Abstract: The invention provides methods for detecting small mutations and structural alterations in DNA by using binding proteins to protect those features while digesting unprotected DNA in a sample. To detect small mutations, a protein that binds exclusively to the mutation of interest, and not to wild-type, is used. For structural alterations, binding proteins are used that flank a breakpoint of the alteration. After digestion of unbound, unprotected nucleic acid in the sample, the mutation- or breakpoint-containing segment remains as an isolated DNA fragment. The sample is then assayed to detect any fragment of DNA and the detection of the fragment indicates the presence of the mutation or breakpoint in the subject.

Abstract: The invention provides methods of detecting a feature of interest in a nucleic acid sample by negatively and positively enriching the sample for segments that contain the feature of interest. Negative enrichment may include digestion of nucleic acids that do not contain the segments, and positive enrichment may include purification of the segments. The methods are useful for diagnostic of genetic elements, e.g., elements indicative of cancer.

Abstract: Provided herein are methods of detecting nucleic acids. The nucleic acid of interest may be detected by using Cas endonuclease to degrade substantially all nucleic acid in a sample except for the nucleic acid of interest, leaving the nucleic acid of interest isolated and amenable to detection. In related methods, Cas endonuclease complexes are used to protect the nucleic acid of interest while unprotected nucleic acid is digested, e.g., by exonuclease, after which the isolated nucleic acid of interest is detected.

Abstract: Provided herein are methods of detecting nucleic acids. The nucleic acid of interest may be detected by using Cas endonuclease to degrade substantially all nucleic acid in a sample except for the nucleic acid of interest, leaving the nucleic acid of interest isolated and amenable to detection. In related methods, Cas endonuclease complexes are used to protect the nucleic acid of interest while unprotected nucleic acid is digested, e.g., by exonuclease, after which the isolated nucleic acid of interest is detected.

Abstract: The invention provides methods for capturing target nucleic acid directly from bodily fluid samples, without the need for certain complex sample preparation steps, using Cas endonuclease to bind to the target nucleic acid sequences. The Cas proteins, along with their sequence-specific guide RNAs, may be introduced directly into the sample, where the Cas proteins bind to ends of a target nucleic acid. The target nucleic acid is thus isolated or enriched in a sequence-specific manner. The target nucleic acid may then be subject to any suitable detection or analysis assay, such as amplification or sequencing. The target nucleic acid may be enriched by digesting other, unbound nucleic acids present in the sample with exonuclease. The bound Cas proteins prevent exonuclease from digesting the target nucleic acid, thereby leaving the only the target nucleic acid substantially present in the sample.

Abstract: The invention provides methods for binding to and protecting target nucleic acid directly from plasma without the need for certain complex sample preparation steps, using catalytically active Cas endonuclease. The catalytically active Cas endonucleases, along with their sequence-specific guide RNAs, may be introduced directly into the plasma sample, where the catalytically active Cas endonucleases bind to ends of a target nucleic acid. The target nucleic acid is thus isolated or enriched in a sequence-specific manner. The target nucleic acid may then be subject to any suitable detection or analysis assay, such as amplification or sequencing. The bound catalytically active Cas proteins prevent exonuclease from digesting the target nucleic acid in a plasma sample leaving only the target nucleic acid substantially present in the enriched sample.

Abstract: The invention provides methods and devices for drawing blood, in which the blood collected into a container closely resembles the blood as it existed before collection (i.e., as it existed in vivo). In some embodiments this may be achieved by modulating a flow rate of blood into the collection container in order to preserve the integrity of the blood. By modulating the flow rate of the blood, shear forces acting on cells and cellular components in the blood are reduced. This preserves the integrity of such cells and cellular components and facilitates more accurate detection and analysis of low-abundance analytes.

Abstract: The invention provides methods for monitoring cancer reoccurrence in an individual. Methods of the invention include identifying passenger mutations specific to an individual and detecting the passenger mutations by capturing target nucleic acid directly from bodily fluid samples, without the need for certain complex sample preparation steps, using Cas endonuclease to bind to the target nucleic acid sequences. The detection of passenger mutations specific to an individual provides the ability to monitor the reoccurrence of cancer in an individual. The presence of passenger mutations in a sample obtained subsequent a treatment, is indicative of the reoccurrence of cancer. Methods of the invention provide Cas proteins, along with their sequence-specific guide RNAs, may be introduced directly into the sample, where the Cas proteins bind to ends of a target nucleic acid. The target nucleic acid is thus isolated or enriched in a sequence-specific manner.

Abstract: The invention provides methods for detecting epigenetic changes, including but not limited to methylation changes, directly from biological samples, without the need for certain complex sample preparation steps. The invention provides Cas protein/guide RNA complexes that may be introduced directly into the sample, where the complexes target and bind the target region. The target region is thus enriched and isolated in a sequence-specific manner. The target region may then be subject to any suitable signal amplification assay to detect the epigenetic change in the target region. Detection of DNA hypermethylation in the target region is indicative of disease, such as cancer.

Abstract: Provided herein are methods of detecting nucleic acids. The nucleic acid of interest may be detected by using Cas endonuclease to degrade substantially all nucleic acid in a sample except for the nucleic acid of interest, leaving the nucleic acid of interest isolated and amenable to detection. In related methods, Cas endonuclease complexes are used to protect the nucleic acid of interest while unprotected nucleic acid is digested, e.g., by exonuclease, after which the isolated nucleic acid of interest is detected.

Abstract: The invention provides methods for capturing target nucleic acid directly from bodily fluid samples, without the need for certain complex sample preparation steps, using Cas endonuclease to bind to the target nucleic acid sequences. The Cas proteins, along with their sequence-specific guide RNAs, may be introduced directly into the sample, where the Cas proteins bind to ends of a target nucleic acid. The target nucleic acid is thus isolated or enriched in a sequence-specific manner. The target nucleic acid may then be subject to any suitable detection or analysis assay, such as amplification or sequencing. The target nucleic acid may be enriched by digesting other, unbound nucleic acids present in the sample with exonuclease. The bound Cas proteins prevent exonuclease from digesting the target nucleic acid, thereby leaving the only the target nucleic acid substantially present in the sample.

Abstract: The invention provides methods for determining the mutation burden of a tumor by assaying tumor DNA that is representative of genetic loci that are themselves representative of genetics of the tumor. The assayed tumor DNA may be itself agnostic as to loci, so long as it is representative of loci that are representative of tumor mutation burden. The invention provides for assays in which the tumor DNA being sequenced or tested can be something other than, and possibly less than, a full panel of oncogenes that is expected to stand for a tumor's mutational load.

Abstract: The invention provides methods of selectively protecting nucleic acids of interest in a sample from damage that occurs during preparative procedures. The methods include binding proteins to ends and to one or more internal regions of a segment of the nucleic acid of interest so that damage to exposed regions of the segment does not lead to degradation of the entire segment.

Abstract: The invention provides methods for detecting small mutations and structural alterations in DNA by using binding proteins to protect those features while digesting unprotected DNA in a sample. To detect small mutations, a protein that binds exclusively to the mutation of interest, and not to wild-type, is used. For structural alterations, binding proteins are used that flank a breakpoint of the alteration. After digestion of unbound, unprotected nucleic acid in the sample, the mutation- or breakpoint-containing segment remains as an isolated DNA fragment. The sample is then assayed to detect any fragment of DNA and the detection of the fragment indicates the presence of the mutation or breakpoint in the subject.

Abstract: The invention provides methods for capturing cfDNA directly from plasma or serum samples, without the need for certain complex sample preparation steps, using sequence-specific DNA-binding proteins such as Cas endonuclease to bind target nucleic acid sequences. The Cas proteins along with their sequence-specific guide RNAs may be introduced directly into blood, plasma, or serum, where the Cas proteins bind to ends of a target nucleic acid. The target nucleic acid is thus isolated or enriched in a sequence-specific manner. The target nucleic acid may then be subject to any suitable detection or analysis assay such as amplification or sequencing. The target nucleic acid may be enriched by digesting other, unbound nucleic acids present in the sample with exonuclease. The bound Cas proteins prevent exonuclease from digesting the target nucleic acid, thereby leaving the only the target nucleic acid substantially present in the sample.

Abstract: Methods and kits are provided that are useful as quality controls for gene editing tools. When a gene editing process is proposed for some subject nucleic acid, the gene editing process may be performed on a representative sample—a sample that represents the subject nucleic acid. Off-target effects may be measured and shown for the representative sample to show a prospective rate of off-target activity were the gene editing process to be performed on the subject nucleic acid.

Abstract: Methods for detecting rare mutations in DNA include obtaining a sample comprising a target nucleic acid, binding a protein to the target nucleic acid in a sequence-specific manner, digesting non-target nucleic acid in the sample, and detecting the target nucleic acid. The method may include amplifying the target nucleic acid with at least one primer with, e.g., a phosphorothioate bond that is resistant to degredation by a nuclease to yield an amplicon that includes a copy of the target nucleic acid and a terminal portion that is resistant to degredation by the nuclease. Preferably digesting the non-target nucleic acid includes exposing amplicons to the nuclease. The nuclease digests the non-target nucleic acid while the amplicon that includes the copy of the target nucleic acid is protected by the terminal portions and the bound protein.

Abstract: The invention provides methods of developing a tumor mutation burden by detecting and quantifying target analytes in a nucleic acid sample. The sample is subjected to enrichment, which may include negative enrichment and positive enrichment. Negative enrichment may include digestion of nucleic acids that do not contain the segments. Positive enrichment may include purification of the segments. Detection and analysis of the segments may include identifying one or more mutations, sequencing, and quantifying the mutations. Relationships between the mutations may be determined and may include determining a ratio between each mutation and the plurality of mutations to develop a tumor mutation burden. The method may include a report from a clinician, where the clinician uses the tumor mutation burden for medical diagnostic purposes.

Abstract: The present disclosure provides biomarkers and methods for providing a clinical assessment of a subject. In particular, the present disclosure provides methods for measuring at least one biomarker to classifying a subject as being test negative, with a high negative predictive value, and recommending that test negative subject be excluded from treatment. The present disclose also provides methods for measuring at least one biomarker in a subject not excluded from treatment and classifying a subject as being test positive, with a high positive predictive value, and recommending that test positive subject receive treatment.

Abstract: The invention generally relates to compositions and methods for targeted delivery of a Cas endonuclease or nucleic acid encoding a Cas endonuclease to a fusion sequence in a cancer cell but not in a healthy cell of a subject. The Cas endonuclease or nucleic acid encoding the Cas endonuclease may be complexed with a guide RNA complementary to a fusion sequence identified based on differences between a mutated sequence obtained from a cancer cell and a wild-type sequence obtained from a healthy cell of the subject. For example, the Cas endonuclease may be a Cas9 and cut DNA or a Cas13a and cut RNA. The Cas endonuclease complexes may induce cell death or cancerous cells or cause other beneficial effects.