The present disclosure generally relates, in certain aspects, to droplet-based microfluidic devices and methods. In certain aspects, target nucleic acids contained within droplets are amplified within droplets in a first step, where multiple primers may be present. However, multiple primers may cause multiple target nucleic acids to be amplified within the droplets, which can make it difficult to identify which nucleic acids were amplified. In a second step, the amplified nucleic acids may be determined. For example, the droplets may be broken and the amplified nucleic acids can be pooled together and sequenced. As an example, new droplets may be formed containing the amplified nucleic acids, and those nucleic acids within the droplets amplified by exposure to certain primers.

CRISPR RNA-guided nucleases are routinely used for sequence-specific manipulation of DNA. While CRISPR-based DNA editing has become routine, analogous methods for editing RNA have yet to be established. Here we repurpose the type III-A CRISPR RNA-guided nuclease for sequence-specific cleavage of the SARS-CoV-2 genome. The type III cleavage reaction is performed in vitro using purified viral RNA, resulting in sequence-specific excision of 6, 12, 18 or 24 nucleotides. Ligation of the cleavage products is facilitated by a DNA splint that bridges the excision and RNA ligase is used to link the RNA products before transfection into mammalian cells. The SARS-CoV-2 RNA is infectious and standard plaque assays are used to recover viral clones. Collectively, this work demonstrates how type III CRISPR systems can be repurposed for sequence-specific editing of RNA viruses including SARS-CoV-2 and more generally for gene therapy.

CRISPR RNA-guided nucleases are routinely used for sequence-specific manipulation of DNA. While CRISPR-based DNA editing has become routine, analogous methods for editing RNA have yet to be established. Here we repurpose the type III-A CRISPR RNA-guided nuclease for sequence-specific cleavage of the SARS-CoV-2 genome. The type III cleavage reaction is performed in vitro using purified viral RNA, resulting in sequence-specific excision of 6, 12, 18 or 24 nucleotides. Ligation of the cleavage products is facilitated by a DNA splint that bridges the excision and RNA ligase is used to link the RNA products before transfection into mammalian cells. The SARS-CoV-2 RNA is infectious and standard plaque assays are used to recover viral clones. Collectively, this work demonstrates how type III CRISPR systems can be repurposed for sequence-specific editing of RNA viruses including SARS-CoV-2 and more generally for gene therapy.

The present disclosure describes a CRISPR nuclease cascade assay that can detect one or more target nucleic acids of interest of interest at attamolar (aM) (or lower) limits in about 10 minutes or less without the need for amplifying the target nucleic acids of interest. The CRISPR cascade assays utilize signal amplification mechanisms comprising various components including CRISPR nucleases, guide RNAs (gRNAs), blocked nucleic acid molecules, blocked primer molecules, and reporter moieties.

The present disclosure provides, among other things, a way to amplify and sequence target sequences in a low-input sample. In some embodiments, the method comprises ligating a double-stranded adaptor onto a population of fragments to produce tagged fragments, and linearly amplifying the tagged fragments.

The present disclosure provides, among other things, a way to amplify and sequence target sequences in a low-input sample. In some embodiments, the method comprises ligating a double-stranded adaptor onto a population of fragments to produce tagged fragments, and linearly amplifying the tagged fragments.

A water-soluble prolamin composition, such as a zein composition, and methods for producing the same. A method for tagging items comprising applying a plurality of non-coding DNA tags in a prolamin composition, such as a zein composition, wherein the selection of the particular taggants corresponds with a binary or nonbinary code sequence containing information about the tagged items.

A water-soluble prolamin composition, such as a zein composition, and methods for producing the same. A method for tagging items comprising applying a plurality of non-coding DNA tags in a prolamin composition, such as a zein composition, wherein the selection of the particular taggants corresponds with a binary or nonbinary code sequence containing information about the tagged items.

A method of distinguishing a subject with pre-clinical Alzheimer's disease from those with similar symptoms but other forms of dementia such as mild cognitive impairment. The blood RNA whole transcriptome profile of a subject with suspected pre-clinical Alzheimer's disease is obtained and analyzed against a reference blood RNA whole transcriptome profile from a subject with another form of dementia such as frontal temporal dementia, CADASIL or mild cognitive impairment (MCI). The blood RNA whole transcriptome profile includes the presence and quantitation of ncRNA. Methods to enhance treatment of epileptic seizures are also discussed.

A method of distinguishing a subject with pre-clinical Alzheimer's disease from those with similar symptoms but other forms of dementia such as mild cognitive impairment. The blood RNA whole transcriptome profile of a subject with suspected pre-clinical Alzheimer's disease is obtained and analyzed against a reference blood RNA whole transcriptome profile from a subject with another form of dementia such as frontal temporal dementia, CADASIL or mild cognitive impairment (MCI). The blood RNA whole transcriptome profile includes the presence and quantitation of ncRNA. Methods to enhance treatment of epileptic seizures are also discussed.