Abstract: The technology described herein is directed to regulated synthetic gene expression systems. In one aspect described herein are synthetic transcription factors (synTFs) comprising a DNA binding domain, a transcriptional activator domain, a transcriptional effector domain (TED), and optionally a regulator protein. In other aspects described herein are gene expression systems comprising said synTFs and methods of treating diseases and disorders using said synTFs.

Abstract: The present disclosure provides Cas9 variants, and base editors comprising these variants, that recognize non-canonical protospacer adjacent motifs (PAMs) and have less restrictive PAM requirements for editing. The present disclosure provides Cas9 protein variants comprising one or more amino acid substitutions relative to wild-type Nme2Cas9. Fusion proteins comprising the Cas protein variants described herein are also provided by the present disclosure. Further provided herein are methods for editing a target nucleic acid using the Cas variants and fusion proteins provided herein. The present disclosure also provides guide RNAs, complexes, polynucleotides, cells, kits, and pharmaceutical compositions. Further described herein are phage-assisted continuous evolution (PACE) systems, vectors, methods, and devices.

Abstract: Provided herein, in various embodiments, are mammalian cells (e.g., immune effector cells) comprising a nucleotide sequence encoding an exogenous fusogen. Also provided herein, in various embodiments, are methods of treating cancer in a subject in need thereof, comprising administering to the subject mammalian cells (e.g., immune effector cells) disclosed herein. Also provided herein, in various embodiments, are methods of killing a cancer cell, comprising contacting the cancer cell with mammalian cells (e.g., immune effector cells) disclosed herein.

Abstract: The methods and compositions described herein are directed to regulated synthetic gene expression systems. In particular, the technology described herein relates to compositions, systems and methods for inducible and transient (e.g., reversible) transcriptional repression of a target transcript of interest (GOI). The methods, compositions and systems described herein relate to engineered synthetic transcription factors (synTF) that are activated by an inducer molecule, which induces the transcription of a repressor or gene editing molecule from a synthetic inducible repressor constructs where the antisense repressor (or gene editing molecule) mediates reversible repression of a target transcript of interest (GOI) in the presence of the inducer.

Abstract: Embodiments disclosed herein provide artificial expression systems comprising multivalent transcription factor complexes for cooperative transcription factor assembly and modulating gene expression. More specifically, engineered synthetic transcription factors are recruited and structurally organized on synthetic gene circuits using molecular clamps, where the strength of intra-complex interactions can be modulated for fine tuning of gene expression as desired.

Abstract: Embodiments disclosed herein provide artificial expression systems comprising multivalent transcription factor complexes for cooperative transcription factor assembly and modulating gene expression. More specifically, engineered synthetic transcription factors are recruited and structurally organized on synthetic gene circuits using molecular clamps, where the strength of intra-complex interactions can be modulated for fine tuning of gene expression as desired.

Abstract: The present disclosure generally relates to, inter alia, a new class of chimeric Notch receptors containing a synthetic zinc finger transcriptional effector (synZTE) module, engineered to modulate gene expression and cellular activities in a ligand-dependent manner. The new Notch receptors surprisingly retain the ability to transduce signals in response to ligand binding despite that the Notch extracellular subunit (NEC), which includes the negative regulatory region (NRR) previously believed to be essential for the functioning of Notch receptors, is partly or completely deleted. In addition, the synZTE is designed to bind orthogonal DNA target sequences in target organisms which in turn facilitates precise regulation of therapeutic gene expression with minimal off-target activity.

Abstract: The technology described herein is directed to regulated synthetic gene expression systems. In one aspect described herein are synthetic transcription factors (synTFs) comprising a DNA binding domain, a transcriptional effector domain, and a regulator protein. In other aspects described herein are gene expression systems comprising said synTFs and methods of treating diseases and disorders using said synTFs.

Abstract: The technology described herein is directed to regulated synthetic gene expression systems. In one aspect described herein are synthetic transcription factors (synTFs) comprising a DNA binding domain, a transcriptional effector domain, and a regulator protein. In other aspects described herein are gene expression systems comprising said synTFs and methods of treating diseases and disorders using said synTFs.

Abstract: The present disclosure generally relates to, inter alia, a new class of chimeric Notch receptors containing a synthetic zinc finger transcriptional effector (synZTE) module, engineered to modulate gene expression and cellular activities in a ligand-dependent manner. The new Notch receptors surprisingly retain the ability to transduce signals in response to ligand binding despite that the Notch extracellular subunit, which includes the negative regulatory region previously believed to be essential for the functioning of Notch receptors, is partly or completely deleted. In addition, the synZTE is designed to bind orthogonal DNA target sequences in target organisms which in turn facilitates precise regulation of therapeutic gene expression with minimal off-target activity.

Abstract: Disclosed herein are a high-throughput continuous culture system and novel methodologies for the experimental evolution of natural and synthetic microbes using the continuous culture system. The microbial culture is exposed to a stress ramp function which is overlaid on top of a culture fitness function. The amount of stress applied to the culture is increased in response to increased fitness of the microbial culture.

Abstract: The technology described herein is directed to regulated synthetic gene expression systems. In one aspect described herein are synthetic transcription factors (synTFs) comprising a DNA binding domain, a transcriptional effector domain, and a regulator protein. In other aspects described herein are gene expression systems comprising said synTFs and methods of treating diseases and disorders using said synTFs.

Abstract: Disclosed herein are a high-throughput continuous culture system and novel methodologies for the experimental evolution of natural and synthetic microbes using the continuous culture system. The microbial culture is exposed to a stress ramp function which is overlaid on top of a culture fitness function. The amount of stress applied to the culture is increased in response to increased fitness of the microbial culture.

Abstract: Embodiments disclosed herein provide artificial expression systems comprising multivalent transcription factor complexes for cooperative transcription factor assembly and modulating gene expression. More specifically, engineered synthetic transcription factors are recruited and structurally organized on synthetic gene circuits using molecular clamps, where the strength of intra-complex interactions can be modulated for fine tuning of gene expression as desired.

Abstract: Provided herein are engineered systems and methods for establishing DNA adenine methylation at specific genomic locations and using DNA adenine methylation as an artificial chemical “handle” on the genome. These systems and methods allow for placing the handle on specific genomic locations as well as molecular technologies to bind, spatially spread, and maintain the handle. The systems described herein comprise, in some embodiments, three functional modules that mediate m6A operations: (1) a synthetic initiator module to place m6A at specific genomic sites; (2) a synthetic readout module to program m6A recognition and m6A-dependent transcriptional logic; and (3) propagation module that implements “read-write,” a mechanism proposed to underlie chromatin spreading and epigenetic maintenance across cellular systems.

Abstract: Microfluidic structures featuring substantially circular channels may be fabricated by embossing polymer sheets.

Abstract: The systems and methods described herein relate to a high-throughput flow apparatus. The apparatus is used with an array of wells, and is configured to impart a predetermined shear stress on cells cultured within each of the wells of the array of wells. The apparatus includes a plurality of mechanical tips. The plurality of mechanical tips each includes a head with a hemispheroid shape. The apparatus also includes a motor associated with at least one of plurality of mechanical tips. The motor is configured to drive the plurality of mechanical tips to impart the shear stress pattern in each of the wells.

Abstract: Embodiments disclosed herein provide artificial expression systems comprising the zinc-finger containing transcription factors and engineered promoters to modulate expression of genes of interest. Engineered zinc-finger transcription factors that interact with engineered promoters constitute synthetic and regulatable expression systems which facilitate the modulation of gene expression as desired.

Abstract: Provided herein are engineered systems and methods for establishing DNA adenine methylation at specific genomic locations and using DNA adenine methylation as an artificial chemical “handle” on the genome. These systems and methods allow for placing the handle on specific genomic locations as well as molecular technologies to bind, spatially spread, and maintain the handle. The systems described herein comprise, in some embodiments, three functional modules that mediate m6A operations: (1) a synthetic initiator module to place m6A at specific genomic sites; (2) a synthetic readout module to program m6A recognition and m6A-dependent transcriptional logic; and (3) propagation module that implements “read-write,” a mechanism proposed to underlie chromatin spreading and epigenetic maintenance across cellular systems.

Abstract: Embodiments disclosed herein provide artificial expression systems comprising the zinc-finger containing transcription factors and engineered promoters to modulate expression of genes of interest. Engineered zinc-finger transcription factors that interact with engineered promoters constitute synthetic and regulatable expression systems which facilitate the modulation of gene expression as desired.