Abstract: Provided herein are methods of detecting nucleic acids. The nucleic acid of interest may be detected by using selective enrichment. At least two Cas endonuclease complexes are introduced to a sample comprising nucleic acid. The Cas endonuclease complexes comprise guide RNAs and Cas endonuclease. The Cas endonuclease complexes attach to a target nucleic acid, thereby protecting the target of interest while unprotected nucleic acid in the sample is degraded, e.g., by exonuclease digestion. Linkers, when added to the sample, will attach to the ends of the target nucleic acid previously protected by the Cas endonuclease and will not attach to the degraded, unprotected nucleic acid in the sample. The target nucleic acid and linkers are then detected.

Abstract: The present disclosure describes a method of treating a sample comprising cells with a process of partial lysing. Cells are exposed to a process such as bead beating that lyses some cells in the mixture. The process generates a resultant sample mixture that is suitable for both cell morphology screening and genetic screening. A first portion of the partially lysed sample can be mounted on a slide and observed for atypical cells and cytologic abnormalities. A second portion of the partially lysed sample can be screened for genetic markers known to correlate with a risk of cervical cancer. The method is particularly useful for cervical screening, where a combination of cytology and genetic screening present a more complete picture of cervical health. The disclosed method streamlines the diagnostic process for protocols that require both types of assays, without compromising screening accuracy.

Abstract: The invention provides methods of detecting residual disease, such as cancer, in a subject. The methods entail amplifying from a sample obtained from the subject one or more nucleic acids having a passenger mutation that contains or results from fusion, such as a translocation, inversion, or deletion.

Abstract: The invention provided methods and devices for performing sequential, regulated multiplex reactions in a single tube without the addition or removal of contents from the tube.

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.

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: 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 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 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: 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.