Methods for depleting unmethylated nucleic acid molecules

Abstract

Disclosed are methods for depleting unmethylated nucleic acid molecules, thereby enriching for target sequences of interest in a sample (e.g., a sample obtained from a subject). Such methods are useful for enriching for a signal in a sample, such as a signal informative for determining presence or absence of cancer in the sample.

Description

METHODS FOR DEPLETING UNMETHYLATED NUCLEIC ACID MOLECULESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 551,886 filed February 9, 2024, the entire disclosure of which is hereby incorporated by reference in its entirety for all purpose.FIELD OF THE INVENTION

[0002] The invention relates generally to methods for depleting unmethylated nucleic acid molecules from a sample e.g., for enriching target nucleic acid sequences.BACKGROUND

[0003] Detecting a target sequence in a nucleic acid can be a challenge when the target sequence is present at a low frequency in the nucleic acid sample. Amplification and / or sequencing of target sequences can fail if such sequences occur at a low frequency. For example, circulating tumor DNA (ctDNA) levels are present at a very low frequency in most early-stage and many advanced stage cancer patients (Bettegowda et al. (2014) Set Trans I Med 6(224): p. 224ra24). Accordingly, a major challenge in the identification of ctDNA is how to identify a trace amount of ctDNAs out of a much larger proportion of total cell free DNA (cfDNA). There is a need in the art for improved techniques for enriching target sequences of interest in a nucleic acid sample.SUMMARY OF THE INVENTION

[0004] The disclosure relates to methods of enriching target sequences in a nucleic acid sample. The target sequence may be indicative of the risk of developing or the presence of cancer in the subject from whom the sample was taken. Disclosed herein are methods that improve detection of nucleic acids containing a target sequence, e.g., rare target sequences, by isolating and / or enriching such target sequences in a nucleic acid sample. For example, the rare target sequence may be in a nucleic acid sequence from a cfDNA sample, such as a cfDNA sample that has been treated with bisulfite or chemical / enzymatic conversion to convert cytosines to uracils to preserve information regarding the methylation status of a particular nucleic acid sequence (e.g., comprising a CpG site), in a subject. In particular embodiments, enriching the target sequences involve depleting other sequences that are not informative or less informative of risk of developing or the presence of cancer. Such other sequences can be e.g., non-methylated nucleic acid sequences.

[0005] Disclosed herein is a method for enriching for a signal in a sample from a subject, the method comprising: contacting a nuclease with a plurality of nucleic acid molecules, wherein afirst subset of the plurality of nucleic acid molecules comprise converted sequences derived from one or more unmethylated CpG sites and wherein a second subset of the plurality of nucleic acid molecules comprise converted sequences derived from one or more methylated CpG sites; selectively cutting the converted sequences of the first subset of the plurality of nucleic acid molecules using the nuclease relative to cutting the converted sequences of the second subset of the plurality of nucleic acid molecules, providing copies of one or more primer sequences, thereby hybridizing at least one copy of the one or more primer sequences to the converted sequences of the second subset of the plurality of nucleic acid molecules, wherein the copies of the one or more primer sequences fail to hybridize with the converted sequences of the first subset of the plurality of nucleic acid molecules due to the cutting; and enriching for the second subset of the plurality of nucleic acid molecules using the hybridized at least one copy of the one or more primer sequences.

[0006] In various embodiments, methods further comprise: selectively cutting the converted sequences of the first subset of the plurality of nucleic acid molecules is performed by a CRISPR-Cas nuclease. In various embodiments, the CRISPR-Cas nuclease is one or more of Casl2, Cas9, and subtypes and / or variants thereof. In various embodiments, the Casl2 is Casl2a. In various embodiments, the Cas9 is Cas9. In various embodiments, selectively cutting the converted sequences of the first subset of the plurality of nucleic acid molecules is performed by a CRISPR-Cas nuclease due to a presence of one or more converted nucleotide bases in the first subset of the plurality of nucleic acid molecules as a result of performing a nucleic acid conversion. In various embodiments, the one or more converted nucleotide bases are present in one or more protospacer adjacent motif (PAM) nucleic acid sequence(s).

[0007] In various embodiments, the one or more PAM nucleic acid sequences comprise TTN or TTTG, where N is one of A, C, T, or G. In various embodiments, the one or more PAM nucleic acid sequences comprise TTTG. In various embodiments, the one or more converted nucleotide bases are present in a seed sequence complementary to a guide RNA sequence. In various embodiments, the one or more converted nucleotide bases are present in a seed sequence less than 7 nucleotide bases from a PAM sequence. In various embodiments, the one or more converted nucleotide bases are present in one or more protospacer adjacent motif (PAM) nucleic acid sequence(s) and further present in a seed sequence complementary to a guide RNA sequence. In various embodiments, the one or more PAM nucleic acid sequences comprise TTN or TTTG, where N is one of A, C, T, or G. In various embodiments, the one or more PAM nucleic acid sequences comprise TTTG. In various embodiments, the one or more converted nucleotide bases are present in a seed sequence less than 7 nucleotide bases from a PAMsequence. In various embodiments, the one or more converted nucleotide bases are not present in the second subset of the plurality of nucleic acid molecules as a result of performing a nucleic acid conversion. In various embodiments, the nucleic acid conversion comprises a bisulfite conversion.

[0008] In various embodiments, the nuclease is associated with a guide RNA (gRNA) comprising a spacer sequence that binds to a target sequence of a converted sequence of the first subset of the plurality of nucleic acids. In various embodiments, the target sequence comprises a sequence or a portion of a sequence of a CGI selected from Tables 1-4. In various embodiments, subsequent to the CRISPR-Cas nuclease recognizing the PAM sequence, the CRISPR-Cas nuclease selectively cuts converted sequences of the first subset of the plurality of nucleic acid sequences. In various embodiments, the CRISPR-Cas nuclease selectively cuts the converted sequences of the first subset of the plurality of nucleic acid sequences, thereby producing a plurality of nucleic acid fragments that fail to hybridize with the one or more primer sequences. In various embodiments, the plurality of nucleic acid fragments lack a complementary sequence to the one or more primer sequences due to the selective cutting, thereby exhibiting reduced binding affinity for the one or more primer sequences.

[0009] In various embodiments, one or more primer sequences comprise indexing adapters. In various embodiments, one or more primer sequences comprise a forward and reverse primer pair. In various embodiments, the sample is a blood or serum sample. In various embodiments, the sample comprises cell free DNA (cfDNA). In various embodiments, the subject is a human. In various embodiments, the method further comprising: obtaining or having obtained nucleic acids from the sample; and performing a conversion of the obtained nucleic acids to generate the converted sequences of the first subset and the second subset of the plurality of nucleic acids. In various embodiments, performing the conversion comprises performing a bisulfite conversion. In various embodiments, enriching for the second subset of the plurality of nucleic acid molecules using the hybridized at least one copy of the one or more primer sequences comprises performing nucleic acid amplification. In various embodiments, the method further comprises sequencing nucleic acids of the enriched second subset of the plurality of nucleic acid molecules. In various embodiments, sequencing nucleic acids comprises performing next generation sequencing. In various embodiments, methods further comprise: performing an additional enrichment step. In various embodiments, the converted sequences of the second subset of the plurality of nucleic acid molecules comprise a target sequence, and wherein the additional enrichment step further enriches at least a subset of the target sequences. In various embodiments, the additional enrichment step comprises hybrid capture. In various embodiments,the additional enrichment step comprises using a nucleic acid binding protein. In various embodiments, the selective cutting is determined by performing a quantitative polymerase chain reaction (qPCR) assay. In various embodiments, the selective cutting is determined by performing a bioinformatics analysis. In various embodiments, the bioinformatics analysis comprises performing a ratio metric calculation. In various embodiments, the ratio metric calculation is a count of reads starting in a 25 base pair window of a cut site divided by the total number of reads in a given nucleic acid region.

[0010] Additionally disclosed herein is a kit comprising reagents for performing a nucleic acid conversion of a sample from a subject; and a nuclease that selectively cuts a converted nucleic acid sequence due to a presence of one or more converted nucleotide bases in the converted nucleic acid as a result of performing or having performed the nucleic acid conversion using the reagents.

[0011] In various embodiments, the nuclease is a CRISPR-Cas nuclease. In various embodiments, the CRISPR-Cas nuclease is one or more of Casl2, Cas9, and subtypes and / or variants thereof. In various embodiments, the Casl2 is Casl2a. In various embodiments, the Cas9 is Cas9, In various embodiments, the one or more converted nucleotide bases are present in one or more protospacer adjacent motif (PAM) nucleic acid sequence(s). In various embodiments, the one or more PAM nucleic acid sequences comprise TTN or TTTG, where N is one of A, C, T, or G. In various embodiments, the one or more PAM nucleic acid sequences comprise TTTG. In various embodiments, the one or more converted nucleotide bases are present in a seed sequence complementary to a guide RNA sequence. In various embodiments, the one or more converted nucleotide bases are present in a seed sequence less than 7 nucleotide bases from a PAM sequence. In various embodiments, the one or more converted nucleotide bases are present in one or more protospacer adjacent motif (PAM) nucleic acid sequence(s) and further present in a seed sequence complementary to a guide RNA sequence. In various embodiments, the one or more PAM nucleic acid sequences comprise TTN or TTTG, where N is one of A, C, T, or G. In various embodiments, the one or more PAM nucleic acid sequences comprise TTTG. In various embodiments, the one or more converted nucleotide bases are present in a seed sequence less than 7 nucleotide bases from a PAM sequence. In various embodiments, the CRISPR-Cas nuclease recognizes one or more protospacer adjacent motif (PAM) nucleic acid sequence(s). In various embodiments, the one or more PAM nucleic acid sequences comprise TTN, where N is one of A, C, T, or G. In various embodiments, the one or more PAM nucleic acid sequences comprise TTG. In various embodiments, the nucleic acid conversion comprises a bisulfite conversion. In various embodiments, a guide RNA (gRNA)comprising a spacer sequence that binds to a target sequence of the converted sequence. In various embodiments, the target sequence comprises a sequence or a portion of a sequence of a CGI selected from Tables 1-4.

[0012] In various embodiments, a kit disclosed herein comprises one or more primer sequences. In various embodiments, the one or more primer sequences comprise indexing adapters. In various embodiments, the one or more primer sequences comprise a forward and reverse primer pair. In various embodiments, the sample is a blood or serum sample. In various embodiments, the sample comprises cell free DNA (cfDNA). In various embodiments, the subject is a human.

[0013] These and other aspects and features of the invention are described in the following detailed description and claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The foregoing and other objects, features and advantages of the invention will become apparent from the following description of preferred embodiments, as illustrated in the accompanying drawings. Like referenced elements identify common features in the corresponding drawings. The drawings are not necessarily to scale, with emphasis instead being placed on illustrating the principles of the present invention, in which:

[0015] It is noted that wherever practicable similar or like reference numbers may be used in the figures and may indicate similar or like functionality. For example, a letter after a reference numeral, such as “CpG site 320A,” indicates that the text refers specifically to the element having that particular reference numeral. A reference numeral in the text without a following letter, such as “CpG site 320” refers to any or all of the elements in the figures bearing that reference numeral (e.g. “CpG site 320” in the text refers to reference numerals “CpG site 320A” and / or “CpG site 302B” in the figures).

[0016] Figure (FIG) 1 shows an example flow diagram for depleting unmethylated nucleic acid molecules, in accordance with an embodiment.

[0017] FIG. 2A depicts an example conversion of nucleic acids, in accordance with an embodiment.

[0018] FIG. 2B shows the results of nitrite conversion on select nucleotides, in accordance with a second embodiment. Figure adapted from Li et al. (2022) Genome Biology 23: 122.

[0019] FIG. 3A is an example diagram depicting selectively cutting of nucleic acids, in accordance with a first embodiment.

[0020] FIG. 3B is an example diagram depicting selectively cutting of nucleic acids, in accordance with a second embodiment.

[0021] FIG. 3 C is an example diagram depicting selectively cutting of nucleic acids, in accordance with a third embodiment.

[0022] FIG. 3D is an example diagram depicting hybridization of primers to uncleaved nucleic acids, in accordance with any one of the embodiments shown in FIGs. 3A, 3B, or 3C.

[0023] FIG. 3E is an example diagram depicting enrichment of uncleaved nucleic acids, in accordance with the embodiment shown in FIG. 3D.

[0024] FIG. 4 shows an example experimental setup involving a hypermethylated library (e.g., hyper gblock library), a hypomethylated library (e.g., hypo gblock library), and a 50:50 hypo / hyper methylated library (e.g., hyperhypo gblock library).

[0025] FIG. 5 shows an example nucleic acid sequence derived from a fully methylated nucleic acid (“Hyper gblock”) and an example nucleic acid sequence comprising a PAM sequence that is derived from a fully unmethylated nucleic acid (“Hypo gblock”).

[0026] FIGs. 6A, 6B, and 6C show qPCR results of the hypermethylated library, hypomethylated library, and 50:50 hypo: hypermethylated library.

[0027] FIG. 7A is an example experimental design for cutting unmethylated nucleic acid molecules prior to library generation and sequencing.

[0028] FIG. 7B shows the differential cutting of hypermethylated and hypomethylated nucleic acids.

[0029] FIG. 7C shows example computational identification and filtering of cut nucleic acids.

[0030] FIG. 8A is an example experimental design for using qPCRto quantify differential amplification of hypermethylated and hypomethylated nucleic acids.

[0031] FIG. 8B shows results of example bisulfite converted sequences derived from cancer or non-cancer samples following depletion using CRISPR Casl2a.

[0032] FIG. 8C shows qPCR results of hypomethylated nucleic acids following depletion using CRISPR Casl2a.

[0033] FIG. 8D shows qPCR results of hypermethylated nucleic acids following depletion using CRISPR Casl2a.

[0034] FIG. 9 is an example experimental design used to evaluate the depletion efficiency of the CRISPR Casl2a-crRNA system under clinically relevant conditions (genomic background).

[0035] FIG. 10 shows DNA gel results demonstrating the presence of both the hypo and hyper gblock with stubby adapters.

[0036] FIG. 11 shows DNA gel results of Cas-dependent cutting of the hypo gblock.

[0037] FIG. 12 shows DNA gel results before indexing PCR with and without Cas presentation.

[0038] FIG. 13 shows bioinformatics results post-iSeq with and without Cas treatment.

[0039] FIG. 14A shows analytical metric to determine enrichment.

[0040] FIG. 14B shows analytical metric to determine enrichment.

[0041] FIG. 14C shows analytical metric to determine enrichment.

[0042] FIG. 15A shows the ratio of AK5m / AK5 of the Hyper gblock in Hypo gDNA based on iSeq results.

[0043] FIG. 15B shows the ratio of AK5m / AK5 of the Hypo gblock in Hyper gDNA based on iSeq results.

[0044] FIG. 15C shows the fold enrichment of the Hyper gblock spiked into Hyper gDNA based on iSeq results.

[0045] FIG. 15D shows the fold enrichment of the Hypo gblock spiked into Hyper gDNA based on iSeq results.

[0046] FIG. 16A shows the ratio of AK5m / AK5 of the Hyper gblock in Hypo gDNA based on Nova seq results.

[0047] FIG. 16B shows the ratio of AK5m / AK5 of the Hypo gblock in Hyper gDNA based on Nova seq results.

[0048] FIG. 16C shows the fold enrichment of the Hyper gblock spiked into Hyper gDNA based on Nova seq results.

[0049] FIG. 16D shows the fold enrichment of the Hypo gblock spiked into Hyper gDNA based on Nova seq results.

[0050] FIG. 17 is an example diagram of the mechanism of crRNA-mediated selective cutting of unmethylated DNA when a common PAM site is present.

[0051] FIG. 18A shows DNA gel results of selective Hypo gblock cutting (Km2).

[0052] FIG. 18B shows the count results of different DNA lengths of Hyper and Hypo gblocks DNA fragments following Hypo crRNA presentation (Km2).

[0053] FIG. 19 shows bioinformatics results of Hypo gblock cutting with and without crRNA (Km2).

[0054] FIG. 20 is an example diagram of the mechanism of PAM-mediated selective cutting of unmethylated DNA.

[0055] FIG. 21A shows DNA gel results of selective Hypo gblock cutting (Km3).

[0056] FIG. 2 IB shows the count results of different DNA lengths of Hyper and Hypo gblocks DNA fragments following Hypo crRNA presentation (Km3).

[0057] FIG. 22 shows bioinformatics results of Hypo gblock cutting with and without crRNA (Km3).DETAILED DESCRIPTIONDefinitions

[0058] The articles “a” and “an” are used herein to refer to one or to more than one (i. e. , to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0059] As used herein, the term “about” or “approximately” can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which can depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. “About” can mean a range of ±20%, ±10%, ±5%, or ±1% of a given value. The term “about” or “approximately” can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value. Where a particular value is described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value can be assumed. The term “about” can have the meaning as commonly understood by one of ordinary skill in the art. The term “about” can refer to ±10%. The term “about” can refer to ±5%.

[0060] It should be understood that the expression of “at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and / or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[0061] As used herein, the term “biological sample,” or “sample” refers to any sample taken from a subject, which can reflect a biological state associated with the subject, and that includescell free DNA. A biological sample can take any of a variety of forms, such as a liquid biopsy (e.g., blood, urine, stool, saliva, or mucous), or a tissue biopsy, or other solid biopsy. Examples of biological samples include, but are not limited to, blood, whole blood, plasma, serum, urine, cerebrospinal fluid, fecal, saliva, sweat, tears, pleural fluid, pericardial fluid, or peritoneal fluid of the subject. A biological sample can include any tissue or material derived from a living or dead subject. A biological sample can be a cell-free sample. A biological sample can comprise a nucleic acid (e.g. , DNA or RNA) or a fragment thereof. The term “nucleic acid” can refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or any hybrid or fragment thereof. The nucleic acid in the sample can be a cell-free nucleic acid. A sample can be a liquid sample or a solid sample (e.g. , a cell or tissue sample). A biological sample can be a bodily fluid, such as blood, plasma, serum, urine, vaginal fluid, fluid from a hydrocele (e.g., of the testis), vaginal flushing fluids, pleural fluid, ascitic fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, discharge fluid from the nipple, aspiration fluid from different parts of the body (e.g., thyroid, breast), etc. A biological sample can be a stool sample. In various embodiments, the majority of DNA in a biological sample that has been enriched for cell-free DNA (e.g., a plasma sample obtained via a centrifugation protocol) can be cell-free (e.g., greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the DNA can be cell-free). A biological sample can be treated to physically disrupt tissue or cell structure (e.g., centrifugation and / or cell lysis), thus releasing intracellular components into a solution which can further contain enzymes, buffers, salts, detergents, and the like which can be used to prepare the sample for analysis.

[0062] As used herein, the terms “nucleic acid” and “nucleic acid molecule” are used interchangeably. The terms refer to nucleic acids of any composition form, such as deoxyribonucleic acid (DNA, e.g., complementary DNA (cDNA), genomic DNA (gDNA) and the like), and / or DNA analogs (e.g., containing base analogs, sugar analogs and / or a non-native backbone and the like), all of which can be in single- or double-stranded form. Unless otherwise limited, a nucleic acid can comprise known analogs of natural nucleotides, some of which can function in a similar manner as naturally occurring nucleotides. A nucleic acid can be in any form useful for conducting processes herein (e.g, linear, circular, supercoiled, single-stranded, double -stranded and the like). A nucleic acid in some embodiments can be from a single chromosome or fragment thereof (e.g. , a nucleic acid sample may be from one chromosome of a sample obtained from a diploid organism). In certain embodiments nucleic acids comprise nucleosomes, fragments or parts of nucleosomes or nucleosome-like structures. Nucleic acids can comprise protein (e.g., histones, DNA binding proteins, and the like). Nucleic acids analyzed by processes described herein can be substantially isolated and are not substantiallyassociated with protein or other molecules. Nucleic acids can also include derivatives, variants and analogs of DNA synthesized, replicated or amplified from single -stranded (“sense” or “antisense,” “plus” strand or “minus” strand, “forward” reading frame or “reverse” reading frame) and double-stranded polynucleotides. Deoxyribonucleotides can include deoxyadenosine, deoxy cytidine, deoxyguanosine and deoxythymidine. A nucleic acid may be prepared using a nucleic acid obtained from a subject as a template.

[0063] As used herein, the terms “template nucleic acid” and “template nucleic acid molecule(s)” are used interchangeably. The terms refer to nucleic acid that has been obtained from a sample and processed to form an immortalized library. The template nucleic acid can be nucleic acid obtained directly from the sample, or nucleic acid that is derived from that obtained directly from the sample. Examples of nucleic acid derived from a sample include DNA that has been reverse-transcribed from RNA obtained directly from a sample, or DNA that has be amplified from DNA obtained directly from a sample, for example, by PCR.

[0064] As used herein, the term “cell-free nucleic acids” refers to nucleic acid molecules that can be found outside cells, in bodily fluids such as blood, whole blood, plasma, serum, urine, cerebrospinal fluid, fecal, saliva, sweat, sweat, tears, pleural fluid, pericardial fluid, or peritoneal fluid of a subject. Cell-free nucleic acids originate from one or more healthy cells and / or from one or more cancer cells, or from non-human sources such bacteria, fungi, viruses. Examples of the cell-free nucleic acids include but are not limited to cell-free DNA (“cfDNA”), including mitochondrial DNA or genomic DNA, and cell-free RNA. In certain embodiments herein, instruments for assessing the quality of the cell-free nucleic acids, such as the TapeStation System from Agilent Technologies (Santa Clara, CA) can be used. Concentrating low- abundance cfDNA can be accomplished, for example using a Qubit™ Fluorometer from Thermofisher Scientific (Waltham, MA).

[0065] As used herein, the term “methylation” refers to a modification of a nucleic acid where a hydrogen atom on the pyrimidine ring of a cytosine base is converted to a methyl group, forming 5 -methylcytosine. Methylation can occur at dinucleotides of cytosine and guanine referred to herein as “CpG sites”. Methylation of cytosine can occur in cytosines in other sequence contexts, for example, 5'-CHG-3' and 5'-CHH-3', where H is adenine, cytosine or thymine. Cytosine methylation can also be in the form of 5 -hydroxymethylcytosine. Methylation of DNA can include methylation of non-cytosine nucleotides, such as N6-methyladenine. Anomalous cfDNA methylation can be identified as hypermethylation or hypomethylation, both of which may be indicative of cancer status. As is well known in the art, DNA methylation anomalies (compared to healthy controls) can cause different effects, which may contribute to cancer.

[0066] Certain portions of a genome comprise regions with a high frequency of CpG sites. A CpG site is portion of a genome that has cytosine and guanine separated by only one phosphate group and is often denoted as “5' — C — phosphate — G — 3'”, or “CpG” for short. Regions with a high frequency of CpG sites are commonly referred to as “CG islands” or “CGIs”. It has been found that certain CGIs and certain features of certain CGIs in tumor cells tend to be different from the same CGIs or features of the CGIs in healthy cells. Herein, such CGIS and features of the genome are referred to herein as “cancer informative CGIs”, which is defined and described in more detail below. An “informative CpG” can be specified by reference to a specific CpG site, or to a collection of one or more CpG sites by reference to a CG island that contains the collection. These cancer informative CGIs tend to have methylation patterns in tumor cells that are different from the methylation patterns in healthy cells. DNA fragments from other CGIs may not express such differences.

[0067] As used herein, “DNA methylation” in mammalian genomes can refer to the addition of a methyl group to position 5 of the heterocyclic ring of cytosine (e.g., to produce 5- methylcytosine) among CpG dinucleotides. Methylation of cytosine can occur in cytosines in other sequence contexts, for example, 5'-CHG-3' and 5'-CHH-3', where H is adenine, cytosine or thymine. Cytosine methylation can also be in the form of 5-hydroxymethylcytosine. Methylation of DNA can include methylation of non-cytosine nucleotides, such as N6- methyladenine.

[0068] The phrase “unmethylated nucleic acid molecules” is broadly used to encompass nucleic acid molecules comprising sequences that include one or more unmethylated CpG sites and / or nucleic acid sequences derived from nucleic acid sequences that include one or more unmethylated CpG sites. For example, “unmethylated nucleic acid molecules” can refer to converted nucleic acid sequences (e.g., bisulfite-converted nucleic acid sequences) that are derived from cell-free DNA that include sequences with one or more unmethylated CpG sites.

[0069] As used herein, the term “amplifying” means performing an amplification reaction. In one aspect, an amplification reaction is “template-driven” in that base pairing of reactants, either nucleotides or oligonucleotides, have complements in a template polynucleotide that are required for the creation of reaction products. In one aspect, template -driven reactions are primer extensions with a nucleic acid polymerase, or oligonucleotide ligations with a nucleic acid ligase. Such reactions include, but are not limited to, polymerase chain reactions (PCRs), linear polymerase reactions, nucleic acid sequence -based amplification (NASBAs), rolling circle amplifications, and the like, disclosed in the following references, each of which are incorporated herein by reference herein in their entirety: Mullis et al., U.S. Pat. Nos. 4,683,195;4,965,188; 4,683,202; 4,800,159 (PCR); Gelfand et al., U.S. Pat. No. 5,210,015 (real-time PCR with “taqman” probes); Wittwer et al., U.S. Pat. No. 6,174,670; Kacian et al., U.S. Pat. No. 5,399,491 (“NASBA”); Uizardi, U.S. Pat. No. 5,854,033; Aono et al., Japanese patent publ. JP 4- 262799 (rolling circle amplification); and the like. In one aspect, the amplification reaction is PCR. An amplification reaction may be a “real-time” amplification if a detection chemistry is available that permits a reaction product to be measured as the amplification reaction progresses, e.g., “real-time PCR”, or “real-time NASBA” as described in Ueone et al., Nucleic Acids Research, 26: 2150-2155 (1998), and like references.

[0070] The terms “fragment” or “segment”, as used interchangeably herein, refer to a portion of a larger polynucleotide molecule. A polynucleotide, for example, can be broken up, or fragmented into, a plurality of segments. Various methods of fragmenting nucleic acid are well known in the art. These methods may be, for example, either chemical or physical or enzymatic in nature. Enzymatic fragmentation may include partial degradation with a DNase; partial depurination with acid; the use of restriction enzymes; intron-encoded endonucleases; DNA- based cleavage methods, such as triplex and hybrid formation methods, that rely on the specific hybridization of a nucleic acid segment to localize a cleavage agent to a specific location in the nucleic acid molecule; or other enzymes or compounds which cleave a polynucleotide at known or unknown locations. Physical fragmentation methods may involve subjecting a polynucleotide to a high shear rate. High shear rates may be produced, for example, by moving DNA through a chamber or channel with pits or spikes, or forcing a DNA sample through a restricted size flow passage, e.g., an aperture having a cross sectional dimension in the micron or submicron range. Other physical methods include sonication and nebulization. Combinations of physical and chemical fragmentation methods may likewise be employed, such as fragmentation by heat and ion-mediated hydrolysis. See, e.g., Sambrook et al., “Molecular Cloning: A Eaboratory Manual,” 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) (“Sambrook et al.) which is incorporated herein by reference for all purposes. These methods can be optimized to digest a nucleic acid into fragments of a selected size range.

[0071] The terms “polymerase chain reaction” or “PCR”, as used interchangeably herein, mean a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA. In other words, PCR is a reaction for making multiple copies or replicates of a target nucleic acid flanked by primer binding sites, such reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates. Usually, the reaction iscycled through different temperatures optimized for each step in a thermal cycler instrument. Particular temperatures, durations at each step, and rates of change between steps depend on many factors that are well-known to those of ordinary skill in the art, e.g., exemplified by the following references: McPherson et al., editors, PCR: A Practical Approach and PCR2: A Practical Approach (IRL Press, Oxford, 1991 and 1995, respectively). For example, in a conventional PCR using Taq DNA polymerase, a double stranded target nucleic acid may be denatured at a temperature>90° C, primers annealed at a temperature in the range 50-75° C, and primers extended at a temperature in the range 72-78° C. The term “PCR” encompasses derivative forms of the reaction, including, but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, and the like. The particular format of PCR being employed is discernible by one skilled in the art from the context of an application. Reaction volumes can range from a few hundred nanoliters, e.g., 200 nL, to a few hundred pL, e.g., 200 pL. “Reverse transcription PCR,” or “RT-PCR,” means a PCR that is preceded by a reverse transcription reaction that converts a target RNA to a complementary single stranded DNA, which is then amplified, an example of which is described in Tecott et al., U.S. Pat. No. 5,168,038, the disclosure of which is incorporated herein by reference in its entirety. “Real-time PCR” means a PCR for which the amount of reaction product, i.e., amplicon, is monitored as the reaction proceeds. There are many forms of real-time PCR that differ mainly in the detection chemistries used for monitoring the reaction product, e.g., Gelfand et al., U.S. Pat. No. 5,210,015 (“taqman”); Wittwer et al., U.S. Pat. Nos. 6,174,670 and 6,569,627 (intercalating dyes); Tyagi et al., U.S. Pat. No. 5,925,517 (molecular beacons); the disclosures of which are hereby incorporated by reference herein in their entireties. Detection chemistries for real-time PCR are reviewed in Mackay et al., Nucleic Acids Research, 30: 1292-1305 (2002), which is also incorporated herein by reference. “Nested PCR” means a two-stage PCR wherein the amplicon of a first PCR becomes the sample for a second PCR using a new set of primers, at least one of which binds to an interior location of the first amplicon. As used herein, “initial primers” in reference to a nested amplification reaction mean the primers used to generate a first amplicon, and “secondary primers” mean the one or more primers used to generate a second, or nested, amplicon. “Asymmetric PCR” means a PCR wherein one of the two primers employed is in great excess concentration so that the reaction is primarily a linear amplification in which one of the two strands of a target nucleic acid is preferentially copied. The excess concentration of asymmetric PCR primers may be expressed as a concentration ratio. Typical ratios are in the range of from 10 to 100. “Multiplexed PCR” means a PCR wherein multiple target sequences (or a single target sequence and one or more reference sequences) are simultaneously carried out in the same reaction mixture, e.g., Bernard et al., Anal. Biochem., 273: 221-228 (1999) (two-color real-time PCR). Usually, distinct sets of primers are employed for each sequence being amplified. Typically, the number of target sequences in a multiplex PCR is in the range of from 2 to 50, or from 2 to 40, or from 2 to 30. “Quantitative PCR” means a PCR designed to measure the abundance of one or more specific target sequences in a sample or specimen. Quantitative PCR includes both absolute quantitation and relative quantitation of such target sequences. Quantitative measurements are made using one or more reference sequences or internal standards that may be assayed separately or together with a target sequence. The reference sequence may be endogenous or exogenous to a sample or specimen, and in the latter case, may comprise one or more competitor templates. Typical endogenous reference sequences include segments of transcripts of the following genes: [3-actin, GAPDH, 2 -microglobulin, ribosomal RNA, and the like. Techniques for quantitative PCR are well-known to those of ordinary skill in the art, as exemplified in the following references, which are incorporated by reference herein in their entireties: Freeman et al., Biotechniques, 26: 112-126 (1999); Becker-Andre et al., Nucleic Acids Research, 17: 9437-9447 (1989); Zimmerman et al., Biotechniques, 21: 268-279 (1996); Diviacco et al., Gene, 122: 3013-3020 (1992); and Becker-Andre et al., Nucleic Acids Research, 17: 9437-9446 (1989).

[0072] The term “primer” as used herein means an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3' end along the template so that an extended duplex is formed. Extension of a primer is usually carried out with a nucleic acid polymerase, such as a DNA or RNA polymerase. The sequence of nucleotides added in the extension process is determined by the sequence of the template polynucleotide. Usually, primers are extended by a DNA polymerase. Primers usually have a length in the range of from 14 to 40 nucleotides, or in the range of from 18 to 36 nucleotides. Primers are employed in a variety of nucleic amplification reactions, for example, linear amplification reactions using a single primer, or polymerase chain reactions, employing two or more primers. Guidance for selecting the lengths and sequences of primers for particular applications is well known to those of ordinary skill in the art, as evidenced by the following reference that is incorporated by reference herein in its entirety: Dieffenbach, editor, PCR Primer: A Eaboratory Manual, 2nd Edition (Cold Spring Harbor Press, New York, 2003).

[0073] As used herein, the term “subject” refers to any living or non-living organism, including but not limited to a human (e.g., a male human, female human, fetus, pregnant female, child, or the like), a non-human animal, a plant, a bacterium, a fungus or a protist. Any human or nonhuman animal can serve as a subject, including but not limited to mammal, reptile, avian,amphibian, fish, ungulate, ruminant, bovine (e.g., cattle), equine (e.g., horse), caprine and ovine (e.g., sheep, goat), swine (e.g., pig), camelid (e.g., camel, llama, alpaca), monkey, ape (e.g., gorilla, chimpanzee), ursid (e.g., bear), poultry, dog, cat, mouse, rat, fish, dolphin, whale and shark. In some embodiments, a subject is a male or female of any age (e.g., a man, a women or a child).

[0074] As used herein, the phrase “selectively cutting” refers to a higher level of cutting of a first plurality of nucleic acids in relation to a lower level of cutting of a second plurality of nucleic acids. In various embodiments, “selectively cutting” encompasses cutting the first plurality of nucleic acids and not cutting the second plurality of nucleic acids. In various embodiments, “selectively cutting” encompasses cutting the first plurality of nucleic acids and cutting the second plurality of nucleic acids at a level less than 30% of the cutting of the first plurality nucleic acids. In various embodiments, “selectively cutting” encompasses cutting the first plurality of nucleic acids and cutting the second plurality of nucleic acids at a level less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the cutting of the first plurality nucleic acids.

[0075] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art.

[0076] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY (1998); Coligan et al., Short Protocols in Protein Science, John Wiley & Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999).

[0077] Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, biochemistry, immunology, molecular biology, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, and chemical analyses.

[0078] Throughout this specification and embodiments, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0079] It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided.

[0080] Any example(s) following the term “e.g.” or “for example” is not meant to be exhaustive or limiting.

[0081] Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0082] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to be inclusive of the numbers defining the range and to encompass any and all subranges subsumed therein. For example, a stated range of “1 to 10” should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g., 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10.

[0083] Where aspects or embodiments of the disclosure are described in terms of a Markush group or other grouping of alternatives, the present disclosure encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups ofthe main group, but also the main group absent one or more of the group members. The present disclosure also envisages the explicit exclusion of one or more of any of the group members in an embodiment of the disclosure.

[0084] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. The materials, methods, and examples are illustrative only and not intended to be limiting.Overview

[0085] Disclosed herein are methods for depleting unmethylated nucleic acid molecules, thereby enriching for target sequences of interest in a sample (e.g., a sample obtained from a subject). Such methods are useful for enriching for a signal in a sample, such as a signal informative for determining presence or absence of cancer in the sample.

[0086] In various embodiments, methods involve obtaining a sample including nucleic acids comprising a first subset of nucleic acids and a second subset of nucleic acids. In various embodiments, the first subset of the nucleic acids and the second subset of the nucleic acids include one or more differentially methylated CpG sites. For example, the first subset of the nucleic acids include unmethylated CpG sites whereas the second subset of the nucleic acids include methylated CpG sites. In various embodiments, methods disclosed herein involve enriching for target sequences, where the target sequence comprises the one or more CpG sites that are differentially methylated in the first subset and the second subset of nucleic acids. Thus, methods disclosed herein leverage the differentially methylated CpG sites to deplete a subset of nucleic acids, thereby enriching for the other subset of nucleic acids. For example, methods involve depleting the first subset of nucleic acids (e.g., nucleic acids including one or more unmethylated CpG sites), thereby enriching for the second subset of nucleic acids (e.g., nucleic acids including one or more methylated CpG sites). Given that target sequences of interest may be present in a sample at low frequency, depleting the first subset of nucleic acids and enriching for the second subset of nucleic acids enables improved detection of the target sequences of interest.

[0087] In various embodiments, depleting the first subset of nucleic acids involves contacting a nuclease with the first subset of nucleic acids. In various embodiments, the nuclease is a CRISPR-Cas nuclease, examples of which are further disclosed herein. Generally, CRISPR-Cas nucleases recognize a protospacer adjacent motif (PAM) sequence. Once the CRISPR-Cas nuclease recognizes a presence of a PAM sequence, the nuclease cuts the nucleic acid at a locus within or near a target sequence located near the PAM sequence. In contrast, if the CRISPR-Casnuclease fails to recognize presence of a PAM sequence, the nuclease does not cut the nucleic acid. In various embodiments, the first subset of nucleic acids comprises a recognizable PAM sequence and is therefore cut by the nuclease, whereas the second subset of nucleic acids does not include a recognizable PAM sequence and therefore is not cut by the nuclease. In various embodiments, the PAM sequence is present in the first subset of nucleic acids arising from a conversion of unmethylated cytosines (e.g., deamination of unmethylated cytosines). In contrast, a PAM sequence is not present in the second subset of nucleic acid sequences as methylated cytosines are protected from conversion. Altogether, the differential methylation of one or more CpG sites in the first subset and second subset of nucleic acids leads to the presence or absence of a PAM sequence, thereby resulting in the cutting and depletion of the first subset of nucleic acids (e.g., nucleic acids including one or more unmethylated CpG sites) while enriching for the second subset of nucleic acids (e.g., nucleic acids including one or more methylated CpG sites). The enriched signal derived from the second subset of nucleic acids can be informative for determining presence or absence of cancer in the sample from where the nucleic acids originated.

[0088] Reference is now made to FIG. 1, which shows an example flow diagram for depleting unmethylated nucleic acid molecules, in accordance with an embodiment. As shown in FIG. 1, step 115 involves obtaining a sample (e.g., a sample from a subject). In various embodiments, a sample is any of a blood sample, a serum sample, a stool sample, a urine sample, a mucous sample, or a saliva sample. In particular embodiments, a sample is a blood sample or a serum sample. The sample can be obtained by the subject or by a third party, e.g., a medical professional. Examples of medical professionals include physicians, emergency medical technicians, nurses, first responders, psychologists, phlebotomists, medical physics personnel, nurse practitioners, surgeons, dentists, and any other medical professional as would be known to one skilled in the art. In various embodiments, the one or more samples can be obtained from the subject by a reference lab.

[0089] In various embodiments, the sample obtained from the subject is a liquid biopsy sample. In various embodiments, the liquid biopsy sample includes cell -free DNA (cfDNA). In particular embodiments, the cfDNA includes genomic sequences corresponding to CpG islands (CGIs) for which methylation states are informative of a health condition. In various embodiments, the cfDNA can be derived from tumor cells and is referred to herein as circulating tumor DNA (ctDNA).

[0090] Step 120 involves converting the nucleic acid from the sample obtained from the subject. In various embodiments, converting the nucleic acid involves converting unmethylatednucleotides (e.g., cytosines) to another nucleotide (a “converted nucleotide”, as used herein). In various embodiments, methylated cytosines are protected from conversion (e.g., deamination) during the conversion step. Further details of performing conversion of nucleic acid molecules (e.g., step 120) are described herein.

[0091] Although not shown in FIG. 1, in various embodiments, after conversion of nucleic acids, the converted nucleic acids undergo library construction. In various embodiments, converted nucleic acids can undergo end-repairing, tailing of 3’ ends, and / or addition of library or sequencing adapters. In various embodiments, converted nucleic acids can undergo biotinylation (e.g., addition of biotin moieties to converted nucleic acids). In various embodiments, barcodes can be incorporated into converted nucleic acids, thereby enabling subsequent sample demultiplexing (e.g., demultiplexing to identify sources of converted nucleic acids or demultiplexing to identify a common source from converted nucleic acids). In various embodiments, one or more washes and / or selections can be performed to remove unwanted DNA fragments, such as single stranded DNA fragments, excess adapters, and other molecules. In particular embodiments, a solid-phase reversible immobilization (SPRI) selection is performed. As used herein, a “nucleic acid template” refers to a nucleic acid derived from the converted nucleic acid (e.g., any of a nucleic acid derived from a converted nucleic acid that underwent library construction, end-repairing, addition of library or sequencing adapters, barcode addition, or any combination thereof).

[0092] Step 125 involves enriching for a subset of nucleic acid molecules. As shown in FIG. 1, step 125 can include subsets 130, 140, 145, and 150. In particular embodiments, enriching for a subset of nucleic acid molecules comprises depleting or reducing a quantity of a different subset of nucleic acid molecules. For example, step 125 involves enriching for a signal from methylated nucleic acid molecules while depleting unmethylated nucleic acid molecules.

[0093] Step 130 involves contacting a nuclease with the nucleic acid molecules (e.g., the first subset and second subset of nucleic acid molecules). In various embodiments, step 130 involves contacting an endonuclease with the nucleic acid molecules. An example nuclease can be an endonuclease, for example, a Cas protein, that is capable of cleaving DNA (e.g., double stranded DNA) to effect a break at the intended locus. In particular embodiments, the Cas protein is a CRISPR Cas 12 protein. Further details of example nucleases are disclosed herein.

[0094] Step 140 involves selectively cutting the first subset of nucleic acid molecules using the nuclease relatively to the second subset of the nucleic acid molecules. As used herein “selectively cutting the first subset of nucleic acid molecules” refers to the increased cutting of nucleic acids of the first subset in relation to other nucleic acids. By selectively cutting the firstsubset of nucleic acid molecules, the remaining nucleic acids of the second subset are enriched. In various embodiments, the first subset of nucleic acid molecules include PAM sequences that are recognizable by the nuclease such that the nuclease cuts nucleic acids of the first subset of nucleic acid molecules. In addition, the first set subset of nucleic acid molecules can also be effectively cut through a TG / CG mismatch being present in the first 7 bases of the crRNA. The second subset of nucleic acid molecules do not include PAM sequences and therefore are not recognized by the nuclease. Nucleic acids of the second subset of nucleic acid molecules are not cut by the nuclease. In various embodiments, the nuclease exhibits reduced cutting of nucleic acids of the second subset in comparison to the first subset of nucleic acids. In various embodiments, the nuclease cuts less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, or less than 0.01% of nucleic acids of the second subset.

[0095] Steps 145 and 150 involve performing an additional enrichment step to further enrich for nucleic acids of the second subset. Step 145 involves providing copies of a primer sequence that hybridizes to the second subset of the nucleic acid molecules. In various embodiments, the copies of the primer sequence are capable of hybridizing to complementary sequences that are present in the nucleic acid molecules of the second subset. In various embodiments, the copies of the primer sequence are incapable of hybridizing to sequences of the nucleic acid molecules of the first subset. For example, as the nucleic acid molecules of the first subset have been cut by the nuclease, a complementary sequence of the primer sequences is not present in nucleic acid molecules of the first subset. Thus, copies of the primer sequence fail to hybridize with sequences of the nucleic acid molecules of the first subset.

[0096] Step 150 involves enriching for the second subset of nucleic acid molecules using the hybridized copies of primer sequences. Here, given that the copies of primer sequences are hybridized to nucleic acid molecules of the second subset and are not hybridized (or are minimally hybridized) to nucleic acid molecules of the first subset, step 150 further increases the quantity of nucleic acid molecules of the second subset (and / or amplicons thereof) in comparison to nucleic acid molecules of the first subset (and / or amplicons thereof). In various embodiments, enriching for the second subset of nucleic acid molecules involves one or more of performing hybrid capture, using DNA-binding proteins to enrich a target sequence or a subset of target sequences, or performing nucleic acid amplification.

[0097] Although not shown in FIG. 1, in various embodiments, after step 150, the enriched nucleic acids undergo one or more washes and / or selections to remove unwanted DNA fragments, such as single stranded DNA fragments, excess primers, excess adapters, and other molecules. In particular embodiments, a solid-phase reversible immobilization (SPRI) selection is performed.

[0098] Although not shown in FIG. 1, in various embodiments, after step 150, the enriched nucleic acids undergo sequencing to determine the sequences of the nucleic acids. In various embodiments, sequencing data can be demultiplexed e.g., using barcode sequences. In various embodiments, sequencing data can be aligned to a reference genome and / or trimmed. In various embodiments, sequencing data can be further analyzed to determine the enriched signal in the sample e.g., for determining presence or absence of cancer in the sample.Methods for Enriching Target Nucleic Acid Sequences a. Exemplary Methods

[0099] As disclosed herein in reference to FIG. 1, methods can involve step 125 for enriching a subset of nucleic acid molecules e.g., nucleic acid molecules comprising target nucleic acid sequences. The steps 130, 140, 145, and 150 are now described in further detail in further reference to FIGs. 3A-3E. Specifically, FIG. 3A is an example diagram depicting selectively cutting of nucleic acids, in accordance with a first embodiment. Here, FIG. 3A shows Cas- mediated selective depletion of unmethylated bisulfite converted DNA via PAM site mismatch. FIG. 3B is an example diagram depicting selectively cutting of nucleic acids, in accordance with a second embodiment. Here, FIG. 3B shows crRNA-mediated selective depletion of unmethylated bisulfite converted DNA. FIG. 3C is an example diagram depicting selectively cutting of nucleic acids, in accordance with a third embodiment. Here, FIG. 3C shows crRNA and Casl2a-mediated selective depletion of unmethylated bisulfite converted DNA via PAM site mismatch. FIG. 3D is an example diagram depicting hybridization of primers to uncleaved nucleic acids, in accordance with any one of the embodiments shown in FIGs. 3A, 3B, or 3C. FIG. 3E is an example diagram depicting enrichment of uncleaved nucleic acids, in accordance with the embodiment shown in FIG. 3D.

[0100] Referring first to FIG. 3 A, the top panel shows a first nucleic acid molecule 315A and a second nucleic acid molecule 315B. In various embodiments, the first nucleic acid molecule 315A and second nucleic acid molecule 315B are in a mixture of nucleic acid molecules. Together, the nucleic acid molecules 315 represent converted nucleic acids that have previously undergone a conversion step that includes treating nucleic acid molecules to capturemethylation modifications. Here, nucleic acid molecule 315A and nucleic acid molecule 315B each include a sequence corresponding to a CpG site, shown as CpG site 320A and CpG site 320B, respectively. Specifically, nucleic acid molecule 315A includes a sequence of “TTCG” at CpG site 320A and nucleic acid molecule 315B includes a sequence of “TTTG” at CpG site 320B. As both nucleic acid molecule 315A and nucleic acid molecule 315B previously underwent a conversion step, the respective sequences at the CpG site 320 may reflect one or more converted nucleotides. In this example, the sequence “TTCG” at CpG site 320A of nucleic acid molecule 315A derives from a previously methylated CpG site, which had the sequence of “TTC*G”, where “C*” refers to a methylated cytosine. In contrast, the sequence TTTG' at CpG site 320B of nucleic acid molecule 315B derives from a previously unmethylated CpG site, which had the sequence of “TTCAG”, where “CA” refers to an unmethylated cytosine.Following conversion, the unmethylated cytosine “CA” may be converted to a uracil nucleotide, resulting in a sequence of “TTUG.” The resulting double-stranded nucleic acid molecule 315B includes the sequence of “TTTG” at the CpG site 320B.

[0101] As further shown in FIG. 3 A, the nucleic acid molecule 315A may include a target sequence 305A and nucleic acid molecule 315B may include a corresponding target sequence 305B. In various embodiments, a target sequence is located upstream to a CpG site. For example, target sequence 305A is located upstream to CpG site 320A and target sequence 305B is located upstream to CpG site 320B. Generally, the distance between the target sequence and the CpG site refers to the number of nucleotides between 1) the most proximal nucleotide of the target sequence relative to the CpG site and 2) the cytosine of the CpG site. For example, the target sequence may be located at most 1 nucleotide, at most 2 nucleotides, at most 3 nucleotides, at most 4 nucleotides, at most 5 nucleotides, at most 6 nucleotides, at most 7 nucleotides, at most 8 nucleotides, at most 9 nucleotides, at most 10 nucleotides, at most 11 nucleotides, at most 12 nucleotides, at most 13 nucleotides, at most 14 nucleotides, at most 15 nucleotides, at most 16 nucleotides, at most 17 nucleotides, at most 18 nucleotides, at most 19 nucleotides, or at most 20 nucleotides upstream of the CpG site, such as CpG site 320A or CpG site 320B. In various embodiments, the target sequence 305A may be located between 1 and 30 nucleotides, between 2 and 25 nucleotides, between 3 and 20 nucleotides, between 4 and 18 nucleotides, between 5 and 15 nucleotides, between 6 and 12 nucleotides, or between 8 and 10 nucleotides upstream of the CpG site, such as CpG site 320A or CpG site 320B.

[0102] The middle panel of FIG. 3A involves providing a nuclease 325 to the nucleic acid molecules 315. In various embodiments, the nuclease 325 is an endonuclease. In various embodiments, the nuclease 325 is a CRISPR Cas endonuclease. In particular embodiments, thenuclease 325 is a CRISPR Casl2 endonuclease or a CRISPR Cas9 endonuclease. In various embodiments, as shown in FIG. 3 A, the middle panel of FIG. 3A further involves providing a guide RNA 328 (as referred to herein as a “crRNA”) to the nucleic acid molecules 315. Generally, the guide RNA 328 is designed to have complementarity to a target sequence, such as target sequence 305A of nucleic acid molecule 315A and / or target sequence 305B of nucleic acid molecule 315B. Hybridization between a target sequence and a guide sequence promotes the formation of a gene editing endonuclease complex (e.g., a CRISPR complex). For example, a nuclease and guide RNA may form a first gene editing endonuclease complex with nucleic acid molecule 315A by binding to target sequence 305A. A nuclease and guide RNA may form a second gene editing endonuclease complex with nucleic acid molecule 315B by binding to target sequence 305B.

[0103] Within the gene editing endonuclease complex, if the nuclease recognizes a sequence (e.g., a protospacer adjacent motif (PAM) sequence), the nuclease may effect a break at the intended locus. In various embodiments, the PAM sequence may be found at CpG site 320 within the converted nucleic acids 310. As one example, the nuclease 325 is a CRISPR Casl2 endonuclease that recognizes a 5’-T-rich PAM, such as “TTN” or “TTTN,” where “N” is any nucleotide. Thus, as shown in FIG. 3 A, the “TTTN” PAM sequence is present at the CpG site 320B of nucleic acid molecule 315B. Therefore, the nuclease 325 cuts the nucleic acid molecule 315B at a locus within or near a target sequence on the opposite strand (e.g., target sequence 305B). In various embodiments, the nuclease 325 cuts the nucleic acid molecule, leaving an overhang. In various embodiments, the overhang is located on the opposite strand of the PAM sequence. In various embodiments, the overhang is a 1 nucleotide, 2 nucleotide, 3 nucleotide, 4 nucleotide, or 5 nucleotide overhang. The bottom panel of FIG. 3A shows the cleaved nucleic acid 340B with a cleavage site 330 at or near the target sequence 305B.

[0104] In contrast, CpG site 320A includes a sequence of “TTCG” which does not correspond to a recognizable PAM sequence. In this scenario, the nuclease 325 does not effect a break at the intended locus. The bottom panel of FIG. 3A shows the uncleaved nucleic acid 340A including the target sequence 305A.

[0105] Reference is now made to FIG. 3B, which shows crRNA-mediated selective depletion of unmethylated bisulfite converted DNA. Here in FIG. 3B, a schematic is presented that outlines a crRNA-dependent mechanism of selective depletion of unmethylated bisulfite converted DNA molecules in cases where the PAM sequence is present in both the nucleic acid molecule 380A (derived from a methylated nucleic acid) and nucleic acid molecule 380B (derived from an unmethylated nucleic acid). In this example, both nucleic acid molecule 380Aand nucleic acid molecule 380B include a “TTTG” nucleic acid sequence, which represent a PAM site (e.g., PAM site 390A and 390B, respectively). Additionally, nucleic acid molecule 380A and nucleic acid molecule 380B may each have a corresponding CpG site (e.g., CpG site 322A and CpG site 322B, respectively). As shown in FIG. 3B, CpG site 322A in nucleic acid molecule 380A has a “CG” sequence which is derived from a methylated CpG site. CpG site 322B in nucleic acid molecule 380B has a “TG” sequence, which is derived from an unmethylated CpG site.

[0106] In each nucleic acid molecule 380, the corresponding CpG site 322 can be located a distance away from the corresponding PAM site 380. For example, as shown in FIG. 3B, CpG site 322A is separated from the PAM site 390A by a gap 385A. Similarly, CpG site 322B is separated from the PAM site 390B by a gap 385B. In various embodiments, the gap (e.g., gap 385A and / or gap 385B) is less than 10 nucleotide bases in length. In various embodiments, the gap (e.g., gap 385A and / or gap 385B) is less than 9 nucleotide bases, less than 8 nucleotide bases, less than 7 nucleotide bases, less than 6 nucleotide bases, less than 5 nucleotide bases, less than 4 nucleotide bases, less than 3 nucleotide bases, or less than 2 nucleotide bases in length. In particular embodiments, the gap (e.g., gap 385A and / or gap 385B) is less than 7 nucleotide bases in length. In particular embodiments, the gap (e.g., gap 385A and / or gap 385B) is less than 6 nucleotide bases in length. In various embodiments, the gap (e.g., gap 385A and / or gap 385B) can be between 0 and 6 nucleotide bases in length. In various embodiments, the gap (e.g., gap 385 A and / or gap 385B) can be between 1 and 5 nucleotide bases in length, between 1 and 4 nucleotide bases in length, between 1 and 3 nucleotide bases in length, or between 1 and 2 nucleotide bases in length.

[0107] In this scenario, specificity can be determined by a crRNA molecule (shown as guide RNA 328 in FIG. 3B) through complementary pairing with a sequence that is dependent on the sequence at the CpG site (e.g., CpG site 322A or CpG site 322B). For example, a crRNA molecule can be designed and introduced such that the crRNA molecule hybridizes with a sequence that is dependent on the sequence at the CpG site (e.g., CpG site 322A or CpG site 322B). In some embodiments, the crRNA molecule shares 100% complementarity with a sequence that comprises the “TG” of the CpG site 322B. Such a crRNA would not share complementarity with a sequence that comprises the “CG” of the CpG site 322A. In some embodiments, the crRNA molecule shares 100% complementarity with a sequence of a strand opposite of the strand that includes the CpG site. Thus, the crRNA molecule can hybridize with the opposite strand that is complementary to a sequence comprising the “TG” of the CpG site 322A. Such a crRNA would not hybridize with an opposite strand that is complementary to asequence comprising the “CG” of the CpG site 322B. Altogether, the crRNA molecule can be specifically designed such that it can hybridize only with a corresponding sequence that is dependent on the sequence derived from an unmethylated CpG site.

[0108] As shown in FIG. 3B, the nuclease 325 and the guide RNA 328 (e.g., crRNA) are introduced. Following hybridization of the guide RNA 328 and associated nuclease binding, the nuclease 325 can selectively cut nucleic acid molecule 380B while not cutting nucleic acid molecule 380A. Specifically, the nuclease 325 can cut a nucleic acid strand opposite to the PAM site 390B, resulting in cut nucleic acid 39 IB. This cut nucleic acid 39 IB will not be amplified in the subsequent steps, effectively allowing for its depletion. In contrast, the nuclease 325 will not cut the nucleic acid molecule 380A, resulting in uncut nucleic acid 391A. Here, the guide RNA 328 will not hybridize due to a hybridization mismatch based on the presence of the “CG” sequence at CpG site 322A. Here, the CpG site 322A is separated from the PAM site 390A by a gap 385A (e.g., in various embodiments, the gap 385A is less than 7 nucleotide bases). As a result, the uncut nucleic acid 391 A will be available for downstream library preparation and DNA amplification steps.

[0109] Reference is now made to FIG. 3C, which is an example diagram depicting selectively cutting of nucleic acids, in accordance with a third embodiment. Here, FIG. 3C shows crRNA and Casl2a-mediated selective depletion of unmethylated bisulfite converted DNA via PAM site mismatch. Specifically, the cutting specificity is determined by a combination of 1) a presence of a PAM sequence arising from a conversion of an unmethylated sequence and 2) a presence of a CpG site located within a threshold number of bases of the PAM site which determines crRNA hybridization specificity.

[0110] At the top of FIG. 3C, nucleic acid molecule 392A includes a “TTCG” sequence at a CpG site 320A and a sequence of “CG” at a CpG site 320C. Here, the “TTCG” sequence at CpG site 320A and “CG” sequence at CpG site 320C are derived from methylated CpG sites. For example, the “TTCG” sequence may derive from a “TTCG” sequence in which the C was methylated. Similarly, the “CG” may derive from a “CG” sequence in which the C was methylated. Nucleic acid molecule 392B includes a TTTG' sequence at a CpG site 320B and a “TG” sequence at CpG site 320D. Here, the TTTG' sequence at CpG site 320B and “TG” sequence at CpG site 320D are derived from unmethylated CpG sites. For example, the TTTG' sequence may derive from a “TTCG” sequence in which the C was unmethylated. Similarly, the “TG” may derive from a “CG” sequence in which the C was unmethylated.

[0111] In each nucleic acid molecule 392, the two CpG sites (e.g., 320A and 320C, or 320B and 320D) can be located a distance away from each other. For example, CpG site 320Ais separated from CpG site 320C by a gap 385 A. Similarly, CpG site 320B is separated from CpG site 320D by a gap 385B. In various embodiments, the gap (e.g., gap 385A and / or gap 385B) is less than 10 nucleotide bases in length. In various embodiments, the gap (e.g., gap 385 A and / or gap 385B) is less than 9 nucleotide bases, less than 8 nucleotide bases, less than 7 nucleotide bases, less than 6 nucleotide bases, less than 5 nucleotide bases, less than 4 nucleotide bases, less than 3 nucleotide bases, or less than 2 nucleotide bases in length. In particular embodiments, the gap (e.g., gap 385A and / or gap 385B) is less than 7 nucleotide bases in length. In particular embodiments, the gap (e.g., gap 385A and / or gap 385B) is less than 6 nucleotide bases in length. In various embodiments, the gap (e.g., gap 385A and / or gap 385B) can be between 0 and 6 nucleotide bases in length. In various embodiments, the gap (e.g., gap 385A and / or gap 385B) can be between 1 and 5 nucleotide bases in length, between 1 and 4 nucleotide bases in length, between 1 and 3 nucleotide bases in length, or between 1 and 2 nucleotide bases in length.

[0112] As shown in FIG. 3C, the nuclease 325 and the guide RNA 328 (e.g., crRNA) are introduced. In various embodiments, the nuclease 325 recognizes a PAM sequence based on the following nucleic acid motif, “TTTN” wherein N can be “A”, “T”, “G”, or “C” Thus, the nuclease 325 does not recognize the sequence at CpG site 320A of “TTCG”. The presence of “CG” in the CpG site 320C disrupts hybridization of the guide RNA 328. In various embodiments, the guide RNA 328 fails to hybridize to the target sequence 305A due to the presence of the “CG” in the CpG site 320C. In various embodiments, the guide RNA 328 fails to hybridize to a sequence of a strand opposite to the target sequence 305A which includes the “CG” in the CpG site 320C. Together, these two mechanisms prevent the nuclease 325 from cutting the nucleic acid molecule 392A. Thus, uncut nucleic acid 393A remains.

[0113] In contrast, the sequence of TTTG' represents a PAM sequence at the CpG site 320B. Thus, this sequence can be successfully recognized by the nuclease 325. Additionally, the guide RNA 328 can successfully hybridize based on the presence of the “TG” sequence of the CpG site 320D. In various embodiments, the guide RNA 328 hybridizes with the target sequence 305B that includes the “TG” sequence of the CpG site 320D. In various embodiments, the guide RNA 328 hybridizes with a sequence of a strand opposite to the target sequence 305B that includes the “TG” sequence of the CpG site 320D. Hybridization of the guide RNA 328 allows for subsequent cutting of the nucleic acid molecule 328B, thereby generating cut nucleic acid 393B.

[0114] Reference is next made to FIG. 3D, which is an example diagram depicting hybridization of primers to uncleaved nucleic acids, in accordance with any one of theembodiments shown in FIGs. 3A-3C. Here in FIG. 3D, the uncleaved nucleic acid 395A can be any of uncut nucleic acid 340A shown in FIG. 3 A, uncut nucleic acid 391 A shown in FIG. 3B, or uncut nucleic acid 393A shown in FIG. 3C. The cleaved nucleic acid 395B can be any of cut nucleic acid 340B shown in FIG. 3A, cut nucleic acid 39 IB shown in FIG. 3B, or cut nucleic acid 393B shown in FIG. 3C. Specifically, as shown in the top panel of FIG. 3D, copies one or more primers 350 are provided to the mixture including the uncleaved nucleic acid 395 A and the now cleaved nucleic acid 395B.

[0115] In various embodiments, primers are oligonucleotides from ten to twenty and / or thirty base pairs in length, but longer sequences can be employed. In various embodiments, pairs of primers are designed and provided to selectively hybridize to particular nucleic acid sequences (e.g., target nucleic acid sequences, or sequences flanking target nucleic acid sequences). In various embodiments, pairs of primers include forward and reverse primer pairs. In various embodiments, the primers include indexing primers that enable subsequent incorporation of library adapters (e.g., P5 / P7 library adapters).

[0116] As shown in the bottom panel of FIG. 3D, copies of the one or more primers successfully hybridize to complementary sequences that are present to the uncleaved nucleic acid 395A. In various embodiments, the copies of the primer sequence are incapable of hybridizing to the cleaved nucleic acid 395B. As the cleaved nucleic acid molecule 395B have been previously cut by the nuclease, a complementary sequence to the primer sequence is not present in cleaved nucleic acid 395B. Thus, copies of the primer sequence fail to hybridize with sequences of the nucleic acid molecules of the first subset. In some embodiments, a first primer of a primer pair can successfully hybridize with the cleaved nucleic acid 395B but a second of the primer pair fails to hybridize with the cleaved nucleic acid 395B.

[0117] Using the hybridized copies of primer sequences, the uncleaved nucleic acids undergo further enrichment relative to the cleaved nucleic acids. Reference is now made to FIG. 3E, which is an example diagram depicting enrichment of uncleaved nucleic acids, in accordance with the embodiment shown in FIG. 3D. FIG. 3E specifically shows an enrichment 360 that involves performing nucleic acid amplification using the hybridized primer sequences. Thus, multiple amplicons of the uncleaved nucleic acid 370 can be generated and recovered following enrichment 360. The cleaved nucleic acid 395B may remain present, but does not undergo amplification. Altogether, amplicons of the uncleaved nucleic acid can be analyzed (e.g., via sequencing methods) to determine the enriched signal informative for determining presence or absence of cancer in the sample.b. Enrichment Steps

[0118] In certain embodiments, the target sequence or a subset of target sequences in the nucleic acid can be enriched using one or more additional enrichment steps. The one or more additional enrichments steps can be performed using any enrichment method known in the art. Non-limiting examples include hybrid capture, use of DNA-binding proteins to enrich a target sequence or a subset of target sequences, and nucleic acid amplification (e.g., polymerase chain reaction). In various embodiments, the additional enrichment step involves performing indexing PCR amplification e.g., using library adapters (e.g., P5 / P7 adapters). Thus, selective amplification nucleic acids with hybridized primer sequences can be performed using the library adapters. In contrast, nucleic acids in which primer sequences are incapable of hybridizing with do not undergo PCR amplification.

[0119] One or more additional enrichment steps can be performed before or after the depletion of the unmethylated nucleic acids, as described herein. For example, in certain embodiments, the method comprises a first step of depleting a first subset of nucleic acids (e.g., unmethylated nucleic acids or converted nucleic acids derived from unmethylated nucleic acids), thereby leaving a second subset of nucleic acids (e.g., methylated nucleic acids or converted nucleic acids derived from methylated nucleic acids). The method comprises a second step of subjecting nucleic acid sequences comprising the target sequence to one or more additional enrichment steps to enrich for at least a subset of the target sequences.

[0120] In certain embodiments, the method comprises a first step of subjecting a plurality of nucleic acid molecules that include target sequences to an enrichment step to enrich for the target sequences. The method further comprises a second step of subjecting the plurality of nucleic acid molecules to the depletion method disclosed herein, which depletes a first subset of nucleic acids (e.g., unmethylated nucleic acids or converted nucleic acids derived from unmethylated nucleic acids) thereby leaving a second subset of nucleic acids (e.g., methylated nucleic acids or converted nucleic acids derived from methylated nucleic acids).

[0121] In certain embodiments, a target sequence or a subset of target sequences in the nucleic acid can be enriched by subjecting the nucleic acid comprising the target sequence or the subset of target sequences to hybrid capture. In hybrid capture, labeled (e.g., biotinylated) capture probes that can bind to one or more target sequences or subsets of target sequences are exposed to the nucleic acid comprising the one or more target sequences. The capture probes are specific to a sequence of interest, for example, a methylation pattern of interest that can be detected as a bisulfite-converted epitype. Examples of such hybrid capture probe sets include the KAPA HyperPrep KAPA HyperCap Workflow with HyperChoice Probes, Twist BioscienceTwist Fast Hybridization Custom Target Enrichment Panel, Integrated DNA technologies xGen Custom Hybridization Capture Panel, and SeqCAP Epi Enrichment System from Roche Diagnostics (Pleasanton, CA).Example Nucleic Acids and Methods for Converting Nucleic Acids

[0122] As discussed herein, step 120 in FIG. 1 involves converting nucleic acid molecules from the obtained sample. In various embodiments, converting nucleic acid molecules includes treating the nucleic acid molecules to capture methylation modifications. In various embodiments, converting nucleic acid molecules involves converting one or more unmethylated nucleotides (e.g., cytosines) to another nucleotide (a “converted nucleotide”, as used herein), e.g., using chemical or enzymatic means. In certain embodiments, one or more unmethylated cytosines are converted to a nucleotide that pairs with adenine (e.g., the unmethylated cytosine may be converted to uracil). In certain embodiments, one or more unmethylated adenines are converted to a base that pairs with cytosine (e.g., the unmethylated adenine may be converted to inosine (I)). In certain embodiments, one or more methylated cytosines (e.g., a 5 -methylcytosine (5mC)) is converted to a thymine, which pairs with adenine. In certain embodiments, methylated cytosines are protected from conversion (e.g., deamination) during the conversion step.

[0123] After a nucleic acid has been treated to convert unmethylated, or, in some cases, methylated nucleotides, into another nucleotide, the nucleic acid may be amplified. During amplification, the converted nucleotide pairs with its complementary nucleotide, and in the next round of amplification, the complementary nucleotide pairs with a replacement nucleotide. For example, following the conversion of an unmethylated cytosine to a uracil, the nucleic acid may be amplified such that an adenine pairs with the uracil in the first round of replication, and in the second round of replication, the adenine pairs with a thymine. Accordingly, the thymine replaces the uracil in the original nucleic acid sequence, and is referred to herein as a “replacement nucleotide”.

[0124] In certain aspects, conversion of the nucleic acids involves selectively deaminating nucleotides. FIG. 2A depicts an example conversion of nucleic acids, in accordance with an embodiment. Selective deamination refers to a process in which unmethylated cytosine residues are selectively deaminated over methylated cytosine (5- methylcytosine) residues. In certain embodiments, deamination of cytosine forms uracil, effectively inducing a C to T point mutation to allow for detection of methylated cytosines. Methods of deaminating cytosine are known in the art, and include chemical conversion (e.g., bisulfite conversion) and enzymatic conversion. In certain embodiments, the enzymaticconversion comprises subjecting the nucleic acid to TET2, which oxidizes methylated cytosines, thereby protecting them, and subsequent exposure to APOBEC, which converts unprotected (i.e., unmethylated) cytosines to uracils.

[0125] In some embodiments, the conversion, for example, bisulfite conversion or enzymatic conversion, uses commercially available kits. Bisulfite conversion can be performed using commercially available technologies, such as EZ DNA Methylation-Gold, EZ DNAMethylation-Direct or an EZ DNAMethylation-Lighting kit (Zymo Research Corp (Irvine, California)) or EpiTect Fast available from Qiagen (Germantown, MD). In another example a kit such as APOBECSeq (NEBiolabs) or OneStep qMethyl-PCR Kit (Zymo Research Corp (Irvine, California)) is used. a. Source of Nucleic Acids

[0126] Nucleic acids used in the methods described herein can be derived from any source, such as a sample taken from the environment or from a subject (e.g., a human subject). A biological sample can be treated to physically disrupt tissue or cell structure (e.g., centrifugation and / or cell lysis), thus releasing intracellular components into a solution which can further contain enzymes, buffers, salts, detergents, and the like which can be used to prepare the sample for analysis. A biological sample can take any of a variety of forms, such as a liquid biopsy (e.g., blood, urine, stool, saliva, or mucous), or a tissue biopsy, or other solid biopsy. Examples of biological samples include, but are not limited to, blood, whole blood, plasma, serum, urine, cerebrospinal fluid, fecal, saliva, sweat, tears, pleural fluid, pericardial fluid, or peritoneal fluid of the subject. A biological sample can include any tissue or material derived from a living or dead subject. A biological sample can be a cell-free sample. A sample can be a liquid sample or a solid sample (e.g., a cell or tissue sample). A biological sample can be a bodily fluid, such as blood, plasma, serum, urine, vaginal fluid, fluid from a hydrocele (e.g., of the testis), vaginal flushing fluids, pleural fluid, ascitic fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, discharge fluid from the nipple, aspiration fluid from different parts of the body (e.g., thyroid, breast), etc.

[0127] The nucleic acid can be of any composition form, such as deoxyribonucleic acid (DNA, e.g., complementary DNA (cDNA), genomic DNA (gDNA) and the like), and / or DNA analogs (e.g., containing base analogs, sugar analogs and / or a non-native backbone and the like), and / or ribonucleic acid (RNA) and / or RNA analogs, all of which can be in single- or doublestranded form. In certain embodiments, single -stranded nucleic acids can be made double stranded prior to cutting with an enzyme. Unless otherwise limited, a nucleic acid can comprise known analogs of natural nucleotides, some of which can function in a similar manner asnaturally occurring nucleotides. A nucleic acid can be in any form useful for conducting processes herein (e.g., linear, circular, supercoiled, single-stranded, double-stranded and the like). A nucleic acid in some embodiments can be from a single chromosome or fragment thereof (e.g., a nucleic acid sample may be from one chromosome of a sample obtained from a diploid organism). In certain embodiments nucleic acids comprise nucleosomes, fragments or parts of nucleosomes or nucleosome-like structures. Nucleic acids can comprise protein (e.g., histones, DNA binding proteins, and the like). Nucleic acids analyzed by processes described herein can be substantially isolated and are not substantially associated with protein or other molecules. Nucleic acids can also include derivatives, variants and analogs of DNA synthesized, replicated or amplified from single-stranded (“sense” or “antisense,” “plus” strand or “minus” strand, “forward” reading frame or “reverse” reading frame) and double-stranded polynucleotides. Deoxyribonucleotides can include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine. A nucleic acid may be prepared using a nucleic acid obtained from a subject as a template.

[0128] In certain embodiments, the nucleic acid is a cell-free nucleic acid, which can be found in bodily fluids such as blood, whole blood, plasma, serum, urine, cerebrospinal fluid, fecal, saliva, sweat, sweat, tears, pleural fluid, pericardial fluid, or peritoneal fluid of a subject. In certain embodiments, a plasma sample can be used directly in the methods disclosed herein (for example, in the cutting step), without prior purification or isolation of nucleic acids in the plasma. Cell-free nucleic acids originate from one or more healthy cells and / or from one or more cancer cells, or from non-human sources such bacteria, fungi, viruses. Examples of the cell-free nucleic acids include but are not limited to cell-free DNA (“cfDNA”), including mitochondrial DNA or genomic DNA, and cell-free RNA. In certain embodiments herein, instruments for assessing the quality of the cell-free nucleic acids, such as the TapeStation System from Agilent Technologies (Santa Clara, CA) can be used. Concentrating low- abundance cfDNA can be accomplished, for example using a Qubit Fluorometer from Thermofisher Scientific (Waltham, MA).

[0129] In various embodiments, the majority of DNA in a biological sample that has been enriched for cell-free DNA (e.g., a plasma sample obtained via a centrifugation protocol) can be cell-free (e.g. , greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the DNA can be cell -free).

[0130] A methylated nucleic acid is a nucleic acid having a modification in which a hydrogen atom on the pyrimidine ring of a cytosine base is converted to a methyl group, forming 5 -methylcytosine. Methylation can occur at dinucleotides of cytosine and guanine referred toherein as “CpG sites”, which can be a target for enrichment. Methylation of cytosine can occur in cytosines in other sequence contexts, for example, 5'-CHG-3' and 5'-CHH-3', where H is adenine, cytosine or thymine. Cytosine methylation can also be in the form of 5- hydroxymethylcytosine. Methylation of DNA can include methylation of non-cytosine nucleotides, such as N6-methyladenine (6mA). Anomalous cfDNA methylation can be identified as hypermethylation or hypomethylation, both of which may be indicative of cancer status. As is well known in the art, DNA methylation anomalies (compared to healthy controls) can cause different effects, which may contribute to cancer.

[0131] In certain embodiments, the nucleic acid comprises a CpG site (i.e., cytosine and guanine separated by only one phosphate group). In certain embodiments, the nucleic acid comprises a CpG island (also referred to as a “CG islands” or “CGI”) or a portion thereof, which is the target for enrichment. Because certain CGIs and certain features of certain CGIs in tumor cells tend to be different from the same CGIs or features of the CGIs in healthy cells, detection of such CGIs can be informative of a health condition. In certain embodiments, the CGI is a “cancer informative CGIs”, which is defined and described in more detail below. In certain embodiments, the CpG is an “informative CpG”, e.g., a “cancer informative CGI”. Such CGIs may have methylation patterns in tumor cells that are different from the methylation patterns in healthy cells. Accordingly, detection of a cancer informative CGI can be informative regarding a subject’s risk of developing cancer or can be indicative that the subject has cancer. Exemplary cancer informative CGIs, which can be target sequences as described herein, are identified in, e.g., Table 1 of U.S. Patent Publication 2020 / 0109456A1, Tables 2 and 3 ofWO2022 / 133315, and TABLES 1-4 provided herein.

[0132] In certain aspects, the nucleic acids of the invention have been treated to convert one or more unmethylated nucleotides (e.g., cytosines) to another nucleotide (a “converted nucleotide”, as used herein, such as a uracil), for example, prior to amplification. In certain embodiments, one or more unmethylated cytosines are converted to a nucleotide that pairs with adenine (e.g., the unmethylated cytosine may be converted to uracil). In certain embodiments, one or more unmethylated adenines are converted to a base that pairs with cytosine (e.g., the unmethylated adenine may be converted to inosine (I)). In certain embodiments, one or more methylated cytosines (e.g., a 5 -methylcytosine (5mC)) is converted to a thymine, which pairs with adenine. In certain embodiments, methylated cytosines are protected from conversion (e.g., deamination) during the conversion step.

[0133] After a nucleic acid has been treated to convert unmethylated, or, in some cases, methylated nucleotides, into another nucleotide, the nucleic acid may be amplified. Duringamplification, the converted nucleotide pairs with its complementary nucleotide, and in the next round of amplification, the complementary nucleotide pairs with a replacement nucleotide. For example, following the conversion of an unmethylated cytosine to a uracil, the nucleic acid may be amplified such that an adenine pairs with the uracil in the first round of replication, and in the second round of replication, the adenine pairs with a thymine. Accordingly, the thymine replaces the uracil in the original nucleic acid sequence, and is referred to herein as a “replacement nucleotide”. b. Bisulfite conversion

[0134] Bisulfite conversion is performed on DNA by denaturation using high heat, preferential deamination (at an acidic pH) of unmethylated cytosines, which are then converted to uracil by desulfonation (at an alkaline pH). Methylated cytosines remain unchanged on the single-stranded DNA (ssDNA) product.

[0135] In some embodiments the methods include treatment of the sample with bisulfite (e.g., sodium bisulfite, potassium bisulfite, ammonium bisulfite, magnesium bisulfite, sodium metabisulfite, potassium metabisulfite, ammonium metabisulfite, magnesium metabisulfite and the like). Unmethylated cytosine is converted to uracil through a three-step process during sodium bisulfite modification. As shown in FIG. 2A, the steps are sulphonation to convert cytosine to cytosine sulphonate, deamination to convert cytosine sulphonate to uracil sulphonate and alkali desulphonation to convert uracil sulphonate to uracil. Conversion on methylated cytosine is much slower and is not observed at significant levels in a 4-16 hour reaction. (See Clark et al., Nucleic Acids Res., 22(15):2990-7 (1994).) If the cytosine is methylated it will remain a methylated cytosine. If the cytosine is unmethylated it will be converted to uracil. When the modified strand is copied, for example, through extension of a locus specific primer, a random or degenerate primer or a primer to an adaptor, a G will be incorporated in the interrogation position (opposite the C being interrogated) if the C was methylated and an A will be incorporated in the interrogation position if the C was unmethylated and converted to U. When the double stranded extension product is amplified those Cs that were converted to Us and resulted in incorporation of A in the extended primer will be replaced by Ts during amplification. Those Cs that were not converted (z.e., the methylated Cs) and resulted in the incorporation of G will be replaced by unmethylated Cs during amplification. c. Enzymatic conversion

[0136] In certain embodiments, the enzymatic treatment with a cytidine deaminase enzyme is used to convert cytosine to uracil. Enzymatic conversion can include an oxidationstep, in which Tet methylcytosine dioxygenase 2 (TET2) catalyzes the oxidation of 5mC to 5hmC to protect methylated cytosines from conversion by subsequent exposure to a cytidine deaminase. Other protection steps known in the art can be used in addition to or in place of oxidation by TET2. After the oxidation step, the nucleic acid is treated with the cytidine deaminase to convert one or more unmethylated cytosines to uracils. As with bisulfite conversion, when the modified strand is copied, a G will be incorporated in the interrogation position (opposite the C being interrogated) if the C was methylated and an A will be incorporated in the interrogation position if the C was unmethylated. When the double stranded extension product is amplified those Cs that were converted to Us and resulted in incorporation of A in the extended primer will be replaced by Ts during amplification. Those Cs that were not modified and resulted in the incorporation of G will remain as C.

[0137] In certain embodiments the cytidine deaminase may be APOBEC. In certain embodiments the cytidine deaminase includes activation induced cytidine deaminase (AID) and apolipoprotein B mRNA editing enzymes, catalytic polypeptide -like (APOBEC). In certain embodiments, the APOBEC enzyme is selected from the human APOBEC family consisting of: APOBEC- 1 (Apol), APOBEC-2 (Apo2), AID, APOBEC-3A, -3B, -3C, -3DE, -3F, -3G, -3H and APOBEC-4 (Apo4). In certain embodiments, the APOBEC enzyme is APOBEC-seq. d. Nitrite Conversion

[0138] In certain embodiments, nitrite treatment is used to deaminate adenine and cytosine. As shown in FIG. 2B, deamination of an A results in conversion to an inosine (I), which is read by a polymerase as a G, whereas deamination of a methylated A (N6- methyladenine (6mA)) results in a nitrosylated 6mA (6mA-N0), which causes the base to be read by a polymerase as an A. Deamination of a C results in conversion to a uracil, which is read by a polymerase as a T, whereas deamination of a A4-mcthylcytosinc (4mC) to 4mC-N0 or a 5 -methylcytosine (5mC) to a T causes the base to be read by a polymerase as a C or a T, respectively. For 5mC bases, the C to T ratio at the 5mC position is about 40% higher than other cytosine positions, allowing 5mC to be differentiated from C. (See, Li et al. (2022) Genome Biology 23: 122.)Guide RNAs (gRNAs, sgRNAs)

[0139] A “guide RNA” (“gRNA”) is a type of RNA that includes a CRISPR RNA sequence (crRNA, also referred to as a “guide sequence” or “spacer”), and, in certain embodiments, a trans-activating CRISPR RNA sequence (tracrRNA). The tracrRNA, if present,binds to an endonuclease (e.g., a CRISPR enzyme) and the crRNA is complementary to a target sequence. In certain embodiments, the guide RNA is referred to as a single guide RNA (sgRNA), which refers to a guide RNA comprising both a crRNA and tracrRNA.

[0140] A guide sequence can be designed to have complementarity to a target sequence of the disclosure, where hybridization between a target sequence and a guide sequence promotes the formation of a gene editing endonuclease complex (e.g., a CRISPR complex). Full complementarity may not be required, provided there is sufficient complementarity to cause hybridization and promote formation of a gene editing endonuclease complex (e.g., a CRISPR complex). In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. In certain embodiments, the guide sequence and the target sequence exhibit full (100%) complementarity .

[0141] Optimal alignment of the polyribonucleotide to the target sequence may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina®, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length.

[0142] The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay. For example, the components of a CRISPR system sufficient to form a CRISPR complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between thetest and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art.Nucleases

[0143] As disclosed herein, methods involve using a nuclease for enriching for target nucleic acids and / or for depleting unmethylated nucleic acids. An example nuclease can be an endonuclease, for example, a Cas protein, that is capable of cleaving DNA (e.g., double stranded DNA) to effect a break at the intended locus. Non-limiting examples of Cas proteins that are capable of cleaving DNA to effect a break at the intended locus, include type V CRISPR enzymes such as Casl2, Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl2fl, Casl2g, Casl2h, Casl2i, homologs thereof, or modified versions thereof, (see, e.g., Liu et al. (2019) supra). For example, in some embodiments, the endonuclease is a Cas 12 endonuclease that effects a break at a locus within or near a target sequence. Cas 12 endonucleases can effect a break, resulting in a 4-base overhang. For example, the 4-base overhang can be present in a strand opposite to a PAM sequence. Cas 12 recognizes a 5’-T-rich PAM, such as “TTN” or “TTTN,” where “N” is any nucleobase. In such embodiments where Casl2 is employed, a target sequence of interest within a nucleic acid can be located upstream of or contain a sequence corresponding to the PAM sequence of “TTN” or “TTTN” where “N” is any nucleobase. For example, the sequence corresponding to the PAM sequence can be a sequence of “TCG” or “TTCG.” In the scenario where the CpG site of “TCG” or “TTCG” is unmethylated, the cytosine is deaminated to a uracil (U) when undergoing conversion, leading to a converted sequence of “TUG” or “TTUG.” The resulting double stranded nucleic acids would include the PAM sequence of “TTG” or “TTTG” and therefore, can be recognized and cut by Cas 12. As another example, the sequence corresponding to the PAM sequence can be a sequence of “TCG” or “TTCG”, where the CpG site of “TCG” or “TTCG” is methylated. When undergoing conversion, the methylated cytosine is protected and does not undergo deamination. Thus, the converted sequence would remain “TCG” or “TTCG.” The resulting double stranded nucleic acids would include the sequence of “TCG” or “TTCG.” Given that the PAM sequence is not present, the Cas 12 would not cut these nucleic acids.

[0144] As another example, the endonuclease is a Cas9 endonuclease that effects a break at a locus within or near a target sequence. Cas9 recognizes a “NGG” (5’ to 3’) PAM sequence, where “N” is any nucleobase. Accordingly, in certain embodiments, the endonuclease is a Cas9 protein.

[0145] Another nuclease capable of cleaving DNA to effect a break at the intended locus is a CasX nuclease. (See, Liu et al. (2019) Nature 566:218-223. CasX recognizes a 5’-TTCN PAM and is capable of creating 10-nt overhangs. (Id.)

[0146] In some embodiments, the endonuclease (e.g., a CRISPR enzyme) directs cleavage at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the endonuclease directs cleavage within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.

[0147] In general, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).Kits

[0148] Also disclosed herein are kits for enriching for a signal in a sample, such as a signal informative for determining presence or absence of cancer in the sample. In various embodiments, kits disclosed herein are useful for enriching for a target nucleic acid and / or for depleting unmethylated nucleic acids. For example, the kit may include a reagents for performing a nucleic acid conversion, and a nuclease that cuts a converted nucleic acid sequence recognizes one or more protospacer adjacent motif (PAM) nucleic acid sequence(s) present in converted nucleic acid sequences as a result of performing or having performed the nucleic acid conversion using the reagents.

[0149] In certain embodiments, the kit includes a nuclease, such as a CRISPR-Cas nuclease. In various embodiments, the CRISPR-Cas nuclease is one or more of Cas 12, Cas9, and subtypes and / or variants thereof. In various embodiments, the CRISPR-Cas nuclease is a Casl2a nuclease. In various embodiments, the CRISPR-Cas nuclease is a Cas9 nuclease. The CRISPR-Cas nuclease recognizes one or more protospacer adjacent motif (PAM) nucleic acid sequence(s).

[0150] In certain embodiments, the kit includes reagents for performing a nucleic acid conversion. Example reagents for performing a nucleic acid conversion may include reagents for performing any of a bisulfite conversion, enzymatic conversion, or nitrite conversion, as disclosed herein. In particular embodiments, the kit includes reagents for performing a bisulfiteconversion, including any of sodium bisulfite, potassium bisulfite, ammonium bisulfite, magnesium bisulfite, sodium metabisulfite, potassium metabisulfite, ammonium metabisulfite, magnesium metabisulfite, and the like.

[0151] Throughout the description, where apparatus, devices, and systems are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are apparatus, devices, and systems of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[0152] Practice of the invention will be more fully understood from the foregoing examples, which are presented herein for illustrative purposes only, and should not be construed as limiting the invention in any way.EXAMPLESExample 1 - Example Methods for Depleting Unmethylated Nucleic AcidsPrepare target specimen

[0153] The target specimen is either the biological source itself (e.g., tissue, blood, plasma, serum, saliva, feces, etc.) or is isolated from a patient’s biological source (e.g., DNA, RNA, protein, exosomes, metabolites, etc.). Bisulfite conversion is performed (e.g., in accordance with the method shown in FIG. 2A) by denaturation using high heat, preferential deamination (at an acidic pH) of unmethylated cytosines, which are then converted to uracil by desulfonation (at an alkaline pH). Methylated cytosines remain unchanged on the single-stranded DNA (ssDNA) product. Subsequently, polymerase chain reaction (PCR) steps convert uracil to thymine.Generate library fragment (no indexing PCR)

[0154] The bisulfite converted ssDNA then undergoes Adaptase® technology from IDT, which is an enzymatic reaction resulting in unbiased addition of a truncated adapter. The Adaptase® enzymatic reaction performs end-repairing, tailing of 3’ ends and ligation of first truncated adapter complement to 3 ’ ends simultaneously. A uracil -free reverse complement to the bisulfite converted ssDNA is then generated using the truncated adapter to prime and extend. A solid-phase reversible immobilization (SPRI) selection follows to remove unwanted ssDNA fragments, excess adapters and molecules. A ligation reaction follows, adding truncated P5 adapter to the 3 ’ end of the uracil-free reverse complement fragment. A solid-phase reversible immobilization (SPRI) selection follows to remove unwanted ssDNA fragments, excess adapters and molecules.Cas-mediated depletion of unmethylated DNA

[0155] Casl2a and crRNA are incubated at room temperature for at least 10 minutes to create Cas complexes. The target specimen is then spiked into the Cas complexes. The Cas complexes cut the specimen at the target sites on unmethylated nucleic acid molecules with a 4- base overhang on the opposite strand to the PAM and the Cas complexes do not cut target sites on methylated nucleic acid molecules, in accordance with the methods shown in FIGs. 3A-3C. This interaction between Cas 12a and the cut site leads to the generation of a cut nucleic acid. In comparison, methylated nucleic acid molecules do not undergo cutting at their CpG site, resulting in an uncut nucleic acid fragment. Cas is inactivated using Proteinase K and incubatingat room temperature for 10 minutes. A solid-phase reversible immobilization (SPRI) selection follows to remove unwanted DNA fragments, excess enzymes, dNTPs and molecules.Indexing PCR of uncut library fragments

[0156] Indexing PCR amplification is performed with a high fidelity DNA polymerase and unique, known 10-bp barcodes ligated to full-length P5 / P7 adapters. Library fragments cut by Cas after ligation will not be able to be amplified by the full-length adapters. In comparison, nucleic acid fragments uncut by Cas 12a will hybridize with adapter primers and subsequently amplify in downstream PCR and enrichment steps. Indices allow for subsequent sample multiplexing for use in the downstream assay. The resulting product is an enriched methylationspecific, bisulfite converted dsDNA library with full length adapters. Post-PCR, a SPRI selection is performed to remove unwanted ssDNA fragments, excess primers, excess adapters and excess molecules. After library construction, the library quality and quantity are evaluated using the Agilent Tape Station and Qubit Fluorometer, respectively.Sequencing of enriched library

[0157] Libraries that pass all quality control checks move forward to sequencing. Sequencing is completed on an iSeq using paired end 150x150 base sequencing with a 5% PhiX spike-in. Sequencing data generated is then demultiplexed utilizing the assigned barcode, aligned to the human genome and trimmed. This cleaned-up data is then processed through a quality pipeline to collapse duplicate reads and evaluate the sequencing data generated.Example 2 -CRISPR-Casl2a and crRNA-Mediated Depletion of Unmethylated Nucleic Acids

[0158] To demonstrate the effectiveness of CRISPR-Casl2a and crRNA in selective depletion of hypomethylated (unmethylated) nucleic acid molecules, an experimental approach was developed leveraging DNA libraries (referred to here as “gblocks”) featuring pure nucleic acid molecules (hypomethylated gblocks, hypermethylated (methylated) gblocks, and 50:50 mixture of hypermethylated and hypomethylated gblocks). FIG. 4 shows the example experimental setup involving a hypermethylated library (e.g., hyper gblock library), a hypomethylated library (e.g., hypo gblock library), and a 50:50 hypo / hyper methylated library (e.g., hyperhypo gblock library). Additionally, all libraries feature the same adapters, thus the primers used in this experiment are universal to all DNA libraries.

[0159] As shown in the right panel of FIG. 4, this experimental setup allowed for the assessment of the selectivity of the approach described herein where CRISPR-Casl2a andcrRNA interact with bisulfite converted nucleic acid molecules to selectively deplete unmethylated gblocks (e.g., hypo gblocks).

[0160] Reference is now made to FIG. 5, which shows an example nucleic acid sequence derived from a fully methylated nucleic acid (“Hyper gblock”) and an example nucleic acid sequence comprising a PAM sequence and CpG converted site within 7 base pairs of the PAM that is derived from a fully unmethylated nucleic acid (“Hypo gblock”). Specifically, the top panel shows the bisulfite converted nucleic acid sequence derived from a fully methylated sense strand. The shown sequence in the top panel is a single contiguous sequence. The bottom panel shows the bisulfite converted nucleic acid sequence derived from a fully unmethylated sense strand. The shown sequence in the bottom panel is a single contiguous sequence.

[0161] In this experiment, hypomethylated gblocks were selectively cut through crRNA and CRISPR-Cas 12a targeting of the unmethylated nucleic acid molecules (hypo gblock), as is shown in the bottom panel of FIG. 5. Casl2a selectively binds to the specific PAM sequence in the unmethylated nucleic acid molecules, due to Casl2a recognition of “TTTG”. In addition, the hypo-specific crRNA hybridizes to the seed region of the unmethylated nucleic acid molecules. The hybridization of the crRNA to the seed region together with Casl2a recognition of the PAM site enables the selective cutting of the opposite strand of the unmethylated nucleic acid molecule. Attention is drawn to the top panel of FIG. 5 which shows that the PAM signature for Casl2a is absent in the methylated nucleic acid molecules (hyper gblock). Additionally, a mismatch occurs between the methylated nucleic acid molecule sequence and the crRNA 6 base pairs from the not PAM sequence, “CG”(top panel of FIG. 5). The resulting mismatch prevents effective hybridization between the crRNA and the methylated nucleic acid molecule, which further prevents Casl2a from cutting.

[0162] Quantitative polymerase chain reaction (qPCR) was utilized to determine the selectivity of Casl2a depletion of hypomethylated nucleic acid molecules based on the aforementioned experimental design. Reference is made to FIGs. 6A, 6B, and 6C, which show qPCR results of the hypermethylated library, hypomethylated library, and 50:50 hypo:hypermethylated library at 1: 10 and 1: 100 dilutions. Each of FIGs. 6A, 6B, and 6C show qPCR results in comparison to a negative control, which involved introduction of a Casl2a without a corresponding crRNA (thus, the negative control is not cut). Notably, as shown in FIG. 6A when Cas complexes (Casl2a and crRNAs) were incubated with the hypermethylated library (hyper gblock), no difference in average cycle threshold (CT) was observed (e.g., 5.42 CT versus 4.87 CT forthe negative control at 1: 10 dilution), indicating Casl2a does not cut the hypermethylated substrate. The hypermethylated nucleic acid molecules do not include arecognizable PAM sequence after being subjected to bisulfite conversion. In contrast, as shown in FIG. 6B, pure hypomethylated libraries that include a recognizable PAM sequence, when exposed to Cas complexes, were selectively cut and depleted, as shown by the increased cycle threshold value (e.g., 10.42 CT versus 4.85 CT for the negative control at 1: 10 dilution). Lastly, when a 50:50 preparation of methylated (hyper) and unmethylated (hypo) nucleic acid molecules were exposed to Cas complexes, a slight depletion of the nucleic acid molecule mixture was observed, as shown by the increased cycle threshold value (e.g., 6.89 versus 5.18 at 1: 10 dilution). Additional analysis of these same experiments enabled assessment of enrichment selectivity of Cas 12a + crRNA of methylated molecules. Enrichment was quantified by raising 2 to the power of the net change in average cycle threshold (CT) between the cut gblock and uncut gblock for a given experiment. Under this analysis approach, enrichment in FIG. 6B was calculated to be 47.6X. Similarly, depletion metrics were calculated by raising 2 to the power of the difference between the average CT of the cut hypo gblock and the cut hyper gblock. Under this analysis method, depletion was calculated to be 32. IX based on the data in FIG. 6A and FIG. 6B. Overall, these experiments demonstrate the depletion and enrichment capacity of Cas complexes including Cas 12a and corresponding crRNA for bisulfite treated unmethylated nucleic acid molecules.Example 3 -Depleting Unmethylated Nucleic Acids Using CRISPR-Casl2a and crRNA for Computational Filtering

[0163] To further validate the selective depletion of hypomethylated nucleic acid molecules, a sequencing and computational approach was implemented. FIG. 7A is an example experimental design for cutting unmethylated nucleic acids followed by library generation and sequencing. In this approach, hypo and hyper gblocks were exposed to Cas complexes (Cas 12a and crRNAs), underwent SPRI enrichment (>100 base pair (bp)), and underwent library preparation.

[0164] FIG. 7B shows the differential cutting of hypermethylated and hypomethylated nucleic acids. Specifically, hypo gblock selective cutting was determined by the percentage of 50-160 bp nucleic acid fragments generated in the library preparation process after exposure to Cas complexes relative to the proportion of hyper gblock nucleic acid fragments generated in the same size range as a result of exposure to Cas complexes. As shown in FIG. 7B, 68.8% of fragments from the hypo gblock were found in the 50-160 bp range whereas only 25.7% of fragments from the hyper gblock were found in the 50-160 range. Additional bioinformatics analyses were performed to confirm the selectivity of Cas complexes for bisulfite converted hypomethylated (hypo gblock) nucleic acid molecules. FIG. 7C shows example computationalidentification and filtering of cut nucleic acids. Specifically, computational filtering of reads was performed starting within a 25 bp window of the cut site on the hypo gblock. Here, the analyses identified 31% of reads resulting from selective cutting in hypo gblocks, which were then computationally identified, thus enabling computational filtering. This is in contrast to 0% of identified reads cutting when the Cas complexes were not present.Example 4 - Depletion of Unmethylated Nucleic Acids Using CRISPR-Casl2a and crRNA Enables Selective Amplification of Target Nucleic Acids

[0165] Experiments were conducted to evaluate whether additional enrichment steps can be performed to further enrich for target nucleic acids relative to unmethylated nucleic acids. For example, to determine if amplification was blocked as a result of Cas 12a and crRNA cutting of hypo gblock nucleic acid molecules (unmethylated), an experimental setup leveraging qPCR was developed . FIG. 8A is an example experimental design for using qPCRto quantify differential amplification of hypermethylated and hypomethylated nucleic acids.

[0166] In this protocol, hyper and hypo gblocks were separately incubated with Cas complexes, where selective cuts would be made on the hypo gblocks. Due to the selective cutting on the hypo gblocks, primers that hybridize with sequences at or beyond the cut site would be prevented from hybridizing (e.g., FIG. 8A shows that the R primer cannot sit). The sequences used include:• Forward primer sequence: GGAAAAGTTTYGTTGATTGTAGGT (SEQ ID: 1),• Probe sequence: YGTTYGGGAGGGGYGGTTAGG (SEQ ID: 2)• Reverse primer sequence: TAACATAAAACCYGAAAATATATAATAAAAY (SEQ ID: 3), where “Y” refers to a pyrimidine (e.g., cytosine or thymidine).

[0167] Reference is now made to FIG. 8B, which shows results of example bisulfite converted sequences derived from cancer or non-cancer samples following depletion using CRISPR Cas 12a. In the bisulfite-converted non-cancer and bisulfite-converted cancer sequences, the lower case letters (e.g., “tg”, “ac”, “eg”, “gc”, etc.) refer to nucleotides corresponding to a CpG site. Thus, bisulfite-converted cancer sequences contain “eg” nucleotide sequences at one or more CpG sites (e.g., as methylated cytosines are not converted and remain as cytosine) whereas bisulfite-converted non-cancer sequences contain “tg” nucleotide sequences at one or more CpG sites (e.g., as non-methylated cytosines are converted to uracil and subsequent PCR steps convert the uracil to thymine). Here, bisulfite converted nucleic acid molecules from non-cancer samples feature the PAM (“TTtg” sequence) + seed sequence(unmethylated), whereas the bisulfite converted nucleic acid molecule from cancer samples do not feature the PAM sequence (“TTcg” sequence) (methylated). The lack of a PAM sequence in the bisulfite converted nucleic acid molecules from cancer samples enables selective cutting on bisulfite converted nucleic acid molecules from non-cancer samples. qPCR was used to confirm the selective cutting and lack of nucleic acid fragment amplification through analysis of cycle threshold.

[0168] FIG. 8C shows qPCR results of hypomethylated nucleic acids following depletion using CRISPR Casl2a. Here, qPCR results confirmed the selective depletion of the hypo gblock in the presence of the guide RNA + Casl2a, cycle threshold = 27.94, as compared to the negative controls (cycle thresholds = 23.50 and 23.48). FIG. 8D shows qPCR results of hypermethylated nucleic acids following depletion using CRISPR Casl2a. Negative controls featured the same input DNA without Cast 2a +crRNA and subsequently underwent same library preparation and clean-up steps. Specificity was further determined by repeating these conditions in the presence of the hyper gblock. Here, no difference was found in cycle threshold between the specificity control (with guide RNA; cycle threshold = 22.41) and negative controls (no guide RNA; cycle thresholds = 21.49 and 22.04). Overall, these results demonstrate that following the selective cutting at the PAM + seed sequence of the hypo gblock, an additional step of nucleic acid amplification further enriches for target nucleic acid sequences through the inability of primers to hybridize to the bisulfite converted nucleic acid fragments of the hypo gblock.Example 5 - Depletion and Enrichment of gblock Spike-Ins in Genomic Background through Casl2a + crRNA

[0169] FIG. 9 shows the experimental strategy that was used to determine selective depletion of unmethylated DNA molecules in a translationally relevant setting, e.g. where multiple DNA molecules are present of various sequence compositions and methylation statuses. Briefly, bisulfite converted hyper and hypo gblocks along with bisulfite converted hyper and hypo genomic DNA (gDNA) libraries underwent library preparation (adaptase, extension, ligation, qPCR). Subsequently, hyper and hypo gblock and gDNA with stubby adapters were spiked into the library. This mixture then was presented with and without Casl2 and crRNAs specific to hypo before undergoing qPCR and indexing PCR where only uncut library fragments were selectively amplified with the full length p5 / p7 adapters. Quantification of the selective enrichment and depletion was achieved through sequencing.

[0170] FIG. 10 is a quality control experiment where the presence of stubby adapters hypo gblock with stubby adapters and hyper gDNA with stubby adapters was confirmed.Specifically, on a DNA gel the presence of both aforementioned nucleic acids was confirmed and annotated. In FIG. 11, a similar quality control experiment demonstrated positive hypo gblock cutting. In this experiment, various percentages of hypo gblock (0, 0.01, 0.1, 1, 5, and 100%) were spiked into a constant amount of hyper genomic DNA (2nM) in the presence and absence of Casl2a. Positive hypo gblock cutting was demonstrated, as noted on the DNA gel in FIG. 11. Lastly, FIG. 12 demonstrates depletion of the hypo gblock is dependent on Cas 12a. This is evidenced by the annotated DNA gel where the reduction in 300 bp DNA products occurred selectively in the presence of Cas 12a, as only uncut DNA molecules were amplified.

[0171] After performing quality control analysis, bioinformatic analysis of iSeq data from the experimental outline described in FIG. 9 revealed the selective targeting of crRNA target region in the hypo gblock under Casl2a conditions (FIG. 13). In this experiment, the crRNA specifically targeted hypo DNA at 52,258,485 bp resulted in minimal hypo gblock Cas 12a presentation as a result of depletion of these molecules. Additionally, hyper gDNA remained uncut with Cas 12a treatment, further demonstrating selectivity of Cas 12a and crRNA for hypomethylated substrates. Lastly, the hypo gblock is annotated in the condition without Cas treatment as it remained uncut. Overall, these bioinformatics analyses collectively demonstrate the selective depletion of the crRNA target region in hypo DNA when Cas 12a is present.

[0172] Additional data analysis methods were developed in FIGs. 14A, 14B, and 14C that were subsequently utilized for the quantification of the enrichment for hypermethylated sequences (e.g., depletion of hypomethylated sequences). FIG. 14A and 14B quantify enrichment with different coverages between cut and uncut samples. Specifically, FIG. 14A refers to the ratio of total K5 counts in cut versus uncut samples. As used herein, “K5” refers to 5 consecutive CpG sites within a target region. FIG. 14B refers to the ratio of fully methylated K5n5 counts in cut versus uncut samples. As used herein “K5n5” refers to 5 consecutive CpG sites that are fully methylated. FIG. 14C combines the metrics described in FIG. 14A and 14B to develop a ratiometric analysis method of determining if there is positive enrichment of fully methylated counts.

[0173] These analytical methods were implemented on sequencing data produced under both iSeq (FIG. 15A-D) and Novaseq (FIG. 16A-16D) conditions and revealed that Cas 12a and crRNA selectively depleted bisulfite converted unmethylated DNA. As shown in FIG. 15 A, Cas 12a treatment did not affect the ratio of AK5m / AK5 (as shown by the data points falling along the line where slope = 1) for hyper gblocks. In contrast, FIG. 15B, shows an increase in the ratio of AK5m / AK5 (shown by data points at higher values of AK5m at lower values of AK5), indicating cutting of hypo gblock. FIG. 15C depicts the quantitative enrichment valuesbased on AK5 counts and AK5m counts when hypergblocks were spiked into hypomethylated gDNA. Here, no fold enrichment was observed. FIG. 15D depicts the quantitative enrichment values based on AK5 counts and AK5m counts when hypo gblocks were spiked into hypermethylated gDNA. Here, significant fold enrichment (e.g., up to 5.5 fold enrichment) was observed across various spiked percentages. Notably, when 0% hypo gblocks were spiked, the fold enrichment was lx, which aligns with the results shown in FIG. 15C. But when hypo gblocks were spiked in, the fold enrichment increased above lx. This indicates that 1) cutting occurs and 2) that the cutting is specific and results in enrichment of fully methylated counts (e.g., due to depletion of unmethylated nucleic acids). Similar results were obtained when the same analysis approach was applied to NovaSeq data (FIG. 16A-D).Example 6 -crRNA-Mediated Selective Depletion of Unmethylated DNA Molecules through crRNA-seed mismatch

[0174] FIG. 17 provides a schematic overview of an experiment that demonstrated the depletion selectivity towards unmethylated DNA molecules mediated by a hypo-specific crRNA when a common PAM sequence is present between methylated and unmethylated DNA molecules. The top panel shows a bisulfite converted nucleic acid sequence derived from a fully methylated sense strand. The shown sequence in the top panel is a single contiguous sequence. The bottom panel shows a bisulfite converted nucleic acid sequence derived from a fully unmethylated sense strand. The shown sequence in the bottom panel is a single contiguous sequence.

[0175] In the top panel of FIG. 17, the PAM sequence, in bold, of the methylated DNA molecule is shown to be “TTTG”, which is recognized by Casl2a. However, depletion of the methylated DNA molecules does not occur due to a mismatch (“CG”), which prevents hybridization of the crRNA (containing the “AC” sequence) to the methylated DNA molecule. Specifically, the mismatch occurs within 4 base pairs of the PAM sequence and within the target region of the crRNA (underlined region). This hybridization error disrupts the complementary binding of the hypo-specific crRNA to the methylated DNA molecule which prevents Casl2a from cutting this DNA molecule. In contrast, the cutting of the unmethylated DNA molecule, bottom panel of FIG. 17, occurs without a hybridization error as the hypo-specific crRNA (containing the “AC” sequence) molecule effectively binds to the target region (containing the “TG” sequence) of the unmethylated DNA molecule. In summary, FIG. 17 demonstrates the method by which depletion specificity towards hypo-methylated DNA molecules is mediated by a hypo-specific crRNA when a common PAM site is present.

[0176] FIG. 18A and FIG 18B present experimental results that demonstrate crRNA- mediated depletion specificity towards unmethylated DNA molecules when a common PAM is present. In FIG. 18A, DNA gel results for the hyper gblock (methylated DNA molecules) and hypo gblock (unmethylated DNA molecules) are displayed. Herein, the hypo gblock featured a clear band of cut DNA molecules following provision of the hypo crRNA to the mixture of hypo gblock and Casl2a. This cut DNA band was noticeably absent in the hyper gblock condition with the hypo crRNA, due to the mismatch that occurred between the hypo-crRNA and the seed of the hyper gblock despite an intact consensus PAM site. To provide quantification of these results, the resulting hypo-crRNA mixtures were analyzed for fragment length (FIG. 18B). The hyper gblock DNA with incubated with Casl2a and hypo crRNA featured minimal (22.6%) 50- 160 nucleotide long DNA fragments (FIG.18A top). Additionally, the full length gblock (180 nucleotides) largely remained intact (75.8% 160-400 nucleotide length) (FIG.18A top). In comparison, hypo gblock incubated with Casl2a and hypo crRNA featured enrichment (71.2%) of the cut length of DNA fragments (50-160 nucleotides) and a reduction in full-length hypo gblock (28.4%) (FIG.18B bottom). Bioinformatic analysis of libraries prepared and sequenced (iSeq) from hypo Km2+ gblocks with and without crRNA samples that underwent SPRI fdtering was performed (FIG. 19). Cut specific reads were elevated in quantity (32.3%) in the presence of the hypo crRNA, compared to 0% without the hypo crRNA (FIG. 19).

[0177] Collectively, these experiments demonstrate the ability of crRNA sequence specificity to direct specific depletion of unmethylated DNA molecules in instances where the a common PAM site is present between methylated and unmethylated bisulfite converted DNA molecules.Example 7 -Casl2a-Mediated Selective Depletion of Unmethylated DNA Molecules through PAM sequence mismatch

[0178] FIG. 20 provides a schematic overview of an experiment that demonstrated the depletion selectivity towards unmethylated DNA molecules mediated by a specific PAM sequence recognized by Casl2a. The top panel shows a bisulfite converted nucleic acid sequence derived from a fully methylated sense strand. The shown sequence in the top panel is a single contiguous sequence. The bottom panel shows a bisulfite converted nucleic acid sequence derived from a fully unmethylated sense strand. The shown sequence in the bottom panel is a single contiguous sequence.

[0179] In the top panel of FIG. 20, the “not PAM” sequence is identified in bold on the methylated DNA molecule. In this example, the fully methylated DNA molecule did not undergo conversion of its methylated cytosines thus the bold “CGGG” region did not undergo conversionto “TGGG”. As a result, a Cast 2a PAM site was not created, as is the case in the unmethylated DNA molecule (bottom panel, FIG. 20). Attention is now directed towards the bottom panel of FIG. 20, where the result of bisulfite conversion created a Casl2a PAM sequence (“TTTG”) in the hypo gblock, allowing Casl2ato bind to the unmethylated DNA molecule and perform selective cutting and depletion.

[0180] In a series of experiments (FIG. 21 A, FIG. 2 IB, and FIG. 22) Casl2a-directed selective depletion of unmethylated DNA molecules through a mismatch in the PAM sequence was empirically demonstrated. As shown in the DNA gel in FIG. 21 A, when hyper and hypo gblocks were separately incubated with Casl2a and either with or without crRNA, selective cutting was restricted to the hypo gblock (rectangle marked by “CUT”, FIG. 21 A) .When quantified, 70% of the full length hyper gblock remained intact (26.6% cut) when presented with Casl2a and hypo crRNA (FIG. 2 IB). This is due to the PAM sequence mismatch, which restricted cutting. In contrast, only 11.9% of the hypo block remained intact (82.4% cut) when presented with Casl2a and hypo crRNA (FIG. 21B). Here, the Casl2a PAM sequence (“TTTN”) was intact and cutting was anticipated. Lastly, hypo Km3+ gblocks and Casl2a with and without hypo crRNA mixtures underwent library preparation, SPRI filtering, and sequencing (iSEQ) to determine specific cutting rate of Casl2a (32.5%; FIG. 22). Here, the median insert size of reads derived from hypo Km3+ gblocks with crRNA was 112bp, which was smaller than the median insert size of reads (130bp) derived from hypo Km3+ gblocks without crRNA. This indicates that increased cutting was achieved for the hypo Km3+ gblocks with crRNA in comparison to hypo Km3+ gblocks without crRNA.

[0181] Collectively, these experiments demonstrate the ability of a mismatch in the PAM to direct the selective depletion of unmethylated sequences.INCORPORATION BY REFERENCE

[0182] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes.EQUIVALENTS

[0183] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.TABLE 1 - List of CGIs ReferencePos (hgl9 coordinates)1 chrl3: 108518334-1085186332 chr6: 137242315-1372454423 chr2: 177016416- 1770166324 chr5:2738953-27412375 chr4: 111553079-1115542106 chrl5:96909815-969100307 chr6:42072032-420727018 chrlO: 123922850-1239235429 chrl6:86612188-8661382110 chrl9:47151768-4715312511 chrl: 110610265-11061330312 chr5:3594467-360305413 chr9: 126773246- 12678095314 chr3: 138656627-13865910715 chr4:4859632-486019116 chrlO: 118895963-11889803717 chr7: 103086344-10308684018 chrl9:407011-40951119 chrl0:22764708-2276705020 chrl6:86549069-8655051221 chr9:96713326-9671818622 chr8 : 139508795 - 13950977423 chr2:73143055-7314826024 chr8:26721642-2672456625 chr9: 129386112-12938923126 chrl2:49483601-4948425527 chrl6:54325040-5432570328 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chrl:21616380-216171011410 chr 1 : 147782066- 1477824731411 chr7:6590563-65909571412 chr7: 97839862-978402221413 chrl2: 113914440-1139146571414 chrl9:7933263-79348981415 chr20:22559553-225600011416 chrl5:53086629-530868581417 chrl0:94180315-941807541418 chr5: 140052059-1400533811419 chrlO: 101287162-1012879201420 chrl4:38677154-386777871421 chr22:39262338-392632111422 chrl8:74153239-741550731423 chrl5:59157045-591575941424 chr4:963804-9641151425 chrl 1:624780-6250531426 chr7: 1362811-13636431427 chrl9:36246328-362479821428 chr5:54528095-545284041429 chrl2:54359658-543599061430 chr2: 127782613-1277828291431 chrl9:406131-4066111432 chrl7:46697413-466977011433 chrl8:43608140-436085101434 chr 16: 23724270-237247751435 chrl8:55922987-559240681436 chrl5:60291879-602921671437 chrl4:92788913-927892041438 chrl9: 1108394-11096101439 chrl 1: 124628367-1246295901440 chrl:32052471-320527711441 chrl9: 11594372-115949871442 chrl9:870774-8713181443 chr2: 54086776-540872661444 chr2:241459632-2414600471445 chr7: 127990926- 1279926161446 chrl:208132327-2081331171447 chr7:90893567-908966831448 chrl:41284847-412851491449 chrl 1:32452144-324527081450 chr5:77146998-771477851451 chrl9:45901452-459016881452 chr7: 6661875-66626951453 chr6: 161188084-1611886391454 chrl7:934417-9350881455 chrl 1:65409636-654101271456 chrl7: 19883325-198836101457 chrl8:77549524-775502991458 chrl:38461584-384619881459 chrl9: 10464666-104649271460 chrl7:70120139-701204421461 chr7:27147589-271483891462 chr2:31806545-318067821463 chrl 1: 119292689-1192928911464 chrl 9: 18979351-189812001465 chr6:42879279-428796231466 chrl2: 130908777-1309091911467 chrl7:46629553-466298161468 chrl:202162958-2021633901469 chrl7:21367114-213675921470 chrl6:84001805-840020111471 chrl:221057463-2210577571472 chrl7:27899511-279000671473 chrl5:40268581-402690611474 chr22:37465056-374653311475 chrl7:77805866-778090461476 chrl9: 13198699-131989991477 chr3: 184056419-1840566711478 chr22:37911979-379122581479 chrl9: 19368708-193696811480 chrl 1:64135815-641363811481 chrl8:77552401-775526031482 chrl9:58554354-585545871483 chr20:57414595-574148961484 chr4: 190938106-1909388481485 chr5: 172110282-1721111661486 chrl6:68480864-684828221487 chr9: 139395020-1393952871488 chrl2: 113515164-1135159701489 chrl:221054554-2210548881490 chr8: 144990270-1450021351491 chr9: 131154346-1311559231492 chr6: 150335525-1503362781493 chr9: 115824684-1158250331494 chrl2:54519768-545204571495 chr6:35479872-354801541496 chrl9:3870788-38710431497 chrl9:48965002-489657921498 chr6:35479388-354796781499 chrl2:52408381-524086751500 chrl:221068782-2210691591501 chr6:46655262-466567381502 chr3:55508336-555087081503 chrl:39980365-399817681504 chrl6:3067521-30683581505 chrl: 1473107-14733421506 chrlO: 105362549-1053628271507 chrl7:46698880-466990831508 chr2: 198029068-1980294381509 chr20: 17209418-172096221510 chrl2:49183049-491832821511 chrl6:58030214-580316331512 chrl0:94820026-948232521513 chrl 1:725596-7268701514 chr6: 170732119-1707324421515 chrl2: 120835586-1208359271516 chr20:36012595-360134391517 chr8: 143545445-1435461781518 chr6:27228100-272283641519 chr21:32624144-326243821520 chr9: 95477296-954777081521 chrlO: 105420685-1054210761522 chrl: 1470604-14714501523 chrl: 146552328-1465525771524 chrl9:33625467-336258051525 chrl 1:64478843-644795981526 chr20:57428308-574285161527 chr7:27182613-271855621528 chrl9: 51815157-518154581529 chrl7:46607804-466083901530 chrl2:52408860-524091211531 chrl9: 10405924-104063981532 chrl 1: 14993452-149936611533 chrl9: 13135317-131361691534 chr7:750788-7512371535 chrl:53742297-537428451536 chrl:200010625-2000108321537 chr5: 139138875-1391392421538 chrl7:45949676-459498851539 chr3: 128722283-1287230361540 chrl5:89312719-893131831541 chr9: 135039673-1350399781542 chrl9: 12831793-128322251543 chr20:51589707-515900201544 chr20:3145121-31457461545 chr8:65710990-657117221546 chr 11 : 128694084-1286946881547 chr2:20870006-208712801548 chrl9: 18977466-189778331549 chr3: 49947621-499484301550 chr6:30139718-301402631551 chrl2: 104697348- 1046979841552 chrlO: 105361784-1053621881553 chr6:29894140-298951171554 chr4: 187219320-1872197451555 chrl5:67073306-670739431556 chr2:220412341-2204126781557 chr6: 170730395-1707308871558 chr9: 115822071-1158234161559 chrl: 10764449-107649251560 chrl7:46627787-466284441561 chrl9:51601822-516022601562 chrl9:55814067-558142781563 chr6: 138745348-1387455931564 chr9: 124987743-1249910861565 chr22:46318693-463190871566 chrl6:3013016-30132281567 chr4: 114900355-1149008101568 chrl9: 1063544-10642651569 chrl9: 1110399-11107011570 chr7: 97841636-978420051571 chr8:57359899-573601141572 chrl7:72915568-729165101573 chrl: 16860873-168622961574 chrl7:75398284-753985271575 chr9: 139397412-1393977101576 chr6:33393592-333939081577 chr6:29595298-295957951578 chrl2:6438272-64389311579 chr3: 113160299-1131606411580 chrl:55505060-555060151581 chrl 1 : 132951692- 1329522601582 chr4:81118137-811186031583 chrl9:38876070-388763321584 chrl9:58549305-585497121585 chrl7:43472527-434743431586 chr9: 139396205-1393970401587 chrl6:3192181-31926691588 chr6:33048416-330488141589 chr7: 128555329-1285566501590 chrl9:46915311-469158021591 chr6:30095173-30095610Table 2: Example CGIsTable 3 : Additional Example CGIsTable 4: Additional Example CGIs

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method for enriching for a signal in a sample from a subject, the method comprising: contacting a nuclease with a plurality of nucleic acid molecules, wherein a first subset of the plurality of nucleic acid molecules comprise converted sequences derived from one or more unmethylated CpG sites and wherein a second subset of the plurality of nucleic acid molecules comprise converted sequences derived from one or more methylated CpG sites; selectively cutting the converted sequences of the first subset of the plurality of nucleic acid molecules using the nuclease relative to cutting the converted sequences of the second subset of the plurality of nucleic acid molecules, providing copies of one or more primer sequences, thereby hybridizing at least one copy of the one or more primer sequences to the converted sequences of the second subset of the plurality of nucleic acid molecules, wherein the copies of the one or more primer sequences fail to hybridize with the converted sequences of the first subset of the plurality of nucleic acid molecules due to the cutting; and enriching for the second subset of the plurality of nucleic acid molecules using the hybridized at least one copy of the one or more primer sequences.

2. The method of claim 1, wherein selectively cutting the converted sequences of the first subset of the plurality of nucleic acid molecules is performed by a CRISPR-Cas nuclease.

3. The method of claim 2, wherein the CRISPR-Cas nuclease is one or more of Casl2, Cas9, and subtypes and / or variants thereof.

4. The method of claim 3, wherein the Casl2 is Casl2a.

5. The method of claim 3, wherein the Cas9 is Cas9,6. The method of any one of claims 1-5, wherein selectively cutting the converted sequences of the first subset of the plurality of nucleic acid molecules is performed by a CRISPR-Cas nuclease due to a presence of one or more converted nucleotide bases in the first subset of the plurality of nucleic acid molecules as a result of performing a nucleic acid conversion.

7. The method of claim 6, wherein the one or more converted nucleotide bases are present in one or more protospacer adjacent motif (PAM) nucleic acid sequence(s).

8. The method of claim 7, wherein the one or more PAM nucleic acid sequences comprise TTN or TTTG, where N is one of A, C, T, or G.

9. The method of claim 7 or 8, wherein the one or more PAM nucleic acid sequences comprise TTTG.

10. The method of any one of claims 6-9, wherein the one or more converted nucleotide bases are present in a seed sequence complementary to a guide RNA sequence.

11. The method of claim 10, wherein the one or more converted nucleotide bases are present in a seed sequence less than 7 nucleotide bases from a PAM sequence.

12. The method of claim 6, wherein the one or more converted nucleotide bases are present in one or more protospacer adjacent motif (PAM) nucleic acid sequence(s) and further present in a seed sequence complementary to a guide RNA sequence.

13. The method of claim 12, wherein the one or more PAM nucleic acid sequences comprise TTN or TTTG, where N is one of A, C, T, or G.

14. The method of claim 12 or 13, wherein the one or more PAM nucleic acid sequences comprise TTTG.

15. The method of any one of claims 12-14, wherein the one or more converted nucleotide bases are present in a seed sequence less than 7 nucleotide bases from a PAM sequence.

16. The method of any one of claims 6-15, wherein the one or more converted nucleotide bases are not present in the second subset of the plurality of nucleic acid molecules as a result of performing a nucleic acid conversion.

17. The method of any one of claims 6-16, wherein the nucleic acid conversion comprises a bisulfite conversion.

18. The method of any one of claims 1-12, wherein the nuclease is associated with a guide RNA (gRNA) comprising a spacer sequence that binds to a target sequence of a converted sequence of the first subset of the plurality of nucleic acids.

19. The method of claim 10, wherein the target sequence comprises a sequence or a portion of a sequence of a CGI selected from Tables 1-4.

20. The method of claims 6-15, wherein subsequent to the CRISPR-Cas nuclease recognizing the PAM sequence, the CRISPR-Cas nuclease selectively cuts converted sequences of the first subset of the plurality of nucleic acid sequences.

21. The method of claim 6, wherein the CRISPR-Cas nuclease selectively cuts the converted sequences of the first subset of the plurality of nucleic acid sequences, thereby producing a plurality of nucleic acid fragments that fail to hybridize with the one or more primer sequences.

22. The method of claim 21, wherein the plurality of nucleic acid fragments lack a complementary sequence to the one or more primer sequences due to the selectivecutting, thereby exhibiting reduced binding affinity for the one or more primer sequences.

23. The method of any one of claims 1-22, wherein the one or more primer sequences comprise indexing adapters.

24. The method of any one of claims 1-22, wherein the one or more primer sequences comprise a forward and reverse primer pair.

25. The method of any one of claims 1-24, wherein the sample is a blood or serum sample.

26. The method of any one of claims 1-25, wherein the sample comprises cell free DNA (cfDNA).

27. The method of any one of claims 1-26, wherein the subject is a human.

28. The method of any one of claims 1-27, the method further comprising: obtaining or having obtained nucleic acids from the sample; and performing a conversion of the obtained nucleic acids to generate the converted sequences of the first subset and the second subset of the plurality of nucleic acids.

29. The method of claim 28, wherein performing the conversion comprises performing a bisulfite conversion.

30. The method of any one of claims 1-29, wherein enriching for the second subset of the plurality of nucleic acid molecules using the hybridized at least one copy of the one or more primer sequences comprises performing nucleic acid amplification.

31. The method of any one of claims 1-30, the method further comprising sequencing nucleic acids of the enriched second subset of the plurality of nucleic acid molecules.

32. The method of any one of claims 1-31, wherein sequencing nucleic acids comprises performing next generation sequencing.

33. The method of any one of claims 1-31, further comprising: performing an additional enrichment step.

34. The method of claim 28, wherein the converted sequences of the second subset of the plurality of nucleic acid molecules comprise a target sequence, and wherein the additional enrichment step further enriches at least a subset of the target sequences.

35. The method of claim 28 or 29, wherein the additional enrichment step comprises hybrid capture.

36. The method of any one of claims 30 or 31, wherein the additional enrichment step comprises using a nucleic acid binding protein.

37. The method of any one of claims 1-36, wherein the selective cutting is determined by performing a quantitative polymerase chain reaction (qPCR) assay38. The method of any one of claims 1-36, wherein the selective cutting is determined by performing a bioinformatics analysis.

39. The method of claim 38, wherein the bioinformatics analysis comprises performing a ratio metric calculation40. The method of claim 39, wherein the ratio metric calculation is a count of reads starting in a 25 base pair window of a cut site divided by the total number of reads in a given nucleic acid region.

41. A kit comprising: reagents for performing a nucleic acid conversion of a sample from a subject; and a nuclease that selectively cuts a converted nucleic acid sequence due to a presence of one or more converted nucleotide bases in the converted nucleic acid as a result of performing or having performed the nucleic acid conversion using the reagents.

42. The kit of claim 41, wherein the nuclease is a CRISPR-Cas nuclease.

43. The kit of claim 42, wherein the CRISPR-Cas nuclease is one or more of Casl2, Cas9, and subtypes and / or variants thereof.

44. The kit of claim 43, wherein the Casl2 is Casl2a.

45. The kit of claim 44, wherein the Cas9 is Cas9,46. The kit of any one of claims 41-45, wherein the one or more converted nucleotide bases are present in one or more protospacer adjacent motif (PAM) nucleic acid sequence(s).

47. The kit of claim 46, wherein the one or more PAM nucleic acid sequences comprise TTN or TTTG, where N is one of A, C, T, or G.

48. The kit of claim 46 or 47, wherein the one or more PAM nucleic acid sequences comprise TTTG.

49. The kit of any one of claims 41-45, wherein the one or more converted nucleotide bases are present in a seed sequence complementary to a guide RNA sequence.

50. The kit of claim 49, wherein the one or more converted nucleotide bases are present in a seed sequence less than 7 nucleotide bases from a PAM sequence.

51. The kit of any one of claims 41-45, wherein the one or more converted nucleotide bases are present in one or more protospacer adjacent motif (PAM) nucleic acid sequence(s) and further present in a seed sequence complementary to a guide RNA sequence.

52. The kit of claim 51, wherein the one or more PAM nucleic acid sequences comprise TTN or TTTG, where N is one of A, C, T, or G.

53. The kit of claim 51 or 52, wherein the one or more PAM nucleic acid sequences comprise TTTG.

54. The kit of any one of claims 51-53, wherein the one or more converted nucleotide bases are present in a seed sequence less than 7 nucleotide bases from a PAM sequence.

55. The kit of claim 42, wherein the CRISPR-Cas nuclease recognizes one or more protospacer adjacent motif (PAM) nucleic acid sequence(s).

56. The kit of claim 55, wherein the one or more PAM nucleic acid sequences comprise TTN, where N is one of A, C, T, or G.

57. The kit of claim 55 or 56, wherein the one or more PAM nucleic acid sequences comprise TTG.

58. The kit of claim 41, wherein the nucleic acid conversion comprises a bisulfite conversion.

59. The kit of any one of claims 41-54, further comprising a guide RNA (gRNA) comprising a spacer sequence that binds to a target sequence of the converted sequence.

60. The kit of claim 59, wherein the target sequence comprises a sequence or a portion of a sequence of a CGI selected from Tables 1-461. The kit of any one of claims 41-60, further comprising one or more primer sequences.

62. The kit of any one of claims 41-61, wherein the one or more primer sequences comprise indexing adapters.

63. The kit of any one of claims 41-62, wherein the one or more primer sequences comprise a forward and reverse primer pair.

64. The kit of any one of claims 41-63, wherein the sample is a blood or serum sample.

65. The kit of any one of claims 41-64, wherein the sample comprises cell free DNA (cfDNA).

66. The kit of any one of claims 41-65, wherein the subject is a human.

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