Patent ID: 12195802

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are methods for quantifying DNA methylation that may be utilized for screening for diseases (e.g., cancer), diagnosing diseases (e.g., cancer type), monitoring progression of a disease, and monitoring response to a treatment regimen. Also disclosed herein is a platform for developing early noninvasive diagnostics that inform novel therapeutic approaches. Also disclosed herein are methods for early detection of highly predictive epigenomic alterations, which include genome-wide misregulation of developmental gene promoters and optimized diagnostics for precise detection at ppm resolution. Also disclosed herein is developmental logic for new molecular therapies, which includes expanding the therapeutic window by targeting unique features of a pan-cancer “cell state.”

As used herein, “CpG” and “CpG dinucleotide” are used interchangeably and refer to a dinucleotide sequence containing an adjacent guanine and cytosine where the cytosine is located 5′ of guanine.

As used herein, “CpG island” or “CGI” refers to a region with a high frequency of CpG sites. The region is at least 200 bp, with a GC percentage greater than 50%, and an observed-to-expected CpG ratio greater than 60%.

As used herein, a “haplotype” refers to a combination of CpG sites found on the same chromosome. Similarly, a “DNA methylation haplotype” represents the DNA methylation status of CpG sites on the same chromosome.

In certain embodiments, a sample (e.g., a fluid sample) is screened. The sample may be screened using whole-genome bisulfite sequencing (WGBS), TCGA Illumina Infinium HumanMethylation450K BeadChip sequencing (TCGA), and/or reduced representation bisulfite sequencing (RRBS), or by other suitable methylation detection assays known in the art. The identified methylated sequences can be analyzed to identify differentially methylated loci and/or regions (e.g., CpG Islands). In some aspects described herein, for WGBS data, a CGI was considered differentially methylated if it was covered by at least 5 CpGs and 80% of them were significantly hyper/hypo-methylated. In some aspects described herein, for TCGA data, a CGI may be considered differentially methylated if 80% of covered CpGs were significantly hyper- or hypo-methylated. In some aspects described herein, for RRBS data, a cut-off of 10% difference in CGI-level methylation was used to identify differential methylation. In some aspects, the sample is also screened using RNA sequencing (RNA-seq) and/or Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq).

In some embodiments, DNA methylation haplotypes corresponding to methylation patterns of CpGs are identified from the screening. The DNA methylation haplotypes may be classified into three groups, concordantly unmethylated haplotypes, disordered haplotypes, and concordantly methylated haplotypes. Haplotypes are also referenced to herein as sequencing reads. In some aspects, the proportion of concordantly unmethylated reads (PUR), proportion of disordered reads (PDR), and proportion of concordantly methylated reads (PMR) are calculated. PMR can be used to quantify DNA methylation (e.g., for diagnosis purposes) as described herein.

In certain embodiments, the inventions disclosed herein relate to methods of using proportion of concordantly methylated reads (PMR) (i.e., fully methylated haplotypes) to detect circulating tumor DNA (ctDNA) in a sample. In certain aspects, a methylation sequence for a sample is obtained and at least one CpG Island (CGI) is identified on the methylation sequence. PMR for the identified CpG Island is calculated and then compared to a control background of a normal tissue or epiblast. The presence of ctDNA is detected in the sample when the PMR of the sample is larger than the control background (e.g., signal is higher by bank sum test).

In some aspects, the sample is selected from the group comprising plasma, urine, stool, menstrual fluid, lymph fluid, or any other body fluid in which ctDNA may be located. The sample may comprise DNA (e.g., cell free DNA (cfDNA)). In some aspects, the sample is obtained from a tumor. It is generally understood that the fraction of ctDNA in the sample (e.g., cfDNA) is usually low. In some aspects, the background noise for detecting ctDNA in the sample may be reduced by using a fully methylated haplotype.

The presence of ctDNA may be detected in the cfDNA with a greater sensitivity and specificity than methods previously known by those of skill in the art. For example, ctDNA may be detected in the sample using PMR with a sensitivity of greater than 75%, 80%, 85%, 90%, 95%, or 99%. In certain aspects, ctDNA is detected in the sample using PMR with 100% sensitivity. ctDNA may be detected in the sample using PMR with a specificity of greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In certain aspects, ctDNA is detected in the sample using PMR with 95% specificity. In some aspects, ctDNA is detected in the sample using PMR with at least 90% sensitivity and at least 90% specificity. In some aspects, ctDNA is detected in the sample using PMR with at least 100% sensitivity and at least 95% specificity.

The amount of ctDNA detected in the sample may be measured and quantified. In some aspects, the sample comprises 0.005% to 1.5% ctDNA, 0.01% to 1% ctDNA, 0.05% to 0.5% ctDNA, 0.1% to 0.3% ctDNA. In some embodiments, the sample comprises 0.01% ctDNA. In certain aspects, the presence of 0.01% ctDNA is detected in cfDNA using PMR with about 100% sensitivity and about 95% specificity, with a p-value cutoff of 10−4.

In certain embodiments, the presence of ctDNA in a sample indicates the presence of cancer. In some aspects, the presence of ctDNA indicates the presence of a tumor. In alternative aspects, the sample is obtained from an individual without a tumor. For example, the sample may be obtained from an individual who is in the early stage of cancer and has not developed a tumor or the individual has a blood cancer (e.g., leukemia). In some aspects, the sample is obtained from an individual diagnosed with, suffering from, at risk of developing, or suspected of having cancer.

As used herein the phrase “cancer” is intended to broadly apply to any cancerous condition. In some aspects, the cancer is selected from the group comprising glioblastoma, colon, lung, breast, and prostate. In certain aspects, the cancer is selected from the group comprising bladder urothelial carcinoma, breast invasive carcinoma, colon adenocardinoma, colorectal adenocarcinoma, oseophageal carcinoma, head and neck squamous cell carcinoma, kidney rental clear cell carcinoma, kidney renal papillar cell carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, prostate adenocarcinoma, stomach and oesophageal carcinoma, thyroid carcinoma, uterine corpus endometrial carcinoma, and chronic lymphocytic leukaemia.

In some embodiments, the inventions disclosed herein relate to methods of screening for cancer by using PMR to detect ctDNA in a sample as described herein, wherein the presence of ctDNA in the sample is indicative of the subject having cancer.

The methods described herein may be applied to a subject who is at risk of cancer or at risk of cancer recurrence. A subject at risk of cancer may be, e.g., a subject who has not been diagnosed with cancer but has an increased risk of developing cancer. Determining whether a subject is considered “at increased risk” of cancer is within the skill of the ordinarily skilled medical practitioner. Any suitable test(s) and/or criteria can be used. For example, a subject may be considered “at increased risk” of developing cancer if any one or more of the following apply: (i) the subject has an inherited mutation or genetic polymorphism that is associated with increased risk of developing or having cancer relative to other members of the general population not having such mutation or genetic polymorphism (e.g., inherited mutations in certain TSGs are known to be associated with increased risk of cancer); (ii) the subject has a gene or protein expression profile, and/or presence of particular substance(s) in a sample obtained from the subject (e.g., blood), that is/are associated with increased risk of developing or having cancer relative to the general population; (iii) the subject has one or more risk factors such as a family history of cancer, exposure to a tumor-promoting agent or carcinogen (e.g., a physical carcinogen, such as ultraviolet or ionizing radiation; a chemical carcinogen such as asbestos, tobacco or smoke components, aflatoxin, arsenic; a biological carcinogen such as certain viruses or parasites); (iv) the subject is over a specified age, e.g., over 60 years of age. A subject suspected of having cancer may be a subject who has one or more symptoms of cancer or who has had a diagnostic procedure performed that suggested or was consistent with the possible existence of cancer. A subject at risk of cancer recurrence may be a subject who has been treated for cancer and appears to be free of cancer, e.g., as assessed by an appropriate method.

In other embodiments, the invention provides methods of treating a subject in need of treatment for cancer. PMR is used to detect ctDNA in a sample as described herein, where the presence of the ctDNA is indicative of the subject having cancer. The individual is then treated for cancer using any methods of treatment generally known to those of skill in the art (e.g., therapeutics or procedures).

For example, therapies or anticancer agents that may be used for treating the subject include anti-cancer agents, chemotherapeutic drugs, surgery, radiotherapy (e.g., γ-radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, and systemic radioactive isotopes), endocrine therapy, biologic response modifiers (e.g., interferons, interleukins), hyperthermia, cryotherapy, agents to attenuate any adverse effects, or combinations thereof, useful for treating a subject in need of treatment for a cancer. Non-limiting examples of cancer chemotherapeutic agents that may be used include, e.g., alkylating and alkylating-like agents such as nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, streptozocin); platinum agents (e.g., alkylating-like agents such as carboplatin, cisplatin, oxaliplatin, BBR3464, satraplatin), busulfan, dacarbazine, procarbazine, temozolomide, thioTEPA, treosulfan, and uramustine; antimetabolites such as folic acids (e.g., aminopterin, methotrexate, pemetrexed, raltitrexed); purines such as cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, thioguanine; pyrimidines such as capecitabine, cytarabine, fluorouracil, floxuridine, gemcitabine; spindle poisons/mitotic inhibitors such as taxanes (e.g., docetaxel, paclitaxel), vincas (e.g., vinblastine, vincristine, vindesine, and vinorelbine), epothilones; cytotoxic/anti-tumor antibiotics such anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone, and valrubicin), compounds naturally produced by various species ofStreptomyces(e.g., actinomycin, bleomycin, mitomycin, plicamycin) and hydroxyurea; topoisomerase inhibitors such as camptotheca (e.g., camptothecin, topotecan, irinotecan) and podophyllums (e.g., etoposide, teniposide); monoclonal antibodies for cancer therapy such as anti-receptor tyrosine kinases (e.g., cetuximab, panitumumab, trastuzumab), anti-CD20 (e.g., rituximab and tositumomab), and others for example alemtuzumab, aevacizumab, gemtuzumab; photosensitizers such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfin; tyrosine and/or serine/threonine kinase inhibitors, e.g., inhibitors of Abl, Kit, insulin receptor family member(s), VEGF receptor family member(s), EGF receptor family member(s), PDGF receptor family member(s), FGF receptor family member(s), mTOR, Raf kinase family, phosphatidyl inositol (PI) kinases such as PI3 kinase, PI kinase-like kinase family members, cyclin dependent kinase (CDK) family members, Aurora kinase family members (e.g., kinase inhibitors that are on the market or have shown efficacy in at least one phase III trial in tumors, such as cediranib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, sorafenib, sunitinib, vandetanib), growth factor receptor antagonists, and others such as retinoids (e.g., alitretinoin and tretinoin), altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase (e.g., pegasparagase), bexarotene, bortezomib, denileukin diftitox, estramustine, ixabepilone, masoprocol, mitotane, and testolactone, Hsp90 inhibitors, proteasome inhibitors (e.g., bortezomib), angiogenesis inhibitors, e.g., anti-vascular endothelial growth factor agents such as bevacizumab (Avastin) or VEGF receptor antagonists, matrix metalloproteinase inhibitors, various pro-apoptotic agents (e.g., apoptosis inducers), Ras inhibitors, anti-inflammatory agents, cancer vaccines, or other immunomodulating therapies, etc. It will be understood that the preceding classification is non-limiting.

The present invention also provides a method of monitoring a subject's response to a cancer treatment comprising using PMR of a first sample obtained prior to a subject receiving a cancer treatment to detect an amount of ctDNA in the first sample, using PMR of a second sample obtained after a subject received the cancer treatment to detect an amount of ctDNA in the second sample, and comparing the amount of ctDNA detected in the first sample and the amount of ctDNA detected in the second sample. In some aspects, the amount of ctDNA detected in the second sample will be less than the amount of ctDNA detected in the first sample indicating the subject's positive response to the cancer treatment (e.g., the treatment is effective). In alternative aspects, the amount of ctDNA detected in the second sample will be greater than or the same as the amount of ctDNA detected in the first sample indicating the subject's negative or neutral response to the cancer treatment (e.g, the treatment is not effective).

In other embodiments, the invention provides a method of monitoring progression or amelioration of cancer in a subject. The method comprises using PMR to identify ctDNA from cfDNA of the subject as described herein, wherein if ctDNA is present the subject is at risk of developing cancer, and monitoring the amount of ctDNA in the cfDNA over time, wherein alternation of the amount of ctDNA in the cfDNA is indicative of progression or amelioration of the cancer.

In still other embodiments, the invent provides a method of assessing cancer in a subject, the method comprising using PMR to identify the presence of ctDNA from cfDNA of the subject as described herein, wherein if ctDNA is present, the subject has or is at risk of developing cancer.

In further embodiments, the invention provides methods of identifying DNA methylation signatures for individual cancer types. For example, methylation patterns of CpGs representing single DNA methylation haplotypes may be quantified for specific cancer types. Examples of cancer types include, but are not limited to, colon, lung, lung (squamous), breast, prostate, glioblastoma, bladder, esophagus, head and neck, kidney (clear), kidney (papillary), liver, and uterine (corpus).

In some aspects, the invention provides predictions of cancer tissues of origin using a DNA methylation signature. For example, a methylation signature may be detected with sensitivity and specificity across a variety of tissue systems. Examples of such tissues include, but are not limited to, adrenal, B cell, bladder, bone/soft tissue, brain, breast, cervix, colon, eye, germ cell, head and neck, kidney, liver, lung, myeloid, mesothelium, neuroendocrine, pancreas, prostate, skin, stomach, thymus, and uterine.

The present invention also provides methods for identifying therapeutic targets within one or more molecular pathways. In some aspects, the molecular pathway is common between a somatic state and a cancer-like state. In some aspects, identifying therapeutic targets includes single animal screening, perturb-seq, identifying candidates, and inhibiting and/or targeting docking sites that misdirect common regulators.

The present invention also provides a method of disrupting methylation of CpG islands comprising reducing expression of PRC2. The present invention also provides a method of disrupting methylation of CpG islands comprising reducing expression of Eed. The present invention further provides a method of disrupting methylation of CpG islands comprising reducing expression of Dnmtl, Dnmt3l, or Dnmt3b. In some aspects, expression is reduced by genomic modification (e.g., using CRISPR/Cas or TALEN systems).

CRISPR/Cas systems can employ a variety of Cas proteins (Haft et al. PLoS Comput Biol. 2005; 1(6)e60). In some embodiments, the CRISPR/Cas system is a CRISPR type I system. In some embodiments, the CRISPR/Cas system is a CRISPR type II system. In some embodiments, the CRISPR/Cas system is a CRISPR type V system. It should be understood that although examples of methods utilizing CRISPR/Cas (e.g., Cas9 and Cpf1) and TALEN are described in detail herein, the invention is not limited to the use of these methods/systems. Other methods of targeting polynucleotide sequences to reduce or ablate expression in target cells known to the skilled artisan can be utilized herein.

The present inventions contemplate altering, e.g., modifying or cleaving, target polynucleotide sequences in a cell for any purpose, but particularly such that the expression or activity of the encoded product is reduced or eliminated. In some embodiments, the alteration results in reduced expression of the target polynucleotide sequence. The terms “decrease,” “reduced,” “reduction,” and “decrease” are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, “decreased,” “reduced,” “reduction,” “decrease” includes a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.

It should be appreciated that CRISPR/Cas systems can cleave target polynucleotide sequences in a variety of ways. In some embodiments, the target polynucleotide sequence is cleaved such that a double-strand break results. In some embodiments, the target polynucleotide sequence is cleaved such that a single-strand break results.

In some embodiments, CRISPR/Cas systems include a Cas protein or a nucleic acid sequence encoding the Cas protein and at least one to two ribonucleic acids (e.g., gRNAs) that are capable of directing the Cas protein to and hybridizing to a target motif of a target polynucleotide sequence. In some embodiments, CRISPR/Cas systems include a Cas protein or a nucleic acid sequence encoding the Cas protein and a single ribonucleic acid or at least one pair of ribonucleic acids (e.g., gRNAs) that are capable of directing the Cas protein to and hybridizing to a target motif of a target polynucleotide sequence. As used herein, “protein” and “polypeptide” are used interchangeably to refer to a series of amino acid residues joined by peptide bonds (i.e., a polymer of amino acids) and include modified amino acids (e.g., phosphorylated, glycated, glycosolated, etc.) and amino acid analogs. Exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, paralogs, fragments and other equivalents, variants, and analogs of the above.

In some embodiments, a Cas protein comprises one or more amino acid substitutions or modifications. In some embodiments, the one or more amino acid substitutions comprise a conservative amino acid substitution. In some instances, substitutions and/or modifications can prevent or reduce proteolytic degradation and/or extend the half-life of the polypeptide in a cell. In some embodiments, the Cas protein can comprise a peptide bond replacement (e.g., urea, thiourea, carbamate, sulfonyl urea, etc.). In some embodiments, the Cas protein can comprise a naturally occurring amino acid. In some embodiments, the Cas protein can comprise an alternative amino acid (e.g., D-amino acids, beta-amino acids, homocysteine, phosphoserine, etc.). In some embodiments, a Cas protein can comprise a modification to include a moiety (e.g., PEGylation, glycosylation, lipidation, acetylation, end-capping, etc.).

In some embodiments, a Cas protein comprises a core Cas protein. Exemplary Cas core proteins include, but are not limited to Cas1, Cast, Cas3, Cas4, Cas5, Cash, Cas7, Cas8 and Cas9. In some embodiments, a Cas protein comprises a Cas protein of anE. colisubtype (also known as CASS2). Exemplary Cas proteins of theE. colisubtype include, but are not limited to Cse1, Cse2, Cse3, Cse4, and Cas5e. In some embodiments, a Cas protein comprises a Cas protein of the Ypest subtype (also known as CASS3). Exemplary Cas proteins of the Ypest subtype include, but are not limited to Csy1, Csy2, Csy3, and Csy4. In some embodiments, a Cas protein comprises a Cas protein of the Nmeni subtype (also known as CASS4). Exemplary Cas proteins of the Nmeni subtype include, but are not limited to Csn1 and Csn2. In some embodiments, a Cas protein comprises a Cas protein of the Dvulg subtype (also known as CASS1). Exemplary Cas proteins of the Dvulg subtype include Csd1, Csd2, and Cas5d. In some embodiments, a Cas protein comprises a Cas protein of the Tneap subtype (also known as CASS7). Exemplary Cas proteins of the Tneap subtype include, but are not limited to, Cst1, Cst2, Cas5t. In some embodiments, a Cas protein comprises a Cas protein of the Hmari subtype. Exemplary Cas proteins of the Hmari subtype include, but are not limited to Csh1, Csh2, and Cas5h. In some embodiments, a Cas protein comprises a Cas protein of the Apern subtype (also known as CASS5). Exemplary Cas proteins of the Apern subtype include, but are not limited to Csa1, Csa2, Csa3, Csa4, Csa5, and Cas5a. In some embodiments, a Cas protein comprises a Cas protein of the Mtube subtype (also known as CASS6). Exemplary Cas proteins of the Mtube subtype include, but are not limited to Csm1, Csm2, Csm3, Csm4, and Csm5. In some embodiments, a Cas protein comprises a RAMP module Cas protein. Exemplary RAMP module Cas proteins include, but are not limited to, Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6.

In some embodiments, the Cas protein is aStreptococcus pyogenesCas9 protein or a functional portion thereof. In some embodiments, the Cas protein is aStaphylococcus aureusCas9 protein or a functional portion thereof. In some embodiments, the Cas protein is aStreptococcus thermophilusCas9 protein or a functional portion thereof. In some embodiments, the Cas protein is aNeisseria meningitidesCas9 protein or a functional portion thereof. In some embodiments, the Cas protein is aTreponema denticolaCas9 protein or a functional portion thereof. In some embodiments, the Cas protein is Cas9 protein from any bacterial species or functional portion thereof. Cas9 protein is a member of the type II CRISPR systems which typically include a trans-coded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas protein. Cas 9 protein (also known as CRISPR-associated endonuclease Cas9/Csn1) is a polypeptide comprising 1368 amino acids. Cas 9 contains 2 endonuclease domains, including a RuvC-like domain (residues 7-22, 759-766 and 982-989) which cleaves target DNA that is noncomplementary to crRNA, and an HNH nuclease domain (residues 810-872) which cleave target DNA complementary to crRNA.

In some embodiments, the Cas protein is Cpf1 protein or a functional portion thereof. In some embodiments, the Cas protein is Cpf1 from any bacterial species or functional portion thereof. In some aspects, Cpf1 is aFrancisella novicidaU112 protein or a functional portion thereof. In some aspects, Cpf1 is anAcidaminococcussp. BV3L6 protein or a functional portion thereof. In some aspects, Cpf1 is aLachnospiraceae bacteriumND2006 protein or a function portion thereof. Cpf1 protein is a member of the type V CRISPR systems. Cpf1 protein is a polypeptide comprising about 1300 amino acids. Cpf1 contains a RuvC-like endonuclease domain. Cpf1 cleaves target DNA in a staggered pattern using a single ribonuclease domain. The staggered DNA double-stranded break results in a 4 or 5-nt 5′ overhang.

As used herein, “functional portion” refers to a portion of a peptide which retains its ability to complex with at least one ribonucleic acid (e.g., guide RNA (gRNA)) and cleaves a target polynucleotide sequence. In some embodiments, the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional portion comprises a combination of operably linked Cpf1 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional domains form a complex. In some embodiments, a functional portion of the Cas9 protein comprises a functional portion of a RuvC-like domain. In some embodiments, a functional portion of the Cas9 protein comprises a functional portion of the HNH nuclease domain. In some embodiments, a functional portion of the Cpf1 protein comprises a functional portion of a RuvC-like domain.

It should be appreciated that the present invention contemplates various ways of contacting a target polynucleotide sequence with a Cas protein (e.g., Cas9). In some embodiments, exogenous Cas protein can be introduced into the cell in polypeptide form. In certain embodiments, Cas proteins can be conjugated to or fused to a cell-penetrating polypeptide or cell-penetrating peptide. As used herein, “cell-penetrating polypeptide” and “cell-penetrating peptide” refers to a polypeptide or peptide, respectively, which facilitates the uptake of a molecule into a cell. The cell-penetrating polypeptides can contain a detectable label.

In certain embodiments, Cas proteins can be conjugated to or fused to a charged protein (e.g., that carries a positive, negative or overall neutral electric charge). Such linkage may be covalent. In some embodiments, the Cas protein can be fused to a superpositively charged GFP to significantly increase the ability of the Cas protein to penetrate a cell (Cronican et al. ACS Chem Biol. 2010; 5(8):747-52). In certain embodiments, the Cas protein can be fused to a protein transduction domain (PTD) to facilitate its entry into a cell. Exemplary PTDs include Tat, oligoarginine, and penetratin. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a cell-penetrating peptide. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a PTD. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a tat domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to an oligoarginine domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a penetratin domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a superpositively charged GFP. In some embodiments, the Cpf1 protein comprises a Cpf1 polypeptide fused to a cell-penetrating peptide. In some embodiments, the Cpf1 protein comprises a Cpf1 polypeptide fused to a PTD. In some embodiments, the Cpf1 protein comprises a Cpf1 polypeptide fused to a tat domain. In some embodiments, the Cpf1 protein comprises a Cpf1 polypeptide fused to an oligoarginine domain. In some embodiments, the Cpf1 protein comprises a Cpf1 polypeptide fused to a penetratin domain. In some embodiments, the Cpf1 protein comprises a Cpf1 polypeptide fused to a superpositively charged GFP.

In some embodiments, the Cas protein can be introduced into a cell containing the target polynucleotide sequence in the form of a nucleic acid encoding the Cas protein (e.g., Cas9 or Cpf1). The process of introducing the nucleic acids into cells can be achieved by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using a viral vector. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises a modified DNA, as described herein. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises a modified mRNA, as described herein (e.g., a synthetic, modified mRNA).

In some embodiments, nucleic acids encoding Cas protein and nucleic acids encoding the at least one to two ribonucleic acids are introduced into a cell via viral transduction (e.g., lentiviral transduction).

In some embodiments, the Cas protein is complexed with one to two ribonucleic acids. In some embodiments, the Cas protein is complexed with two ribonucleic acids. In some embodiments, the Cas protein is complexed with one ribonucleic acid. In some embodiments, the Cas protein is encoded by a modified nucleic acid, as described herein (e.g., a synthetic, modified mRNA).

The methods of the present invention contemplate the use of any ribonucleic acid that is capable of directing a Cas protein to and hybridizing to a target motif of a target polynucleotide sequence. In some embodiments, at least one of the ribonucleic acids comprises tracrRNA. In some embodiments, at least one of the ribonucleic acids comprises CRISPR RNA (crRNA). In some embodiments, a single ribonucleic acid comprises a guide RNA that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell. In some embodiments, at least one of the ribonucleic acids comprises a guide RNA that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell. In some embodiments, both of the one to two ribonucleic acids comprise a guide RNA that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell. The ribonucleic acids of the present invention can be selected to hybridize to a variety of different target motifs, depending on the particular CRISPR/Cas system employed, and the sequence of the target polynucleotide, as will be appreciated by those skilled in the art. The one to two ribonucleic acids can also be selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence. In some embodiments, the one to two ribonucleic acids hybridize to a target motif that contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids hybridize to a target motif that contains at least one mismatch when compared with all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids are designed to hybridize to a target motif immediately adjacent to a deoxyribonucleic acid motif recognized by the Cas protein. In some embodiments, each of the one to two ribonucleic acids are designed to hybridize to target motifs immediately adjacent to deoxyribonucleic acid motifs recognized by the Cas protein which flank a mutant allele located between the target motifs.

One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The details of the description and the examples herein are representative of certain embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention. It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

The articles “a” and “an” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention provides all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. It is contemplated that all embodiments described herein are applicable to all different aspects of the invention where appropriate. It is also contemplated that any of the embodiments or aspects can be freely combined with one or more other such embodiments or aspects whenever appropriate. Where elements are presented as lists, e.g., in Markush group or similar format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. For example, any one or more active agents, additives, ingredients, optional agents, types of organism, disorders, subjects, or combinations thereof, can be excluded.

Where ranges are given herein, the invention includes embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also understood that where a series of numerical values is stated herein, the invention includes embodiments that relate analogously to any intervening value or range defined by any two values in the series, and that the lowest value may be taken as a minimum and the greatest value may be taken as a maximum. Numerical values, as used herein, include values expressed as percentages. For any embodiment of the invention in which a numerical value is prefaced by “about” or “approximately”, the invention includes an embodiment in which the exact value is recited. For any embodiment of the invention in which a numerical value is not prefaced by “about” or “approximately”, the invention includes an embodiment in which the value is prefaced by “about” or “approximately”.

“Approximately” or “about” generally includes numbers that fall within a range of 1% or in some embodiments within a range of 5% of a number or in some embodiments within a range of 10% of a number in either direction (greater than or less than the number) unless otherwise stated or otherwise evident from the context (except where such number would impermissibly exceed 100% of a possible value). It should be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited, but the invention includes embodiments in which the order is so limited. It should also be understood that unless otherwise indicated or evident from the context, any product or composition described herein may be considered “isolated”.

As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

As used herein the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

It is to be understood that the inventions disclosed herein are not limited in their application to the details set forth in the description or as exemplified. The invention encompasses other embodiments and is capable of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

While certain compositions, methods and assays of the present invention have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the methods and compositions of the invention and are not intended to limit the same.

EXAMPLES

Example 1: Epigenetic Restriction of Extraembryonic Lineages Mirrors the Somatic Transition to Cancer

In mammals, the canonical somatic DNA methylation landscape is established upon specification of the embryo proper and subsequently disrupted within many cancer types1-4. However, the underlying mechanisms that direct this genome-scale transformation remain elusive, with no clear model for its systematic acquisition or potential developmental utility5,6. Here, global remethylation was analyzed from the mouse preimplantation embryo into the early epiblast and extraembryonic ectoderm. It was shown that these two states acquire highly divergent genomic distributions with substantial disruption of bimodal, CpG density-dependent methylation in the placental progenitor7,8. The extraembryonic epigenome includes specific de novo methylation at hundreds of embryonically protected CpG island promoters, particularly those that are associated with key developmental regulators and are orthologously methylated across most human cancer types9. The data suggest that the evolutionary innovation of extraembryonic tissues may have required co-option of DNA methylation-based suppression as an alternative to regulation by Polycomb-group proteins, which coordinate embryonic germ-layer formation in response to extraembryonic cues10. Moreover, it was established that this decision is made deterministically, downstream of promiscuously used—and frequently oncogenic—signaling pathways, via a novel combination of epigenetic cofactors. Methylation of developmental gene promoters during tumorigenesis may therefore reflect the misappropriation of an innate trajectory and the spontaneous reacquisition of a latent, developmentally encoded epigenetic landscape.

To compare how epigenetic landscapes evolve during early mammalian development, whole-genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) datasets were generated from mouse precompacted 8-cell stage embryos, inner cell mass (ICM) and trophectoderm from embryonic day (E)3.5 blastocysts, as well as epiblast and extraembryonic ectoderm (ExE) from E6.5 conceptuses, the latest stage at which these progenitors remain largely homogeneous and undifferentiated (FIG.1A,FIG.5). Holistically, the time series captures the expected transition through the indistinguishably hypomethylated—but transcriptionally distinct—blastocyst-stage tissues, followed by a considerable departure at implantation, at which approximately 80% of the genome becomes differentially methylated (FIG.6A). Specifically, the extraembryonic lineage lacks canonical bimodality: most CpGs are incompletely methylated in comparison to the epiblast and 1.36% are methylated in the ExE (FIG.1B,FIG.6B). ExE-specific hypo- or hypermethylated CpGs segregate into distinct genomic compartments by CpG density and location, with de novo methylation preferentially enriched for CpG islands (CGIs) near transcription start sites (TSSs) and 5′ exons (FIG.1C,FIGS.6C-6F). Once established, these two alternative landscapes are largely preserved across embryonic tissues or in the midgestation placenta, respectively11,12(FIG.6G).

Notably, ExE-methylated CGIs (ExE hyper CGIs) frequently overlap with Polycomb repressive complex 2 (PRC2)-regulated genes, including master transcription factors that direct germ-layer and body-axis formation (FIGS.7A-7B). Although the majority of targeted genes are not yet expressed in the epiblast, ExE-specific promoter methylation is generally associated with repression, including of many pluripotency-specific regulators, as well as concurrent loss of chromatin accessibility (FIGS.7-8). Moreover, the global relationship between promoter methylation and gene repression is more pronounced in the ExE than in the epiblast (FIG.8C). DNA methylation surrounding these promoters is largely dispersive, with flanking regions less methylated in the ExE than in the epiblast, but with a maximal increase specifically at the TSS (FIGS.1D-1E). ExE hyper CGIs only reach methylation levels of ˜0.25, but methylated CpGs are distributed across 80% of the sequencing reads that fall within them and have a median per-read methylation status that matches the unphased measurement (FIG.4D). The consistency between per-molecule and aggregate methylation is most likely to be explained by population-wide recruitment of de novo methyltransferases, followed by stochastic gains at individual CpGs in phase, similar to a variety of cancer systems13,14. Importantly, the higher CpG density of ExE-targeted regions leads invariably to a higher local methylation density, even though the per-CpG methylation status is intermediate (FIG.1E).

Suppression overlaps with WNT pathway effectors that are induced in the proximal epiblast to promote primitive streak formation (FIG.2A). However, the ExE expresses alternative WNT proteins, suppresses fibroblast growth factor (Fe promoters by de novo methylation, and specifically expresses receptors for epiblast-secreted factors (FIG.2B,FIG.9A). The extraembryonic landscape may proceed deterministically from these two major signaling pathways, which are used promiscuously in many downstream developmental processes and frequently misregulated in cancers. To investigate this hypothesis, the ICM was selected as a model because it is indistinguishably hypomethylated from the trophectoderm and can be cultured independently of FGFs, whereas extraembryonic development rapidly attenuates15. ICMs were cultured in four conditions using combinations of FGF4, the mitogen-activated protein kinase kinase (MAPKK or MEK) inhibitor PD0325901, and the GSK3β inhibitor, WNT agonist CHIR99021 (CHIR) (FIG.2C,FIG.9B). Isolated outgrowths were dually assayed by a combined RNA-seq and reduced representation bisulfite sequencing (RRBS) approach (FIG.10, Methods). Those cultured in FGF4 plus CHIR progressively diverged into two separate, morphologically distinguishable interior and exterior tissues that were independently isolated.

In combination, PD0325901 and CHIR comprise the ‘2i’ condition, an FGF-impeded, WNT-activated state that maintains preimplantation like global hypomethylation16. Alternatively, exogenous FGF is sufficient to drive genome and CGI methylation to higher than physiological levels (FIG.2D). Surprisingly, when coupled with FGF, WNT agonism effectively blocks genome remethylation but redirects CGI-level methylation to a greater subset of extraembryonic targets (FIGS.2E-2F). CGI-targeting is specific to the FGF plus CHIR outgrowth exterior, which establishes an asymmetric Fgfr2 and Fgf4 expression pattern with the interior, similar to what occurs in vivo. The specific overlap between in vitro and ExE-methylated CGI promoters appears to reflect progressive restriction of potential targets over early development: those shared across conditions have early developmental functions and are often expressed in the ICM and the 2i condition such as Prdm14; those methylated in the ExE and in FGF plus CHIR, but not in FGF alone, generally encompass neuroectodermal regulators such as Otx2 and Pax6; and ExE-exclusive targets are often endodermal and induced by dual FGF and WNT activity such as FoxA2 and Sox17 (FIG.9C). Seemingly, ExE-like global hypomethylation and CGI methylation can be recapitulated in vitro by WNT and FGF, but target specificity can be modulated to include multiple discrete developmental programmes.

The configuration of epigenetic regulators that specifically execute this transition was next investigated. Whereas Dnmt1 and Dnmt3b are expressed in both tissues, Dnmt3l and Dnmt3a isoform 2 are reciprocally expressed in either the ExE or the epiblast and regulated by de novo promoter methylation in the other (FIGS.11A-11D). A truncated, non-catalytic isoform of the histone 3 lysine 36 (H3K36) demethylase Kdm2b is expressed during preimplantation and within the ExE, whereas a longer Jumonji demethylase domain containing isoform is specifically induced in the epiblast″ (FIG.11E). Otherwise, epigenetic regulator expression appears relatively stable between the two tissues at this time, such that their specific integration could explain the assembly of such profoundly different landscapes. To compare their capacity to direct both global and CGI methylation, we acutely disrupted Dnmt1, Dnmt3a, Dnmt3b, and Dnmt3l, the essential PRC2 component Eed, and Kdm2b by zygotic CRISPR-Cas9 injection (Methods). We found that Dnmt1, Dnmt3b, and Dnmt3l ablation substantially disrupt the ExE methylome, including at CGI targets, but show no obvious specificity for these regions or corresponding changes in expression (FIGS.3A-3B,FIGS.11F-11H). The near complete loss of methylation in Dnmt1-null ExE compared to sample-matched epiblast indicates diminished de novo activity, and greater reliance on epigenetic maintenance, despite prolonged Dnmt3l expression (FIGS.3A-3B). Alternatively, Eed-null ExE disrupts CGI methylation without affecting global levels, suggesting that PRC2 may specifically coordinate repression upstream of DNMT3B as part of a novel developmental pathway (FIGS.3B-3D,FIGS.11F-11G). Consistently, Eed-null ExE fails to suppress associated genes, which are induced to similar levels to those of the sample-matched epiblast (FIG.11H).

The data indicates a point in early development at which sensitivity to promiscuously used growth factors instructs a distinct epigenome that is not observed during downstream ontogeny. However, de novo CGI methylation is also a general feature of tissue culture, cancer cell lines, and primary tumours, indicating a latent vulnerability in somatic cells5,18(FIG.4,FIGS.12-13). To investigate a possible link with the subsequent re-emergence of this landscape in cancer, orthologous CGIs were mapped to compare patient-matched DNA methylation profiles from The Cancer Genome Atlas (TCGA) project, an age-matched chronic lymphocytic leukaemia (CLL) cohort, as well as data from the Encyclopedia of DNA Elements (ENCODE) and the Roadmap Epigenomics Project14,19-21. Of the 16 cancer types with sufficient normal biopsied samples, 15 significantly methylate ExE hyper CGIs (FIGS.4A-4B). The signal is surprisingly robust and segregates cancer and normal tissue when measured as a feature across patients or when examining CGI-level changes (FIG.4B,FIG.12). 84% of ExE hyper CGIs are methylated in at least one cancer type, and they are more frequently shared as conserved, pan-cancer targets (FIGS.4C-4D,FIGS.14A-14B). Some direct and indirect evidence was found that CGI methylation can be influenced by FGF sensing. For example, matched mutational and methylation analyses of the entire TCGA dataset (n=10,629 cancers) show a 19.3% increase in the average methylation of ExE hyper CGIs when any FGF pathway member is mutated (from 0.275 to 0.328,FIG.14C). Similarly, statistical assessment of the connectivity between the ExE hyper CGIs and the 10 most mutated pathways in cancer reveals a notable enrichment for FGFR signaling in disease (enrichment z-score=3.88,FIGS.14D-14E). Over the more expansive, but less internally controlled, ENCODE and Roadmap data, cancers and immortalized cell lines are clearly separated from primary tissues by their ExE hyper CGI methylation status (FIG.4E). Notably, mature adaptive immune cells and endodermal lineages are generally more susceptible to low-level methylation within these regions, suggesting a pre-existing heterogeneity even in normal populations.

The developmental acquisition of an epigenetic landscape that partitions extraembryonic tissues within the embryo human cancers is presented. This landscape co-occurs with the establishment of the first major signaling axes, can be partially directed from the hypomethylated ICM in vitro, and appears to be determined by disparate regulation of the DNMTs and associated cofactors. Notably, de novo methylation of CGIs in the ExE requires PRC2, which indicates a transient, biochemical interaction with DNMT3B or an upstream role in either determining the ExE state or priming CGIs for suppression. The coordination of this alternative, and presumably more permanent, repressive mechanism warrants further investigation and shares features with the somatic transition to cancer. Most obviously, FGF sensing passes through RAS/MAPK/ERK signaling, which has extensive oncogenic potential and putative roles in the establishment of the cancer methylome22-24. Similarly, the ExE displays attenuated de novo methylation activity directed wholly by DNMT3B, broadly resembling the high frequency of somatic DNMT3A mutations in acute myeloid leukemia and myelodysplastic syndrome or DNMT3B-directed CGI methylation during colorectal transformation25-28. Transgenic mouse cancer models confirm conserved ExE hyper CGI methylation in similar contexts (FIG.14F). The extraembryonic landscape depends on extrinsic cues with numerous downstream developmental functions, which may provide a latent opportunity for spontaneous state transition without genetic perturbation in later development. If so, the likelihood of such a transition may relate to how closely a given regulatory network resembles the one governing extraembryonic specification. Whether or not additional morphological and molecular features of placental development that appear analogous to cancer hallmarks29,30—such as immunosuppression, tissue invasion, and angiogenesis—proceed as part or downstream of this primary epigenetic switch remains unexplored, but would provide a parsimonious developmental foundation for their systematic emergence during transformation.

Additional details and supplemental information are provided in Smith et al., “Epigentic restriction of extraembryonic lineages mirrors the somatic transition to cancer”Nature549, 543-547 (28 Sep. 2017), incorporated herein by reference in its entirety, including the extended data and supplementary information.

Example 2: Haplotype-Level Methylation Significantly Reduces Background Noise in Normal Cells

It has recently been demonstrated that disordered methylation is frequently observed in cancer. This is one of the reasons why single CpG-based diagnosis has low sensitivity because methylation may occur in nearby CpG sites. For example, the overall sensitivity of single CpG-based diagnosis is only 60% for SEPT9 in colorectal cancer. Moreover, diagnosis of early cancers (with <0.1% ctDNA) requires nearly zero background. However, normal cells acquire low-level methylation (˜1%) due to stochastic processes when measured at single CpG sites.

It was found that DNA methylation haplotypes provide a better choice for diagnosis purposes. Here, a haplotype refers to a combination of CpG sites found on the same chromosome. Similarly, a DNA methylation haplotype represents the DNA methylation status of CpG sites on the same chromosome. In bulk bisulfate sequencing, DNA methylation status of thousands/millions of cells was measured. Though fragments of DNA were sequenced, every single fragment is guaranteed to come from a single chromosome and a single cell. Thus, the methylation pattern of CpGs on each fragment represents a single DNA methylation haplotype.

For example, as shown inFIG.15A, for a locus with 4 CpG sites, if it is determined that 14 reads cover this locus, then it was stated that 14 DNA methylation haplotypes were observed. Traditionally, only the aggregated methylation in these 4 sites is measured as a fraction of methylated CpGs divided by all CpGs measured (0.57, 0.43, 0.43 and 0.5 in this example) (FIG.15A). In contrast, haplotypes were checked instead. Specifically, these haplotypes were classified into three groups, concordantly un-methylated haplotypes, disordered haplotypes and concordantly methylated haplotypes. Since next generation sequencing is usually applied for DNA methylation, sequencing reads and haplotypes were utilized interchangeably.

To normalize the total sequencing depth, the proportion of concordantly unmethylated reads (PUR), proportion of disordered reads (PDR) and proportion of concordantly methylated reads (PMR) was calculated. So PUR, PDR and PMR always range from 0 to 1, and PUR+PDR+PMR=1. Currently, PUR/PDR/PMR is calculated for regions with at least 4 CpG sites and covers at least 20×.

PUR/PDR/PMR can also be calculated with other parameter settings too. For example, with advances in sequencing technologies, longer reads that cover more CpGs may be sequenced. Alternatively, utilizing current technology, in CpG dense regions (CpG Islands, promoters, enhancers), a shorter read may cover more CpG sites. It also can be used on sequencing reads generated by all sequencing platforms, including Sanger sequencing, next generation sequencing and single-molecular sequencing.

Cancer-like tissues (ExE) and normal-like tissues (Epi) were checked, and it was found that fully methylated reads/haplotypes are very rare in normal cells and thus significantly reduce background noise (FIG.15C). In contrast, notable background methylation is observed when current method mean methylation is used (FIG.15B).

Example 3: Detection of Low Frequency Tumors (0.01%) from cfDNA Through Simulation

It has been shown that in early stages of cancer, ctDNA only represents 0.01% to 1% of cfDNA from plasma. This is challenging in view of traditional methods of methylation analysis. Established herein is a novel way to predict ctDNA from cfDNA with a resolution as high as 0.01%, in which five copies of tumor DNA are present (FIG.16). According to the simulations, the presence of 0.01% ctDNA can be predicted with 100% sensitivity and 95% specificity, with a p-value cutoff of 10−4.

A simulation was performed by mixing sequencing reads from tumor-like tissues (ExE), with fractions ranging from 1%, 0.1% and 0.01%, with reads from normal-like tissues (Epi). (Note: only reads that locate in a CpG Island and that distinguish tumor and normal were sampled (see Example 1)). 0.01% of tumor DNA mimic the fraction of circulating tumor DNA (ctDNA) among cell free DNA in early cancer patients. Random dropout of tumor reads in simulation mimiced the experimental dropout during sample preparation or sequencing. In every simulated sample (mixture of tumor-like reads and norm-like reads), PMR was calculated for each CpG Island, and then compared to background (pure normal-like tissue). If the signal was significantly higher in the simulated sample (by rank sum test), it was concluded that this sample contained tumor DNA.

Methods

Data Reporting

No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.

Sample Isolation and Library Preparation

Preparation of preimplantation and post implantation samples was performed as described in ref 31. In brief, B6D2F1 hybrid females between 5 and 8 weeks old (Charles River) were serially primed with 5 IU pregnant mare gonadotropin (Sigma) followed by 5 IU human chorionic gonadotropin (Millipore) after 46 h, and subsequently mated with B6D2F1 male mice ≤6 months old. For preimplantation time points, zygotes from mated females were isolated from the oviduct the following morning (E0.5) and cultured in KSOM media (Millipore) droplets under mineral oil until E2.25. The 8-cell sample was collected by careful monitoring of 4-cell embryos from ˜E2 onwards, and emergent 8-cell embryos were swapped into KSOM supplemented with 1 μg ml−1aphidicolin (Sigma) to ensure synchronization and minimal entry into the fourth replication cycle. 8-cell embryos were collected within 4 h of the first apparent embryo of this stage. Prior to collection, embryos were serially transferred through Acidic Tyrode's solution (Sigma) to remove the zona pellucida and carefully pipetted with a drawn glass capillary through 0.25% Trypsin-EDTA (Life Technologies) to remove maternal polar bodies. E3.5 blastocysts were also treated with Acidic Tyrode's solution to remove the zona, and the ICM and trophectoderm of matched samples were dissected using standard micromanipulation equipment (Eppendorf) and a Hamilton Thorne XYClone laser with 300 μs pulsing at 100% intensity. Isolation of post implantation tissues was performed as described32. The deciduae of mated female mice were isolated on the morning of E6.5 and the conceptuses removed. Then, under a stereomicroscope, the embryo was carefully bisected along the extraembryonic-embryonic axis, removing the ectoplacental cone from the extraembryonic ectoderm when apparent. After separation, the epiblast and the ExE were incubated for 15 min at 4° C. in 0.5% trypsin, 2.5% pancreatin dissolved in PBS and allowed to rest for 5-10 min in KSOM at room temperature. Finally, the visceral endoderm was removed by drawing the embryo through a narrow, flame-drawn glass capillary and only samples with no apparent contamination were collected. On average, matched ExE and epiblast or ICM and trophectoderm samples from 5-10 embryos or from 20 or more 8-cell embryos were collected per assay.

DNA for whole-genome bisulfite sequencing was isolated as described previously33, and libraries were prepared using the Accel-NGS Bisulfite DNA library kit (Swift Biosciences) according to the manufacturer's protocol. Final libraries were generated from 10-12 PCR cycles. RNA was purified using the RNeasy Micro Kit (Qiagen) and RNA-seq libraries were generated using the SMRTseq v4 Ultra Low Input Kit (Clontech) according to the manufacturer's protocol with 10-11 long-distance PCR cycles. Libraries were generated from 150 pg of the subsequent cDNA using the Nextera XT DNA library preparation kit (Illumina) and 12 PCR cycles. ATAC-seq libraries were generated as described previously34using a 10 μl reaction and incubation with the TN5 transposase mixture (Nextera DNA library preparation kit, Illumina) for 45 min. The reaction was stopped according to the protocol described previously35and purified using silane beads (Thermo Fisher). Tagmented DNA was amplified for 12-14 cycles to generate the library. WGBS libraries were sequenced as a pool using the HiSeq X Ten platform (Illumina), and RNA-seq and ATAC-seq data were sequenced using the HiSeq 2500 (Illumina).

Outgrowth Experiments

To generate controlled outgrowth data, ICM were immunosurgically isolated from BDF1×129S1/SvImJ strain blastocysts at 96 h post fertilization as described31. In brief, oocytes were isolated by hormone priming from B6D2F1 females 12-14 h after administration of human chorionic gonadotropin and fertilized by intracytoplasmic sperm injection using piezo-actuated injection of 129S1/SvImJ strain sperm36. At 96 h post-fertilization, blastocysts were stripped of their zona pellucida by brief incubation in Acidic Tyrode's solution and incubated for 30 min in 1:10 diluted whole mouse antisera (Sigma) in CO2-equilibrated KSOM, followed by destruction of the trophectoderm by culture in 1:10 diluted guinea pig complement sera (Sigma). After 15 min at 37° C., the ICM separates from the complement-lysed trophectoderm and could be cleanly isolated by brief pulsing through a narrow glass capillary. ICM were isolated in batches of ˜12 per drop. Once isolated, ICM were then plated into basal N2/B27 media supplemented with 1,000 U ml−1leukaemia inhibitory factor (made in house) and one of the following conditions; ‘2i’ supplemented with 1 μM PD0325901 and 3 μM CHIR99021 (Reagents Direct)37; ‘PD0325901’ supplemented with 1 μM PD0325901 and 10 ng ml−1BMP4 to promote outgrowth expansion (Peprotech)38; ‘FGF plus CHIR’ supplemented with 25 ng ml−1mouse recombinant FGF4 (R&D systems) and 3 μM CHIR99021; and ‘FGF’ supplemented with 25 ng ml−1FGF4 only. FGF4 was selected because it is the most highly expressed FGF family member in the preimplantation embryo and we sought to direct specific remethylation changes as is observed in vivo. ICM were placed into gelatin-treated tissue culture dishes plated with irradiated CF-1 strain embryonic fibroblasts to promote attachment. The primary outgrowth from the ICM, characterized as a centrally expanding, three-dimensional mass, was isolated after four days of culture. In all cases but the 2i condition, an outer layer of differentiated cells was apparent and removed using an identical strategy to that of removal of the visceral endoderm from E6.5 samples described above. However, under the FGF plus CHIR condition, the ‘outer layer’ was often of the same size or larger than the internal outgrowth, and only became defined during the latter portion of culture (seeFIG.9B). As such, we collected both interior and exterior portions as they could clearly be distinguished as mutually ICM-derived. After incubation and either isolation or removal of external cells, outgrowths were serially washed through several KSOM drops under mineral oil before being snap-frozen in minimal volume for RNA-seq and RRBS profiling.

Generation of Knockout Embryos by Zygotic CRISPR-Cas9 Injection

Zygotic injection was performed essentially as described39. To improve the efficiency with which null alleles were generated, three separate single-guide RNA (sgRNA) sequences were designed per target, prioritizing highly scored protospacer sequences with no high scoring off-target sites using the CHOPCHOP web tool40and as 5′ as possible given these constraints to disrupt the coding frame Protospacer sequences were input into the following oligonucleotide primer pair and used to amplify off of the pX300 plasmid (Addgene): forward primer, AGTCAGTTAATACGACTCACTATAGN19GTTTTAGAGCTAGAAATAGCAAG (SEQ ID NO: 1); reverse primer, AAAAAAAGCACCGACTCGGTGCCAC (SEQ ID NO: 2). Protospacer sequences that did not begin with a G to initiate T7 transcription were inserted and an additional 5′ G was added. 200 ng of gel-purified, T7 promoter-containing sgRNA templates were used to generate sgRNAs by in vitro transcription using the MEGAshortscript T7 transcription kit (Thermo Fisher), followed by purification with phenol:chloroform and ethanol precipitation. Translation-competent spCas9 RNA was in vitro transcribed from a similarly designed, T7 promoter-driven template using the mMESSAGE mMACHINE T7 Ultra kit (Thermo Fisher) and purified using the RNA Clean and Concentrator Kit (Zymo Research). RNA was resuspended in an injection buffer comprising 5 mM Tris-HCl and 0.1 mM EDTA at pH 7.4. Zygotes were isolated from hormone-primed B6D2F1 females mated with B6D2F1 males as described above. Shortly after the formation of visible pronuclei (pronuclear stage 3), zygotes were cytoplasmically injected with 100 ng μl−1of all three targeted sgRNAs pooled 1:1:1 and 200 ng−1Cas9 mRNA. At E3.5, cavitated blastocysts were transferred in clutches of 10-15 into one uterine horn of pseudopregnant CD-1 strain mice (Charles River) that had been mated with vasectomized male Swiss-Weber strain mice (Taconic) two days previously. To account for the ˜1 day offset in developmental progression that results from uterine transfer, appropriately E6.5 stage conceptuses were isolated 4 days after uterine transfer and epiblast and extraembryonic ectoderm tissue were isolated as described above before snap-freezing in minimal volume. Each replicate consisted of at least 4 embryos and all experimental series include replicates generated from at least 2 rounds of zygotic injection. Care was taken to ensure epiblast and extraembryonic ectoderm tissue from matched embryos were included for each replicate set, and RRBS data in which both fractions did not cover >1 million CpGs at ≥5× coverage each were excluded from further analysis. Disruption of the target allele was confirmed by PCR amplification from the primary cDNA using primers that flank all three protospacer sequences to capture multiple simultaneous perturbations or truncations in phase.

Dual RNA-Seq and RRBS Profiling

Genomic DNA and mRNA purifications from low input samples were performed as described previously with modifications41. In brief, the cells were mixed with 15 μl of RLT plus buffer (Qiagen) containing 1 U μl−1of SUPERase. In RNase inhibitor (ThermoFisher), 1% β-mercaptoethanol (Sigma), and were then transferred to 1 well in a 96-well DNA LoBind plate (Eppendorf). After adding 10 μl of M-280 streptavidin bead-conjugated reverse transcription primer to each sample, the reaction was incubated at 72° C. for 3 min in a thermocycler followed by incubation at room temperature for 25 min with gentle rotation. The genomic DNA and mRNA were separated in a DynaMag-96 Side Magnet (Thermo Fisher). The bead-tagged mRNA was subjected to reverse transcription as described previously41and the genomic DNA in the supernatant was transferred to a fresh 96-well DNA LoBind plate. After reverse transcription, the cDNA was PCR amplified and the RNA-seq library was generated according to the Smart-seq2 protocol42. Indexed RNA-seq libraries were pooled and sequenced in an Illumina Hiseq2500 sequencer.

Genomic DNA was isolated using 1× Agencourt AMPure beads (Beckman Coulter) and was eluted with 15 μl of low Tris-EDTA buffer. The RRBS library was generated as reported previously with modifications43. We used the CutSmart buffer (New England Biolabs) for all three enzymatic reactions including MspI digestion, end-repair/A-tailing and T4 DNA ligation. To minimize DNA loss, the DNA purification step was eliminated after each enzymatic reaction. In brief, the genomic DNA was digested by 16 units of MspI (New England Biolabs) for 80 min at 37° C., and followed by heat inactivation at 65° C. for 15 min. The digested DNA fragments were end-repaired and A-tailed by adding 4 units of Klenow fragment (3′→5′ exo-) (New England Biolabs), 0.03 mM dCTP, 0.03 mM dGTP and 0.3 mM dATP; the reaction was carried out at 30° C. for 25 min and 37° C. for 25 min, followed by incubation at 70° C. for 10 min to inactive the enzyme. We then ligated the A-tailed DNA fragments with indexed adapters overnight at 16° C., by adding 2,000 U of T4 DNA ligase, 0.75 mM ATP and 7 nM of the adapters. The T4 ligase was heat-inactivated at 65° C. for 15 min before pooling libraries together. To remove adaptor dimers, the library pool was cleaned up using 1.8× AMPure beads and the adaptor-tagged DNA fragments were eluted to 30 μl of low Tris-EDTA buffer. The bisulfite conversion of the adaptor-tagged DNA fragments was conducted using a Qiagen EpiTect Fast Bisulfite Conversion Kit following the manufacturer's instructions with a minor modification. We extended the bisulfite conversion time from 2 cycles of 10 min to 2 cycles of 20 min to achieve bisulfite conversion rates >99%. The bisulfite-converted DNA fragments were PCR amplified according to the following thermocycler settings: 98° C. for 45 s, 6 cycles of 98° C. for 20 s, 58° C. for 30 s, 72° C. for 1 min, and then 8-10 cycles of 98° C. for 20 s, 65° C. for 30 s, 72° C. for 1 min, followed by a final extension cycle of 5 min at 72° C. The PCR-amplified library DNA was cleaned up using 1.3× AMPure beads and the RRBS libraries were paired-end sequenced for 2×100 cycles. Only instances in which the matched pool of Epiblast and ExE from a given replicate both had >1 million CpGs covered at ≥5× were included for downstream analysis.

For each sample, 10 μl of M-280 streptavidin beads (Thermo Fisher) were prepared as per the manufacturer's recommendations. Specifically, after washing with Solution A (0.1 M NaOH, 0.05 M NaCl) and B (0.1 M NaCl) sequentially, the beads were resuspended in 10 μl of 2× binding and washing buffer (10 mM Tris-HCl, 1 mM EDTA, 2 M NaCl) and then mixed with an equal volume of 2 μM of reverse transcription primer41. The mixture was incubated for 15 min at room temperature with gentle rotation. The bead-bound reverse transcription primer was collected using a magnet and was subsequently resuspended in 10 μl of binding buffer (10 mM Tris-HCl (pH 8.0), 167 mM NaCl, 0.05% Tween-20).

Estimating Methylation Levels

The methylation level of each sampled cytosine was estimated as the number of reads reporting a C, divided by the total number of reads reporting a C or T. Single CpG methylation levels were limited to those CpGs that had at least fivefold coverage. For 100 bp tiles, reads for all the CpGs that were covered more than fivefold within the tile were pooled and used to estimate the methylation level as described for single CpGs. The CpG density for a given single CpG is the number of CpGs 50 bp up- and downstream of that CpG. The CpG density for a 100 bp tile is the number of CpGs in the tile. The methylation level reported for a sample is the average methylation by pooling all reads across replicates.

Genomic Features

LINE, LTR and SINE annotations were downloaded from the UCSC (University of California, Santa Cruz) browser (mm9) RepeatMasker tracks. CGI annotations were downloaded from the UCSC browser (mm9) CpG Islands track. Gene annotations (exon, 5′ exon, intron) were downloaded from the UCSC browser (mm9) RefSeq track. Promoters (TSSs) are defined as ±2 kb of the RefSeq annotation. Corresponding human annotations were downloaded from the UCSC browser for hg19. In each case, the methylation level of an individual feature is estimated by averaging methylation for all CpGs within the feature that are covered greater than fivefold. Assignment of CGIs to a given TSS (CGI promoters) included annotated CGIs that fell within this boundary. Methylation was estimated for ‘core TSS’ sequences defined as ±1 kb of the RefSeq annotation and only included CpGs measured at ≥5× in both samples (WGBS) or pooled samples (RRBS). ForFIG.2B,FIG.7F, andFIG.9C, promoters for all isoforms are included and the maximally different alternative TSS was reported. The methylation levels of all annotated TSSs were calculated and reported in this manner, with the mean transcripts per million (TPM) estimate for the gene reported for all associated TSSs.

Identification of Differentially Methylated Loci and Regions

For WGBS data, identification of differentially methylated loci was performed using the DSS package, which uses biological replicates and information from CpG sites across the genome to stabilize the estimation of the dispersion parameters44. Only CpGs that were covered at least fivefold across all samples were considered for a given comparison. A false discovery rate (FDR) cut-off of 5% was used to identify differentially methylated CpGs. A CGI was called as differentially methylated if it was covered by at least 5 CpGs and 80% of them were significantly hyper/hypo methylated. For TCGA Illumina Infinium HumanMethylation450K BeadChip data, given that most cancer types have more than 20 cancer and normal samples, Wilcoxon rank-sum test was used to identify differentially methylated CpGs, with a FDR cut-off of 5%. All statistical tests throughout this study are two-sided. A CGI was called as differentially methylated if 80% of covered CpGs were significantly hyper/hypo methylated. For RRBS data, a simple cut-off of 10% difference in CGI-level methylation was used to call differential methylation.

Gene Expression Analysis

Alignment was performed using TopHat2 against mouse genome assembly mm9 with default settings. Isoform-level expression was quantified by kallisto, which performs pseudoalignment of reads against cDNA sequence of transcripts. Gene-level expression was estimated as the sum of expression of associated isoforms. Refseq mRNA sequences were downloaded from the UCSC genome browser. Expression levels were reported as transcripts per million (TPM).

Pathway Enrichment

Pathway enrichment was performed by a hypergeometric test using the GSEA online tool. The P value was adjusted for multiple hypothesis testing according to Benjamini and Hochberg, with 5% as a cut-off. Regulation by PRC2 in human ES cells taken from ref. 45.

Connectivity Analysis

We used GRAIL (gene relationships across implicated loci)46to test whether a query gene is functionally related to a set of seed genes. GRAIL uses text-mining to quantify the relatedness between two genes in the genome, by which a global gene network is built. It has been demonstrated that genes that function in the same pathway tend to distribute in a coherent subnetwork. In this study, we built a subnetwork using ExE hyper CGI-associated genes, which were significantly enriched in several pathways. To predict whether a query gene is functionally related to the ExE hyper subnetwork, we project this gene to the global network, and test whether connection of this gene to the subnetwork is random or statistically significant.

ATAC-Seq Data Processing

Reads were aligned to mouse genome mm9 using BWA with default parameters. Duplicates were removed by the function MarkDuplicates from the Picard tool kit. Reads with low mapping quality (<10) or in the mitochondrial chromosome were removed. NucleoATAC was used to generate insert density, which was normalized by the total number of insertions in each sample47.

Orthology Mapping Between Human and Mouse

Mouse mm9 CGIs were mapped to human hg19 segments using liftOver with chain file mm9ToHg19.over.chain. Then human orthologous CGIs were defined as the nearest CGIs to the mapped segments.

Data Availability

All datasets have been deposited in the Gene Expression Omnibus and are accessible under GSE84236. Additional data include: Roadmap and ENCODE samples from RnBeads Methylome Resource (rnbeads.mpiinf.mpg.de/methylomes.php), mouse adult tissues from GSE42836, and CLL and normal B lymphocytes from GSE58889.

TABLE 1Characterization of genomic location of the feature, the number ofcancer types as reported in the disclosure in which they are specificallymethylated, and one of two additional designations - their orthology toregions that are methylated during mouse development (Mouse ExEortholog) or their specific methylation in human placenta and at least8 of the 15 human cancer types examined.Human CGI (hg19)# CancersGroupchr1: 1181756-11824703Mouse ExE orthologchr1: 1470604-14714500Mouse ExE orthologchr1: 2772126-27726651Mouse ExE orthologchr1: 4713989-47165559Mouse ExE orthologchr1: 18436551-184376735Mouse ExE orthologchr1: 18956895-1895982912Mouse ExE orthologchr1: 18962842-189634817Mouse ExE orthologchr1: 18967251-189681190Mouse ExE orthologchr1: 19203874-192042341Mouse ExE orthologchr1: 21616380-216171010Mouse ExE orthologchr1: 25255527-252590055Mouse ExE orthologchr1: 29585897-295865981Mouse ExE orthologchr1: 34628783-346309767Mouse ExE orthologchr1: 39980365-3998176813Mouse ExE orthologchr1: 40235767-402371900Mouse ExE orthologchr1: 41831976-418325420Mouse ExE orthologchr1: 46951168-4695179212Mouse ExE orthologchr1: 47909712-4791102013Mouse ExE orthologchr1: 53742297-537428452Mouse ExE orthologchr1: 55505060-555060153Mouse ExE orthologchr1: 61515875-615168311Mouse ExE orthologchr1: 63782394-637904712Mouse ExE orthologchr1: 65731411-657318497Mouse ExE orthologchr1: 66258440-6625891812Mouse ExE orthologchr1: 77747314-777482247Mouse ExE orthologchr1: 91172102-9117277110Mouse ExE orthologchr1: 91176404-9117670114Mouse ExE orthologchr1: 92945907-929526091Mouse ExE orthologchr1: 115880167-1158813326Mouse ExE orthologchr1: 116380359-1163823643Mouse ExE orthologchr1: 156105707-1561061715Mouse ExE orthologchr1: 156338758-1563392510Mouse ExE orthologchr1: 156358050-15635825210Mouse ExE orthologchr1: 156390403-1563915811Mouse ExE orthologchr1: 160340604-16034084313Mouse ExE orthologchr1: 161695637-1616972980Mouse ExE orthologchr1: 177133392-17713384610Mouse ExE orthologchr1: 180198119-1802049750Mouse ExE orthologchr1: 197887088-1978877918Mouse ExE orthologchr1: 201252452-2012536483Mouse ExE orthologchr1: 202678881-2026797696Mouse ExE orthologchr1: 214156000-21415685110Mouse ExE orthologchr1: 214158726-2141590809Mouse ExE orthologchr1: 221057463-22105775711Mouse ExE orthologchr1: 221067447-22106818512Mouse ExE orthologchr1: 226075150-22607568012Mouse ExE orthologchr1: 248020330-2480212525Mouse ExE orthologchr10: 50602989-506067838Mouse ExE orthologchr10: 50817601-5082035611Mouse ExE orthologchr10: 71331926-713333927Mouse ExE orthologchr10: 88122924-881273645Mouse ExE orthologchr10: 94820026-9482325211Mouse ExE orthologchr10: 101279941-1012803826Mouse ExE orthologchr10: 101281181-1012821168Mouse ExE orthologchr10: 102419147-10241966811Mouse ExE orthologchr10: 102473206-1024740269Mouse ExE orthologchr10: 102484200-10248447611Mouse ExE orthologchr10: 102489343-1024910112Mouse ExE orthologchr10: 102507482-1025096464Mouse ExE orthologchr10: 102893660-10289505913Mouse ExE orthologchr10: 102896342-10289666513Mouse ExE orthologchr10: 102899822-10290026313Mouse ExE orthologchr10: 102975969-1029780964Mouse ExE orthologchr10: 105361784-1053621880Mouse ExE orthologchr10: 105420685-1054210766Mouse ExE orthologchr10: 106399567-10640281213Mouse ExE orthologchr10: 118899247-11890032914Mouse ExE orthologchr10: 119000435-1190015307Mouse ExE orthologchr10: 119311204-11931210410Mouse ExE orthologchr10: 119312766-1193135635Mouse ExE orthologchr10: 124905634-12490616112Mouse ExE orthologchr10: 124907283-12491103511Mouse ExE orthologchr10: 129534410-12953736610Mouse ExE orthologchr11: 725596-7268706Mouse ExE orthologchr11: 8190226-819067110Mouse ExE orthologchr11: 17740789-177437797Mouse ExE orthologchr11: 20181200-2018232514Mouse ExE orthologchr11: 20622720-2062339913Mouse ExE orthologchr11: 31825743-3182696714Mouse ExE orthologchr11: 31839363-3183981314Mouse ExE orthologchr11: 31848487-3184877614Mouse ExE orthologchr11: 32452144-3245270813Mouse ExE orthologchr11: 32454874-324573119Mouse ExE orthologchr11: 36397926-363993980Mouse ExE orthologchr11: 44327240-443279322Mouse ExE orthologchr11: 46299544-463002160Mouse ExE orthologchr11: 46366876-463671013Mouse ExE orthologchr11: 64136814-641381870Mouse ExE orthologchr11: 65352231-6535313413Mouse ExE orthologchr11: 69517840-695199290Mouse ExE orthologchr11: 69831571-698324844Mouse ExE orthologchr11: 70672834-706730558Mouse ExE orthologchr11: 72532612-725337742Mouse ExE orthologchr11: 79148358-791522004Mouse ExE orthologchr11: 124629723-1246299266Mouse ExE orthologchr12: 3475010-34756543Mouse ExE orthologchr12: 5018585-50211719Mouse ExE orthologchr12: 6438272-64389312Mouse ExE orthologchr12: 15475318-154759018Mouse ExE orthologchr12: 29302034-293029542Mouse ExE orthologchr12: 45444202-454453868Mouse ExE orthologchr12: 49183049-491832820Mouse ExE orthologchr12: 49371690-493755501Mouse ExE orthologchr12: 49484920-494851787Mouse ExE orthologchr12: 53491572-534919550Mouse ExE orthologchr12: 54338761-5433916811Mouse ExE orthologchr12: 54366815-543691032Mouse ExE orthologchr12: 54378696-543801021Mouse ExE orthologchr12: 54423427-5442371211Mouse ExE orthologchr12: 54440642-544415439Mouse ExE orthologchr12: 54447744-5444809110Mouse ExE orthologchr12: 54519768-545204572Mouse ExE orthologchr12: 57618769-576194024Mouse ExE orthologchr12: 58003880-580042497Mouse ExE orthologchr12: 58158855-581600000Mouse ExE orthologchr12: 63543636-6354496712Mouse ExE orthologchr12: 75602991-7560334413Mouse ExE orthologchr12: 99139386-9913976910Mouse ExE orthologchr12: 101109863-10111162211Mouse ExE orthologchr12: 106979429-10698108612Mouse ExE orthologchr12: 113590806-1135913040Mouse ExE orthologchr12: 113900750-1139064425Mouse ExE orthologchr12: 113908887-1139106819Mouse ExE orthologchr12: 113913615-11391432213Mouse ExE orthologchr12: 114878143-11487915512Mouse ExE orthologchr12: 114886354-11488657911Mouse ExE orthologchr12: 115109503-1151100617Mouse ExE orthologchr12: 117798076-1177994489Mouse ExE orthologchr12: 120835586-12083592711Mouse ExE orthologchr12: 122016170-1220176934Mouse ExE orthologchr12: 130387609-13038913913Mouse ExE orthologchr12: 130908777-1309091910Mouse ExE orthologchr13: 27334226-273352058Mouse ExE orthologchr13: 28498226-2849904614Mouse ExE orthologchr13: 36049570-360501598Mouse ExE orthologchr13: 36052553-360531197Mouse ExE orthologchr13: 79182859-791838802Mouse ExE orthologchr13: 84453664-8445389714Mouse ExE orthologchr13: 108518334-1085186339Mouse ExE orthologchr13: 109147798-10914901913Mouse ExE orthologchr14: 36974548-369754259Mouse ExE orthologchr14: 36986362-369905764Mouse ExE orthologchr14: 37049333-370517266Mouse ExE orthologchr14: 37116188-371176281Mouse ExE orthologchr14: 38678245-386809377Mouse ExE orthologchr14: 54418677-544188819Mouse ExE orthologchr14: 57274607-5727684013Mouse ExE orthologchr14: 57283967-5728455811Mouse ExE orthologchr14: 69256676-692570365Mouse ExE orthologchr14: 74706188-7470819211Mouse ExE orthologchr14: 95237622-9523821113Mouse ExE orthologchr14: 105167663-1051681290Mouse ExE orthologchr15: 33009530-330116967Mouse ExE orthologchr15: 40268581-402690616Mouse ExE orthologchr15: 45408573-454095287Mouse ExE orthologchr15: 47476369-4747749910Mouse ExE orthologchr15: 49254984-492555641Mouse ExE orthologchr15: 60287107-602876638Mouse ExE orthologchr15: 60296135-602985205Mouse ExE orthologchr15: 67073306-670739430Mouse ExE orthologchr15: 74419870-744230443Mouse ExE orthologchr15: 79724099-797256436Mouse ExE orthologchr15: 89914363-8991506112Mouse ExE orthologchr15: 89920793-899227687Mouse ExE orthologchr15: 89949373-8995113012Mouse ExE orthologchr15: 91642908-916437022Mouse ExE orthologchr15: 96873408-968777212Mouse ExE orthologchr16: 2228190-22309460Mouse ExE orthologchr16: 3013016-30132283Mouse ExE orthologchr16: 3190765-31913890Mouse ExE orthologchr16: 22824616-2282645912Mouse ExE orthologchr16: 48844551-4884526413Mouse ExE orthologchr16: 49311413-4931230811Mouse ExE orthologchr16: 49314037-493165439Mouse ExE orthologchr16: 49872449-4987292611Mouse ExE orthologchr16: 51147490-511479448Mouse ExE orthologchr16: 51168266-5116911013Mouse ExE orthologchr16: 54970301-549728467Mouse ExE orthologchr16: 55513220-555135267Mouse ExE orthologchr16: 58030214-580316330Mouse ExE orthologchr16: 62069121-6207063411Mouse ExE orthologchr16: 67208067-672086780Mouse ExE orthologchr16: 67571252-675727281Mouse ExE orthologchr16: 68480864-684828221Mouse ExE orthologchr16: 86530747-865329947Mouse ExE orthologchr16: 86549069-865505126Mouse ExE orthologchr16: 86612188-866138218Mouse ExE orthologchr16: 88943427-889436690Mouse ExE orthologchr17: 12568667-125693353Mouse ExE orthologchr17: 14248391-142487210Mouse ExE orthologchr17: 32484007-3248428015Mouse ExE orthologchr17: 35291899-353008755Mouse ExE orthologchr17: 37764092-377643043Mouse ExE orthologchr17: 40937258-409374809Mouse ExE orthologchr17: 43472527-434743430Mouse ExE orthologchr17: 45949676-459498852Mouse ExE orthologchr17: 46607804-466083905Mouse ExE orthologchr17: 46620367-466213731Mouse ExE orthologchr17: 46631800-466322126Mouse ExE orthologchr17: 46669434-466698118Mouse ExE orthologchr17: 46691520-466920977Mouse ExE orthologchr17: 48194634-481950850Mouse ExE orthologchr17: 50235175-5023646612Mouse ExE orthologchr17: 59485573-5948578011Mouse ExE orthologchr17: 59528979-5953026614Mouse ExE orthologchr17: 70116274-701199981Mouse ExE orthologchr17: 70120139-701204427Mouse ExE orthologchr17: 72855621-728580120Mouse ExE orthologchr17: 72915568-729165104Mouse ExE orthologchr17: 74017769-740186585Mouse ExE orthologchr17: 77805866-778090460Mouse ExE orthologchr17: 79314962-793206530Mouse ExE orthologchr17: 79859808-798609631Mouse ExE orthologchr18: 19744936-197523631Mouse ExE orthologchr18: 30349690-303523022Mouse ExE orthologchr18: 35144907-351476286Mouse ExE orthologchr18: 55103154-551088536Mouse ExE orthologchr18: 55922987-559240681Mouse ExE orthologchr18: 59000683-5900169212Mouse ExE orthologchr18: 74153239-741550730Mouse ExE orthologchr18: 74961556-7496382213Mouse ExE orthologchr19: 407011-4095110Mouse ExE orthologchr19: 1063544-10642654Mouse ExE orthologchr19: 1108394-11096102Mouse ExE orthologchr19: 1748167-17502431Mouse ExE orthologchr19: 2424005-24279830Mouse ExE orthologchr19: 7933263-79348980Mouse ExE orthologchr19: 11594372-115949871Mouse ExE orthologchr19: 13135317-131361698Mouse ExE orthologchr19: 13198699-131989992Mouse ExE orthologchr19: 13213450-132138212Mouse ExE orthologchr19: 18979351-1898120013Mouse ExE orthologchr19: 19368708-193696810Mouse ExE orthologchr19: 30715549-307157530Mouse ExE orthologchr19: 35633409-356336971Mouse ExE orthologchr19: 36336275-363371382Mouse ExE orthologchr19: 36500169-365005301Mouse ExE orthologchr19: 38876070-388763320Mouse ExE orthologchr19: 42891311-428916460Mouse ExE orthologchr19: 45898879-459003154Mouse ExE orthologchr19: 48965002-489657922Mouse ExE orthologchr19: 50881418-508816643Mouse ExE orthologchr19: 50931270-509316383Mouse ExE orthologchr19: 51169659-511720239Mouse ExE orthologchr19: 55815940-558162770Mouse ExE orthologchr19: 56598038-566002962Mouse ExE orthologchr2: 3750828-37519278Mouse ExE orthologchr2: 30453566-304556551Mouse ExE orthologchr2: 38301276-383045180Mouse ExE orthologchr2: 45155195-4515704912Mouse ExE orthologchr2: 45395869-453981865Mouse ExE orthologchr2: 50574045-5057481710Mouse ExE orthologchr2: 66808568-6680940413Mouse ExE orthologchr2: 71787430-717878973Mouse ExE orthologchr2: 73143055-731482601Mouse ExE orthologchr2: 80529677-8053084611Mouse ExE orthologchr2: 102803672-1028045562Mouse ExE orthologchr2: 105459127-10546177012Mouse ExE orthologchr2: 105468851-1054734884Mouse ExE orthologchr2: 108602824-10860346711Mouse ExE orthologchr2: 119599458-11960096612Mouse ExE orthologchr2: 137522460-1375236968Mouse ExE orthologchr2: 142887724-1428885537Mouse ExE orthologchr2: 144694666-1446951803Mouse ExE orthologchr2: 157185557-1571863557Mouse ExE orthologchr2: 162273294-1622737258Mouse ExE orthologchr2: 176949511-1769497959Mouse ExE orthologchr2: 176964062-17696550912Mouse ExE orthologchr2: 176969217-1769698956Mouse ExE orthologchr2: 176977284-17697754013Mouse ExE orthologchr2: 176982107-17698240212Mouse ExE orthologchr2: 177036254-17703721314Mouse ExE orthologchr2: 177042751-1770434442Mouse ExE orthologchr2: 182321761-18232302910Mouse ExE orthologchr2: 182521221-1825219272Mouse ExE orthologchr2: 219736132-2197365928Mouse ExE orthologchr2: 219848919-2198505416Mouse ExE orthologchr2: 219857682-2198589171Mouse ExE orthologchr2: 220299483-22030024310Mouse ExE orthologchr2: 220412341-2204126780Mouse ExE orthologchr2: 223183013-2231854680Mouse ExE orthologchr2: 237071794-2370787627Mouse ExE orthologchr2: 241758141-2417607833Mouse ExE orthologchr20: 3145121-31457465Mouse ExE orthologchr20: 21485932-214967145Mouse ExE orthologchr20: 21686199-2168768912Mouse ExE orthologchr20: 22557517-225592408Mouse ExE orthologchr20: 33296514-332982420Mouse ExE orthologchr20: 37352130-3735737213Mouse ExE orthologchr20: 39994545-399958107Mouse ExE orthologchr20: 44657463-4465924312Mouse ExE orthologchr20: 44685771-446876109Mouse ExE orthologchr20: 51589707-515900204Mouse ExE orthologchr20: 52789252-527909863Mouse ExE orthologchr20: 57415135-574171535Mouse ExE orthologchr21: 31311386-3131210611Mouse ExE orthologchr21: 32624144-326243820Mouse ExE orthologchr21: 38065179-3806618510Mouse ExE orthologchr22: 19967279-199678080Mouse ExE orthologchr22: 29709281-297120130Mouse ExE orthologchr22: 31198491-311990331Mouse ExE orthologchr22: 31500396-3150123912Mouse ExE orthologchr22: 37212769-372134670Mouse ExE orthologchr22: 37911979-379122581Mouse ExE orthologchr22: 38476836-384788396Mouse ExE orthologchr22: 42305617-423072540Mouse ExE orthologchr22: 42322043-423229093Mouse ExE orthologchr22: 44726724-447275901Mouse ExE orthologchr22: 46318693-463190871Mouse ExE orthologchr22: 46440393-464410191Mouse ExE orthologchr3: 3840513-38427728Mouse ExE orthologchr3: 6902823-690351613Mouse ExE orthologchr3: 13114627-1311524511Mouse ExE orthologchr3: 19189688-191901006Mouse ExE orthologchr3: 49947621-499484302Mouse ExE orthologchr3: 55508336-555087084Mouse ExE orthologchr3: 62354291-6235501213Mouse ExE orthologchr3: 62357639-6235977411Mouse ExE orthologchr3: 71834068-718346534Mouse ExE orthologchr3: 87841796-878425636Mouse ExE orthologchr3: 137482964-1374844547Mouse ExE orthologchr3: 137489594-13749100410Mouse ExE orthologchr3: 147108511-14711170313Mouse ExE orthologchr3: 147113608-14711447915Mouse ExE orthologchr3: 147130342-14713057715Mouse ExE orthologchr3: 147131066-14713133314Mouse ExE orthologchr3: 154146347-15414696513Mouse ExE orthologchr3: 157821232-15782160415Mouse ExE orthologchr3: 170303044-17030324912Mouse ExE orthologchr3: 172165372-17216673814Mouse ExE orthologchr4: 4868440-48691739Mouse ExE orthologchr4: 25090106-2509051012Mouse ExE orthologchr4: 41749184-4174981113Mouse ExE orthologchr4: 47034427-470349407Mouse ExE orthologchr4: 54966163-549680633Mouse ExE orthologchr4: 81119095-8111939111Mouse ExE orthologchr4: 90228714-902290101Mouse ExE orthologchr4: 94755786-9475631013Mouse ExE orthologchr4: 100870377-1008719940Mouse ExE orthologchr4: 107956555-10795745311Mouse ExE orthologchr4: 109093038-1090945463Mouse ExE orthologchr4: 114900355-1149008102Mouse ExE orthologchr4: 122301567-12230229010Mouse ExE orthologchr4: 128544031-12854490310Mouse ExE orthologchr4: 144620822-1446222189Mouse ExE orthologchr4: 147559205-14756190114Mouse ExE orthologchr4: 156680095-1566813866Mouse ExE orthologchr4: 164264821-16426577210Mouse ExE orthologchr4: 172733734-17273511811Mouse ExE orthologchr4: 174430386-17443086114Mouse ExE orthologchr4: 185939222-1859427474Mouse ExE orthologchr5: 1879689-187992810Mouse ExE orthologchr5: 1881924-18877438Mouse ExE orthologchr5: 2748368-27570247Mouse ExE orthologchr5: 37834671-3783512812Mouse ExE orthologchr5: 38257825-382591364Mouse ExE 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