Abstract: High throughput methods utilizing spatially segregated detection systems provide a robust CRISPR-based diagnostic enabling highly sensitive detection of both DNA and RNA target molecules, with applications in multiple scenarios in human health including, for example, viral detection. Kits comprising the systems allow for point-of care applications with high-throughput processing of samples.

Abstract: The subject matter disclosed herein is generally directed to methods and systems for screening phenotypes associated with genetic elements and identifying genetic elements at the single-cell level using optical barcodes. A major advantage offered by this approach is the ability to screen for any cellular phenotype that can be identified by high-resolution microscopy—including live-cell phenotypes, protein localization, or highly multiplexed expression profile and mRNA localization in conjunction with a large array of genetic elements applied as a pool in a single test volume.

Abstract: Compositions and methods for inducing and isolating virus-like particles (VLPs), and for allowing real-time assessment of VLP-captured analytes obtained from targeted living mammalian cells, are provided.

Abstract: Method of generating a barcoded library, comprising delivering a polynucleotide into a cell, each polynucleotide comprising: (i) a sequence encoding a barcoding construct operably linked to a first promoter that is an antisense promoter, wherein the barcoding construct comprises a trans-splicing element and a barcode sequence; and a sequence encoding a perturbation element operably linked to a second promoter; generating RNA transcripts of the polynucleotide delivered into the cell, wherein the RNA transcripts comprise the barcoding construct and the perturbation element; and splicing the barcoding sequence onto endogenous RNA molecules in the cell, thereby generating a barcoded library, each member of the barcoded library comprising the barcode sequence and the endogenous RNA molecule attached with the barcode sequence.

Abstract: The present invention provides for methods to obtain transcriptome-wide multiple information-rich samples from living cells while minimally disrupting the cell. The subject matter disclosed herein is generally related to nucleic acid constructs for continuous monitoring of live cells. Specifically, the subject matter disclosed herein is directed to nucleic acid constructs that encode a fusion protein and a construct RNA sequence that induce live cells to self-report cellular contents while maintaining cell viability. The present invention may be used to monitor gene expression in single cells while maintaining cell viability.

Abstract: It has been discovered that lung tumor growth is associated with a dysregulation of the local microbiota, including an increased total bacterial load and reduced bacterial diversity in the airway. In the lungs, commensal bacteria, which are otherwise non-pathogenic and colonize pulmonary tissue at a much lower density in healthy individuals, provoke chronic inflammation and exacerbation of lung cancer through tumor-infiltrating immune cells. Thus, targeting the lung microbiota and its responding immune pathways is useful in treating lung cancer. Disclosed are compositions and methods targeting the lung microbiota and its responding immune pathways in a subject by specific targeting of commensal bacteria in the subject. Typically, the methods involve administering an effective amount of one or more therapeutics such as an antibiotic that reduces the local bacterial load, blocks or depletes tumor-infiltrating immune cells, and/or locally inhibits one or more cytokines or chemokines.

Abstract: RNA targeting proteins are utilized to provide a robust massively multiplexed CRISPR-based diagnostic by detection in droplets with attomolar sensitivity. Detection of both DNA and RNA with comparable levels of sensitivity at nanoliter volumes can differentiate targets from non-targets based on single base pair differences, with applications in multiple scenarios in human health including, for example, viral detection, bacterial strain typing, and sensitive genotyping.

Abstract: A system and method for isolating target substrates includes a microfluidic chip, comprising a plurality of processing units, each processing unit comprising: an inlet port, a plurality of first chambers connected to the inlet port by a fluid channel, the fluid channel comprising a plurality of valves, a plurality of second chambers, each of the second chambers connected to a respective first chamber by a fluid channel, each fluid channel including a controllable blocking valve, and a plurality of respective outlet ports, each outlet port in fluid communication with a respective one of said second chambers and each outlet port including a blocking valve. A magnet is adjacent the microfluidic chip and is movable relative to the microfluidic chip. A valve control is capable of actuating certain ones of the controllable blocking valves in response to a control signal.

Abstract: The invention provides for use of CRISPR-Cas systems to sort barcoded cells or molecules. Cells or nucleic acid molecules may be sorted from a heterogenous population by targeting a barcode of interest specific for a cell or cell progeny or nucleic acid molecule of interest.

Abstract: The disclosure provides Cas9 protein variants that are thermostable at elevated temperatures (e.g., at least 70° C. or above). A Cas9 protein may have at least 75% sequence identity to a wild-type Cas9 protein (e.g., a wild-type Cas9 protein having the sequence of SEQ ID NO:1) and/or one or more amino acid substitutions relative to the wild-type Cas9 protein.

Abstract: Embodiments disclosed herein are directed to a new genetic perturbation and screening method that combines advantages of pooled perturbation with imaging assays for complex phenotypes. Specifically, the method may be used to screen pooled genomic perturbations to identify phenotypes and to identify perturbed genes at the single-cell level using optical barcodes. A major advantage offered by this approach is the ability to screen for any cellular phenotype that can be identified by high-resolution microscopy—including live-cell phenotypes, protein localization, or highly multiplexed expression profile and mRNA localization by RNA-FISH—in conjunction with a large array of genetic perturbations applied as a pool in a single test volume.

Abstract: The present invention relates to compositions which may comprise a non-naturally occurring or engineered artificial transcription factor, wherein the transcription factor may comprise a sequence specific DNA binding domain, a sliding domain, and one or more linkers, wherein the DNA binding domain and the sliding domain are operably connected by the one or more linkers, and uses thereof. Methods involving the use of a non-naturally occurring or engineered artificial transcription factors and pharmaceutical compositions, methods for treating cancer, a degenerative disease, a genetic disease or an infectious disease as well as diagnostic methods are also contemplated by the present invention.

Abstract: Embodiments disclosed herein are directed to a new genetic perturbation and screening method that combines advantages of pooled perturbation with imaging assays for complex phenotypes. Specifically, the method may be used to screen pooled genomic perturbations to identify phenotypes and to identify perturbed genes at the single-cell level using optical barcodes. A major advantage offered by this approach is the ability to screen for any cellular phenotype that can be identified by high-resolution microscopy—including live-cell phenotypes, protein localization, or highly multiplexed expression profile and mRNA localization by RNA-FISH—in conjunction with a large array of genetic perturbations applied as a pool in a single test volume.

Abstract: The subject matter disclosed herein is generally directed to methods and compositions for tagging cells of interest, tracking evolution of the tagged cells, and recovering the original tagged cells for further study. Specifically, cells are tagged with a DNA construct encoding a barcode sequence comprising a guide sequence. Barcoded cells can then be recovered using a reporter construct having CRISPR target sequences specific for the cell having a barcode of interest.

Abstract: Provided herein are methods for screening biological functions of microscale biological systems comprises segregating each microscale biological system from a set of microscale biological systems to be screened into individual discrete volumes, the individual discrete volume comprising a first polymer. The first polymer is then forced or allowed to polymerize to form a set of polymerized beads that encapsulate an individual microscale biological system. The polymerized beads are further encapsulated in a second droplet comprising a second polymer and one or more reporter elements. The reporter elements are configured to produce a readout upon detecting the absence or presence of a biological function to be screened. The second polymer is then forced or allowed to polymerize to form an outer capsule around each individual bead thereby forming a set of encapsulated beads. One or more biological functions of the double-encapsulated system are identified by detecting the readout of the reporters.

Abstract: Embodiments disclosed herein are directed to microfluidic devices that allow for scalable on-chip screening of combinatorial libraries and methods of use thereof. Droplets comprising individual molecular species to be screened are loaded onto the microfluidic device. The droplets are labeled by methods known in the art, including but not limited to barcoding, such that the molecular species in each droplet can be uniquely identified. The device randomly sorts the droplets into individual microwells of an array of microwells designed to hold a certain number of individual droplets in order to derive combinations of the various molecular species. The paired droplets are then merged in parallel to form merged droplets in each microwell, thereby avoiding issues associated with single stream merging. Each microwell is then scanned, e.g., using microscopy, such as high content imaging microscopy, to detect the optical labels, thereby identifying the combination of molecular species in each microwell.

Abstract: The present invention relates to novel hybridization probes useful for rapid hybridization to realize a practicable and affordable pathogen diagnostic based on RNA signature detection, technology and hardware. In particular, the present invention relates to a nucleic acid structure which may comprise nucleic acid tiles, wherein each nucleic acid tile comprises nucleic acid oligomers, wherein each nucleic acid oligomer hybridizes to each other thereby forming a nucleic acid tile.

Abstract: Embodiments disclosed herein are directed to microfluidic devices that allow for scalable on-chip screening of combinatorial libraries and methods of use thereof. Droplets comprising individual molecular species to be screened are loaded onto the microfluidic device. The droplets are labeled by methods known in the art, including but not limited to barcoding, such that the molecular species in each droplet can be uniquely identified. The device randomly sorts the droplets into individual microwells of an array of microwells designed to hold a certain number of individual droplets in order to derive combinations of the various molecular species. The paired droplets are then merged in parallel to form merged droplets in each microwell, thereby avoiding issues associated with single stream merging. Each microwell is then scanned, e.g., using microscopy, such as high content imaging microscopy, to detect the optical labels, thereby identifying the combination of molecular species in each microwell.

Abstract: A system and method for isolating target substrates includes a microfluidic chip, comprising a plurality of processing units, each processing unit comprising: an inlet port, a plurality of first chambers connected to the inlet port by a fluid channel, the fluid channel comprising a plurality of valves, a plurality of second chambers, each of the second chambers connected to a respective first chamber by a fluid channel, each fluid channel including a controllable blocking valve, and a plurality of respective outlet ports, each outlet port in fluid communication with a respective one of said second chambers and each outlet port including a blocking valve. A magnet is adjacent the microfluidic chip and is movable relative to the microfluidic chip. A valve control is capable of actuating certain ones of the controllable blocking valves in response to a control signal.

Abstract: The present disclosure relates to compositions and methods for detection of single-stranded breaks (SSBs) in dsDNA, including in genomic DNA. Measurement of the precise location of SSB damage in DNA, e.g.