Abstract: Viral infection is a persistent cause of human disease. Guided nuclease systems of the invention target the genomes of viral infections, rendering the viruses incapacitated. The invention further provides delivery methods and compositions for antiviral therapeutics. Methods and compositions are provided for targeted delivery of antiviral therapeutics into cells of interest using, for example, viral vectors such as adenovirus, AAV, and replication incompetent HSV. These and other delivery systems can be used as vehicles to deliver DNA vectors encoding a nuclease or a cell-killing gene. These delivery methods can also be used to deliver naked DNA or RNA, protein products, plasmids containing a promoter that is active only in a latent viral state which drives a cell-killing gene, or other therapeutic agents.

Abstract: The invention generally relates to methods for assessing the health of a tissue by characterizing circulating nucleic acids in a biological sample. According to certain embodiments, methods for assessing the health of a tissue include the steps of detecting a sample level of RNA in a biological sample, comparing the sample level of RNA to a reference level of RNA specific to the tissue, determining whether a difference exists between the sample level and the reference level, and characterizing the tissue as abnormal if a difference is detected.

Abstract: The invention generally relates to methods for assessing the health of a tissue by characterizing circulating nucleic acids in a biological sample. According to certain embodiments, methods for assessing the health of a tissue include the steps of detecting a sample level of RNA in a biological sample, comparing the sample level of RNA to a reference level of RNA specific to the tissue, determining whether a difference exists between the sample level and the reference level, and characterizing the tissue as abnormal if a difference is detected.

Abstract: The invention generally relates to methods for assessing the health of a tissue by characterizing circulating nucleic acids in a biological sample. According to certain embodiments, methods for assessing the health of a tissue include the steps of detecting a sample level of RNA in a biological sample, comparing the sample level of RNA to a reference level of RNA specific to the tissue, determining whether a difference exists between the sample level and the reference level, and characterizing the tissue as abnormal if a difference is detected.

Abstract: A static fluid and a second fluid are placed into contact along a microfluidic free interface and allowed to mix by diffusion without convective flow across the interface. In accordance with one embodiment of the present invention, the fluids are static and initially positioned on either side of a closed valve structure in a microfluidic channel having a width that is tightly constrained in at least one dimension. The valve is then opened, and no-slip layers at the sides of the microfluidic channel suppress convective mixing between the two fluids along the resulting interface. Applications for microfluidic free interfaces in accordance with embodiments of the present invention include, but are not limited to, protein crystallization studies, protein solubility studies, determination of properties of fluidics systems, and a variety of biological assays such as diffusive immunoassays, substrate turnover assays, and competitive binding assays.

Abstract: The invention provides methods for determining the presence of a disease by comparing a sequence from a single target molecule with a predetermined sequence that is associated with a specific disease.

Abstract: The invention provides transgenic organisms that include a transgene that codes for a product that can be used to digest foreign nucleic acid. The transgene can code for a targeting nuclease, a guide sequence, or other components of a guided nuclease system. Expression of the transgene causes the organism to express an active targeting nuclease that targets and digests foreign nucleic acid. The targeting nuclease targets the foreign nucleic acid specifically and avoids targeting the organism's native genetic material.

Abstract: Methods for treating latent viral infections using a gene for a nuclease that is expressed in the presence of a latent viral infection, allowing the nuclease to digest viral nucleic acid. The gene is controlled by a switch that turns expression on in the presence of viral transcripts. The switch may be an engineered sequence that, in the absence of a viral transcript, forms a duplex structure to inhibit translation. The viral transcript hybridizes to the switch and disrupts the duplex structure, allowing translation to occur. A nucleic acid encodes a nuclease and a switch that causes the nuclease to be expressed in the presence of a viral nucleic acid. A portion of the switch may be complementary to at least a portion of a latency associated transcript such as an HHV latency associated transcript that, when present, interacts with the switch to initiate translation of the nuclease.

Abstract: The invention provides compositions and methods that can be used to regulate viral transcription. Using a catalytically inactive nuclease such as deactivated Cas9, or dCas9, a guide RNA can be designed that recognizes a regulatory element within a viral nucleic acid. The dCas9 may function as an RNA-dependent DNA-binding protein that binds to a viral promoter and upregulates or down-regulates transcription. For example, the dCas9 with a viral promoter-specific gRNA may hybridize to a promoter within a viral genome within a host cell and inhibit transcription by, for example, sterically blocking recruitment of the transcription machinery.

Abstract: The invention provides methods and compositions that remove target genetic material from a subject by delivery of an enzyme that degrades the target genetic material. The methods include delivering a composition of a nucleic acid to a tissue, such as skin, of a subject along with various types of energy to enhance permeability of the tissue and cause the nucleic acid to enter cells of the tissue, wherein the nucleic acid comprises a gene for an enzyme that cuts target genetic material. The nucleic acid may be a plasmid comprising a cas9 gene and at least one gene for a short guide RNA (sgRNA) and the target genetic material may be viral genome, i.e., with the sgRNA complementary to a portion of the viral genome.

Abstract: Viral infection is a persistent cause of human disease. Fusion polypeptide systems target the genomes of viral infections, rendering the viruses incapacitated.

Abstract: Nucleic acid from cells and viruses sampled from a variety of environments may purified and expressed utilizing microfluidic techniques. In accordance with one embodiment of the present invention, individual or small groups of cells or viruses may be isolated in microfluidic chambers by dilution, sorting, and/or segmentation. The isolated cells or viruses may be lysed directly in the microfluidic chamber, and the resulting nucleic acid purified by exposure to affinity beads. Subsequent elution of the purified nucleic acid may be followed by ligation and cell transformation, all within the same microfluidic chip. In one specific application, cell isolation, lysis, and nucleic acid purification may be performed utilizing a highly parallelized microfluidic architecture to construct gDNA and cDNA libraries.

Abstract: Disclosed is a method to achieve digital quantification of DNA (i.e., counting differences between identical sequences) using direct shotgun sequencing followed by mapping to the chromosome of origin and enumeration of fragments per chromosome. The preferred method uses massively parallel sequencing, which can produce tens of millions of short sequence tags in a single run and enabling a sampling that can be statistically evaluated. By counting the number of sequence tags mapped to a predefined window in each chromosome, the over- or under-representation of any chromosome in maternal plasma DNA contributed by an aneuploid fetus can be detected. This method does not require the differentiation of fetal versus maternal DNA. The median count of autosomal values is used as a normalization constant to account for differences in total number of sequence tags is used for comparison between samples and between chromosomes.

Abstract: A method of fabricating an elastomeric structure, comprising: forming a first elastomeric layer on top of a first micromachined mold, the first micromachined mold having a first raised protrusion which forms a first recess extending along a bottom surface of the first elastomeric layer; forming a second elastomeric layer on top of a second micromachined mold, the second micromachined mold having a second raised protrusion which forms a second recess extending along a bottom surface of the second elastomeric layer; bonding the bottom surface of the second elastomeric layer onto a top surface of the first elastomeric layer such that a control channel forms in the second recess between the first and second elastomeric layers; and positioning the first elastomeric layer on top of a planar substrate such that a flow channel forms in the first recess between the first elastomeric layer and the planar substrate.

Abstract: The invention relates to systems and methods for removing viral genetic sequences from host genomes by using a computer system to read a nucleotide string next to a protospacer adjacent motif (PAM) in the viral sequence, determine that the host genome lacks any region that matches the nucleotide string according to a predetermined similarity criteria and is adjacent to the PAM, and provide a guide sequence at least partially complementary to the nucleotide string. Providing the guide sequence may include synthesizing a guide RNA that includes a portion that is complementary to the nucleotide string.

Abstract: The invention relates to methods for generating viral-free cells using nucleases for use in transplantation. The nucleases may be CRISPR/Cas9 complexes with guided RNA to target and inactivate viral genomes within cells. The nucleases degrade or destroy the viruses within the cells prior to transplantation.

Abstract: The invention relates to methods for treating cytomegalovirus (CMV) in cells and tissues using targeted nucleases. The nucleases may be CRISPR/Cas9 complexes with guided RNA to target and inactivate CMV genomes within cells and tissues. The nucleases degrade or destroy the CMV genomes within the cells and tissues. Methods of the invention can be used to treat tissues, such as whole organs, prior to transplantation.

Abstract: The invention provides compositions and methods for treating human papillomavirus (HPV) infections using a targetable nuclease, which compositions and methods can be used to selectively target the HPV genome or selectively express the targetable nuclease within cells that infected by HPV. By selectively targeting cells infected by HPV, the HPV genome within infected cells, or both, the nuclease is able to cleave the HPV genome thereby inactivating it and rendering it inoperable, interfering with the virus's ability to propagate even where the virus is in a latent stage of infection. Since latent HPV can be cleaved and eradicated from the host cells, compositions and methods of the invention may be used to treat HPV infections and potentially prevent many of the adverse health consequences associated with the papillomavirus.

Abstract: Herpes simplex virus (HSV) including herpes simplex virus 1 and 2 (HSV-1 and HSV-2) are a persistent cause of human disease with no known cure. Guided nuclease systems target specific regions of the HSV-1 and HSV-2 genomes, disrupting the virus' nucleic acid and rendering even latent viruses incapacitated.

Abstract: The invention relates to a microfabricated device for the rapid detection of DNA, proteins or other molecules associated with a particular disease. The devices and methods of the invention can be used for the simultaneous diagnosis of multiple diseases by detecting molecules (e.g. amounts of molecules), such as polynucleotides (e.g., DNA) or proteins (e.g., antibodies), by measuring the signal of a detectable reporter associated with hybridized polynucleotides or antigen/antibody complex. In the microfabricated device according to the invention, detection of the presence of molecules (i.e., polynucleotides, proteins, or antigen/antibody complexes) are correlated to a hybridization signal from an optically-detectable (e.g. fluorescent) reporter associated with the bound molecules. These hybridization signals can be detected by any suitable means, for example optical, and can be stored for example in a computer as a representation of the presence of a particular gene.