and analysis. A solid support, such as a plastic substrate, is contacted with a formulation containing a plurality of nucleic acid probes, a plurality of bifunctional polymer linkers, such as oligothymidine linkers, and a solvent mixture of water and a water-miscible liquid. The bifunctional polymer linkers are attached to the solid support and the water is evaporated. Then the nucleic acid probes are attached to the bifunctional polymer linker.

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).

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).

Provided herein are methods of identifying a cell uptake modulator of a molecule that include (a) contacting a plurality of cells of a cell-containing biological sample with a plurality of gene-editing agents, wherein a gene-editing agent from the plurality of gene-editing agents recognizes and alters a target gene of at least one cell of the plurality of cells; (b) contacting the plurality of cells with a plurality of molecules, wherein at least one molecule of the plurality of molecules is transported into at least one cell of the plurality of cells; and (c) detecting a presence of the at least one molecule in the plurality of cells, thereby identifying the cell uptake modulator of the molecule.

Provided herein are methods and compositions for batch production of producer cells using fluorescence activated cell sorting (FACS). In some aspects, the disclosure provides a drug-selection-free method for batch production of producer cells using FACS. Such batch production methods and compositions can be further utilized to generate clonal populations of producer cells, e.g., for large-scale manufacturing of a polypeptide of interest.

Tobacco etch virus protease (TEV) is one of the most widely used proteases in biotechnology because of its exquisite sequence-specificity. A limitation of TEV is its slow catalytic rate, which limits product generation and therefore signal output. Provided is a generalizable yeast-based platform for directed evolution of protease catalytic properties. Protease activity is determined via proteolytic release of a membrane-anchored transcription factor, and access to TEV's cleavage site is temporally regulated using a photosensory LOV domain. By gradually decreasing light exposure time, faster variants of TEV were selected over multiple rounds of selection. The mutant TEV proteases and the directed evolution platform are useful in a wide range of biotechnology applications, such as FLARE and SPARK tools.

The disclosure provides methods for the concurrent assessment of large numbers of genome engineering proteins, including CRISPR nucleases and base editors. Specifically, the disclosure provides methods of providing a plurality of individual discrete samples comprising populations of cells, wherein each population of cells overexpresses both (i) a single genome engineering protein or a variant thereof and (ii) a reporter protein, lysing the cells to release the proteins; normalizing levels of the genome engineering proteins or variants thereof; allowing the genome engineering proteins or variants thereof to combine with a guide RNA under conditions sufficient to form ribonucleoprotein complexes in each sample; contacting each sample with a plurality of analysis substrates, determining levels of each of the analysis substrate in each sample at a plurality of times; and calculating rate of depletion or enrichment of each of the analysis substrates from each sample.

Embodiments herein describe systems and methods to identify genetic silencers. Many embodiments screen one or more DNA fragments to identify sequences that are capable of silencing gene expression. Once identified, silencer elements can be utilized for many applications, including precision medicine.