IDENTIFICATION OF GENOMIC STRUCTURAL VARIANTS USING LONG-READ SEQUENCING
20230028445 · 2023-01-26
Inventors
Cpc classification
C12N2310/20
CHEMISTRY; METALLURGY
C12Q1/6811
CHEMISTRY; METALLURGY
C12Q2537/159
CHEMISTRY; METALLURGY
C12Q2537/159
CHEMISTRY; METALLURGY
C12Q1/6806
CHEMISTRY; METALLURGY
C12Q1/6883
CHEMISTRY; METALLURGY
International classification
Abstract
Provided herein are systems and methods for detecting genomic structural variants using a non-application gene-editing sample preparation followed by long-read sequencing.
Claims
1. A method for identifying a set of guide RNAs (gRNAs) that are hybridizable to a genomic region of interest in a genome comprising designing a plurality of gRNAs, wherein: at least one gRNA is hybridizable to a target site within the genomic region of interest and is configured to produce a genomic variant that comprises at least 1000 bp; and said plurality of gRNAs comprises a plurality of CRISPR RNAs (crRNAs), wherein said plurality of crRNAs comprises a GC of at least about 40% to about 80%.
2. A method for identifying a set of guide RNAs (gRNAs) that are hybridizable to a genomic region of interest in a genome comprising designing a plurality of gRNAs, wherein: at least one gRNA is hybridizable to a target site within the genomic region of interest and is configured to produce a genomic variant that comprises at least 1000 bp; and said plurality of gRNAs comprises a plurality of CRISPR RNAs (crRNAs), wherein said plurality of crRNAs comprises a self-complementarity score of zero.
3. A method for identifying a set of guide RNAs (gRNAs) that are hybridizable to a genomic region of interest in a genome comprising designing a plurality of gRNAs, wherein: at least one gRNA is hybridizable to a target site within the genomic region of interest and is configured to produce a genomic variant that comprises at least 1000 bp; and said plurality of gRNAs comprises a plurality of CRISPR RNAs (crRNAs), wherein said plurality of crRNAs comprises an efficiency score of about 0.2.
4. A method for identifying a set of guide RNAs (gRNAs) that are hybridizable to a genomic region of interest in a genome comprising designing a plurality of gRNAs, wherein: at least one gRNA is hybridizable to a target site within the genomic region of interest and is configured to produce a genomic variant that comprises at least 1000 bp; and said plurality of gRNAs comprises a plurality of CRISPR RNAs (crRNAs), wherein said plurality of crRNAs comprises a mismatch profile of MM0=0 or MM0=1, MM1=0 or MM1=1, MM2=0 or MM2=2, and MM3<21.
5. The method of claim 4, wherein said plurality of crRNAs comprises a mismatch profile of MM3<5.
6. A method of detecting a genomic variant in a sample, the method comprising: (a) enriching said sample for a genomic region of interest comprising said genomic variant using a gene-editing based approach; and (b) sequencing said enriched sample comprising said genomic region of interest using long-read sequencing.
7. The method of any of claims 1-6, wherein said genomic variant comprises a structural variant.
8. The method of claim 5, wherein said genomic variant comprises at least 50 bp.
9. The method of claim 5, wherein said genomic variant comprises at least 1000 bp.
10. The method of any of claims 1-5, wherein said gene-editing based approach comprises use of a clustered regularly interspersed short palindromic repeats (CRISPR)-Cas system.
11. The method of claim 9, wherein said CRISPR-Cas system comprises Cas 9.
12. The method of claim 5, wherein step (a) of enriching said sample further comprises an amplification of said genomic region of interest.
13. The method of claim 5, wherein step (a) of enriching said sample does not require an amplification of said genomic region of interest.
14. The method of claim 5, wherein step (a) of enriching said sample further comprises coupling a sequence of dAMPs to said genomic variant.
15. The method of claim 5, wherein step (a) of enriching said sample further comprises coupling a plurality of barcode molecules to said genomic variant.
16. The method of claim 5, wherein step (a) of enriching said sample further comprises coupling said genomic variant to a magnetic bead.
17. The method of any of claims 1-5, wherein said long-read sequencing comprises nanopore sequencing.
18. The method of any of claims 1-5, wherein said long-read sequencing comprises single molecule, real-time (SMRT) sequencing.
19. The method of any of claims 1-5, wherein said CRISPR-Cas system further comprises a crRNA that is hybridizable to a sequence listed in Tables 1-117.
20. The method of any of claims 1-5, wherein said genomic region of interest comprises two or more repeat regions.
21. The method of any of claims 1-5, wherein said sample comprises at least 10 genomic regions of interest.
22. The method of any of claims 1-5, wherein said genomic variant is associated with a disorder and drug response (pharmacogenomics).
23. The method of claim 22, wherein said disorder is selected from the group consisting of acute lymphoblastic leukemia (ALL), alpha-thalassemia, ataxia-telangiectasia (AT), autosomal recessive deafness 16, autosomal recessive deafness 22, beta-thalassemia, breast cancer, Canavan disease, cancer, celiac disease, chronic myeloid leukemia (CML), cystic fibrosis, cystinosis, deafness infertility syndrome (DIS), Duchenne muscular dystrophy, Ehlers-Danlos syndrome type III and IV, Ellis-van Creveld syndrome, Fabry disease, familial adenomatous polyposis (FAP), familiar cutaneous melanoma, Fragile X, gastric cancer (including hereditary diffuse gastric cancer), Gaucher disease, hereditary predisposition to develop cancer, Huntington disease, hypophosphatasia (HPP), incontinentia pigmenti, Krabbe disease, Leber congenital amaurosis (LCA), Loeys-Dietz syndrome, Long QT syndrome, Lynch syndrome, Marfan syndrome, mental disorder, medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency, MUTYH-associated polyposis, neuroblastoma, neuronal ceroid-lipofuscinoses (NCLs), Niemann-Pick Type C disease, pancreatic cancer syndromes, papillary renal carcinoma, Parkinson disease, phenylketonuria, Pompe disease, propiopnic acidemia, rheumatoid arthritis, solid tumors, spinal muscular atrophy, spinocerebellar ataxia, susceptibility to breast cancer, Tay-Sachs disease, very long-chain acyl-coenzyme A dehydrogenase deficiency, Von Hippel-Lindau syndrome, Wilms tumor, Wilson disease, Wolfram syndrome type 1, X-linked creatine deficiency syndrome, X-linked hemophilia A, and X-linked retinitis pigmentosa.
24. A method of designing a probe to target a genomic region of interest, the method comprising: (c) designing a plurality of nucleic acid probe options to target said genomic region of interest; (d) selecting a first set of candidates from said plurality of nucleic acid probe options with a GC content of at least 20%; (e) selecting a second set of candidates from said first set of candidates with a self-complementarity score of zero or a complementarity score of 1; (f) selecting a third set of candidates from said second set of candidates with an efficiency greater than or equal to 0.2; and (g) selecting a fourth set of candidates from said third set of candidates with a mismatch profile of MM0=0 or MM0=1, MM1=0 or MM1=1 or MM1=2, MM2=0 or MM2=1 or MM2=2, and MM3<21, wherein said fourth set of candidates comprises said probe to target a genomic region of interest.
25. The method of claim 24, wherein fourth set of candidates comprises a mismatch profile of MM3<5.
26. The method of claim 24, wherein said designing comprises using CHOPCHOP.
27. The method of claim 24, wherein said first set of candidates have a GC content of about 40% to about 80%.
28. The method of claim 24, wherein said nucleic acid probe of interest comprises a crRNA.
29. The method of claim 27, wherein a probability of said crRNA cutting said genomic region of interest is greater than or equal to 80%.
30. The method of claim 21, further comprising estimating on-target value of said crRNA.
31. The method of claim 29, further comprising estimating off-target value of said crRNA.
32. A kit comprising a set of guide RNAs (gRNAs) that are hybridizable to a genomic region of interest in a genome comprising designing a plurality of gRNAs, wherein: at least one gRNA is hybridizable to a target site within the genomic region of interest and is configured to produce a genomic variant that comprises at least 1000 bp; and said plurality of gRNAs comprises a plurality of CRISPR RNAs (crRNAs), wherein said plurality of crRNAs comprises a GC of at least about 40% to about 80%.
33. A kit comprising a set of guide RNAs (gRNAs) that are hybridizable to a genomic region of interest in a genome comprising designing a plurality of gRNAs, wherein: at least one gRNA is hybridizable to a target site within the genomic region of interest and is configured to produce a genomic variant that comprises at least 1000 bp; and said plurality of gRNAs comprises a plurality of CRISPR RNAs (crRNAs), wherein said plurality of crRNAs comprises a self-complementarity score of zero.
34. A kit comprising a set of guide RNAs (gRNAs) that are hybridizable to a genomic region of interest in a genome comprising designing a plurality of gRNAs, wherein: at least one gRNA is hybridizable to a target site within the genomic region of interest and is configured to produce a genomic variant that comprises at least 1000 bp; and said plurality of gRNAs comprises a plurality of CRISPR RNAs (crRNAs), wherein said plurality of crRNAs comprises an efficiency score of about 0.2.
35. A kit comprising a set of guide RNAs (gRNAs) that are hybridizable to a genomic region of interest in a genome comprising designing a plurality of gRNAs, wherein: at least one gRNA is hybridizable to a target site within the genomic region of interest and is configured to produce a genomic variant that comprises at least 1000 bp; and said plurality of gRNAs comprises a plurality of CRISPR RNAs (crRNAs), wherein said plurality of crRNAs comprises a mismatch profile of MM0=0 or MM0=1, MM1=0 or MM1=1, MM2=0 or MM2=2, and MM3<21.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:
[0027]
[0028]
[0029]
[0030]
[0031]
[0032]
DETAILED DESCRIPTION OF THE INVENTION
[0033] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.
[0034] Where values are described as ranges, it will be understood that such disclosure includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.
[0035] The terms “a,” “an,” and “the,” as used herein, generally refers to singular and plural references unless the context clearly dictates otherwise.
[0036] The term “subject,” as used herein, generally refers to an animal, such as a mammal (e.g., human) or avian (e.g., bird), or other organism, such as plant. For example, the subject can be a vertebrate, a mammal, a rodent (e.g., a mouse), a primate, a simian, or a human. Animals may include, but are not limited to, farm animals, sport animals, and pets. A subject may be a healthy or asymptomatic individual, an individual that has or is suspected of having a disease (e.g., a genetic disorder) or a pre-disposition to a disease, and/or an individual that is in need of therapy or suspected of needing therapy. A subject can be a patient.
[0037] The term “genome,” as used herein, generally refers to genomic information from a subject, which may be, for example, at least a portion or an entirety of a subject's hereditary information. A genome can be encoded either in DNA or in RNA. A genome can include the sequence of all chromosomes together in an organism. For example, the human genome ordinarily has a total of 46 chromosomes. The sequence of all these together may constitute a human genome.
[0038] The term “sequencing,” as used herein, generally refers to methods and technologies for determining the sequence of nucleotide bases in one or more polynucleotides. The polynucleotides can be, for example, nucleic acid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (e.g., single stranded DNA). Sequencing can be performed by various systems currently available, such as, without limitation, sequencing system by Illumina®, Pacific Biosciences (PacBio®), Oxford Nanopore®, Life Technologies (Ion Torrent®), Roche®, Genapsys®, and MGI Tech®. Sequencing may be performed without using nucleic acid amplification. Alternatively, or in addition, sequencing may be performed using nucleic acid amplification, polymerase chain reaction (PCR) (e.g. digital PCR, quantitative PCR, or real time PCR), or isothermal amplification. Such systems may provide a plurality of raw genetic data corresponding to the genetic information of a subject (e.g., human), as generated by the systems from a sample provided by the subject. In some examples, such systems provide sequencing reads (also “reads” herein). A read may include a string of nucleic acid bases corresponding to a sequence of a nucleic acid molecule that has been sequenced. In some situations, systems and methods provided herein may be used with proteomic information.
[0039] The term “sample,” as used herein, generally refers to a biological sample of a subject. The biological sample may comprise any number of macromolecules, for example, cellular macromolecules. The sample may be a cell sample. The sample may be a cell line or cell culture sample. The sample can include one or more cells. The sample may include one or more microbes. The biological sample may be a nucleic acid sample or protein sample. The biological sample may also be a carbohydrate sample or a lipid sample. The biological sample may be derived from another sample. The sample may be a tissue sample, such as a biopsy, core biopsy, needle aspirate, of fine needle aspirate. The sample may be a fluid sample, such as blood sample, urine sample, or saliva sample. The sample may be a skin sample. The sample may be a cheek swab. The sample may be a plasma or serum sample. The sample may include cells or may be cell-free. A cell-free sample may include extracellular polynucleotides. Extracellular polynucleotides may be isolated from a bodily sample that may be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool and tears.
[0040] The term “short read,” as used herein, generally refers to a read length of a DNA or RNA polynucleotide of about 100 to about 600 bp.
[0041] The term “long read,” as used herein, generally refers to a read length of a DNA or RNA polynucleotide of greater than 1 Kbp.
[0042] The term “ribonucleoprotein (RNP),” as used herein is a ribonucleoprotein is a ribonucleic acid (RNA)-protein complex.
[0043] The term “CRISPR-Cas system,” as used herein, generally refers the clustered regularly short palindromic repeats (CRISPR system) which comprises an array of two types of DNA sequences: (i) repetitive, flanking DNA sequences; and (ii) spacer sequences that are endogenously derived from a virus, and can be used to target DNA or RNA sequences for cleaving using the CRISPR-associated (Cas) enzyme (ribonucleoprotein) complex that are used to cleave the CRISPR sites that are complementary to those in spacer regions.
[0044] The term “barcoding,” as used herein, is the ligation of known, unique sequences to target DNA molecules, between the adapter and the ROI in order for the target sequence recognition in the downstream analysis, i.e. post-base calling.
[0045] The term “multiplexing,” as used herein, is the running of multiple samples in a single flow cell, identifying each sample's DNA molecules through unique ‘barcode’ molecules that have been attached to the DNA ends. The decoded sequences of a sample's DNA will be identified downstream once the sequences have been basecalled.
[0046] The term “crRNA,” as used herein, are the RNA sequences that recognize the target site. Together with the tracrRNA, this forms a single guide RNA (sgRNA) and when several are used together, gRNA.
[0047] The term “tracrRNA,” as used herein, refers to trans-activating-crRNA specific to Type II Cas/CRISPR system. It is used to process the pre-crRNA along with an RNase III. The tracrRNA provides structural support to the ribonucleoprotein and anneals to the pre-crRNA for processing via the internal endonuclease activity of the Cas protein. Non-limiting examples of Cas enzymes can include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1, c2c1, c2c3, Cas9HiFi, homologues thereof, or modified versions thereof. In some cases, a catalytically dead Cas protein can be used, for example a dCas9. An unmodified CRISPR enzyme can have DNA cleavage activity, such as Cas9.
[0048] The term “protospacer,” as used herein, refers to a sequence acquired from a pathogenic organism's DNA molecule. The sequence is converted into DNA and forms the gene of the crRNA, which, along with the PAM in the substrate sequence, directs the Cas-crRNA-tracrRNA ternary complex to cleave target molecule.
[0049] The term “protospacer adjacent motif (PAM),” as used herein, refers to pathogenic sequences from host sequences i.e. the crRNA gene. It is adjacent to the 3′ end of the protospacer and facilitates the pathogen's sequence to be cut by the Cas-crRNA-tracrRNA ternary complex. In Type II CRISPR systems, NGG (where N is any nucleotide as per FASTA conventions), defines the sequences that will be cleaved. If a PAM is 3′ of crRNA sequence in the DNA, the crRNA-tracrRNA-Cas9 ternary complex will not cleave the host DNA/genome sequence.
[0050] The term “untranslated region (UTR),” as used herein, refers to the untranslated region (UTR) of a mRNA transcript and is present both at the 5′ and 3′ ends of the protein coding region. It is not translated via the protein synthesis process by the ribosome.
[0051] The human genome has many different types of genomic variations that range in size and type.
[0052] The types of genomic variants can be categorized based on the number of nucleotides involved. Single nucleotide variants (SNVs) affect a single nucleotide or base pair. Small insertions and deletions, commonly called indels, are shorter than 50 nucleotides in length. Structural variants are changes in the structure of chromosome and generally affect 50 or more nucleotides. The typical human genome has about 8 million bases that differ from a reference due to SNVs and indels. The typical human genome has about 20,000 structural variants that differ from the reference and affects about 10 million bases.
[0053] In one aspect, provided herein are systems and methods to detect one or more genomic variants in a sample, comprising (a) preparing a sample for sequencing using a non-amplification-based, gene-editing based approach, and (b) long-read sequencing, as described herein elsewhere.
[0054] In another aspect, provided herein are systems and methods for conducting a diagnostic assay for a genetic disorder, comprising (a) preparing a sample for sequencing using a non-amplification-based, gene-editing based approach, and (b) long-read sequencing, as described herein elsewhere.
[0055] In some cases, the one or more genomic variants comprise one or more structural variants. In some cases, the one or more genomic variants comprise at least one structural variant. In some cases, the structural variant is about 30 bp to about 1,000 bp. In some cases, the structural variant is about 30 bp to about 50 bp, about 30 bp to about 100 bp, about 30 bp to about 500 bp, about 30 bp to about 750 bp, about 30 bp to about 1,000 bp, about 50 bp to about 100 bp, about 50 bp to about 500 bp, about 50 bp to about 750 bp, about 50 bp to about 1,000 bp, about 100 bp to about 500 bp, about 100 bp to about 750 bp, about 100 bp to about 1,000 bp, about 500 bp to about 750 bp, about 500 bp to about 1,000 bp, or about 750 bp to about 1,000 bp. In some cases, the structural variant is about 30 bp, about 50 bp, about 100 bp, about 500 bp, about 750 bp, or about 1,000 bp. In some cases, the structural variant is at least about 30 bp, about 50 bp, about 100 bp, about 500 bp, or about 750 bp. In some cases, the structural variant is at most about 50 bp, about 100 bp, about 500 bp, about 750 bp, or about 1,000 bp. In some cases, the structural variant is about 1 Kbp to about 1,000 Kbp. In some cases, the structural variant is about 1 Kbp to about 50 Kbp, about 1 Kbp to about 100 Kbp, about 1 Kbp to about 250 Kbp, about 1 Kbp to about 500 Kbp, about 1 Kbp to about 750 Kbp, about 1 Kbp to about 1,000 Kbp, about 50 Kbp to about 100 Kbp, about 50 Kbp to about 250 Kbp, about 50 Kbp to about 500 Kbp, about 50 Kbp to about 750 Kbp, about 50 Kbp to about 1,000 Kbp, about 100 Kbp to about 250 Kbp, about 100 Kbp to about 500 Kbp, about 100 Kbp to about 750 Kbp, about 100 Kbp to about 1,000 Kbp, about 250 Kbp to about 500 Kbp, about 250 Kbp to about 750 Kbp, about 250 Kbp to about 1,000 Kbp, about 500 Kbp to about 750 Kbp, about 500 Kbp to about 1,000 Kbp, or about 750 Kbp to about 1,000 Kbp. In some cases, the structural variant is about 1 Kbp, about 50 Kbp, about 100 Kbp, about 250 Kbp, about 500 Kbp, about 750 Kbp, or about 1,000 Kbp. In some cases, the structural variant is at least about 1 Kbp, about 50 Kbp, about 100 Kbp, about 250 Kbp, about 500 Kbp, or about 750 Kbp. In some cases, the structural variant is at most about 50 Kbp, about 100 Kbp, about 250 Kbp, about 500 Kbp, about 750 Kbp, or about 1,000 Kbp. In some cases, the structural variant is about 1 Mbp to about 10 Mbp. In some cases, the structural variant is at least about 1 Mbp. In some cases, the structural variant is at most about 10 Mbp. In some cases, the structural variant is about 1 Mbp to about 2 Mbp, about 1 Mbp to about 3 Mbp, about 1 Mbp to about 4 Mbp, about 1 Mbp to about 5 Mbp, about 1 Mbp to about 6 Mbp, about 1 Mbp to about 7 Mbp, about 1 Mbp to about 8 Mbp, about 1 Mbp to about 9 Mbp, about 1 Mbp to about 10 Mbp, about 2 Mbp to about 3 Mbp, about 2 Mbp to about 4 Mbp, about 2 Mbp to about 5 Mbp, about 2 Mbp to about 6 Mbp, about 2 Mbp to about 7 Mbp, about 2 Mbp to about 8 Mbp, about 2 Mbp to about 9 Mbp, about 2 Mbp to about 10 Mbp, about 3 Mbp to about 4 Mbp, about 3 Mbp to about 5 Mbp, about 3 Mbp to about 6 Mbp, about 3 Mbp to about 7 Mbp, about 3 Mbp to about 8 Mbp, about 3 Mbp to about 9 Mbp, about 3 Mbp to about 10 Mbp, about 4 Mbp to about 5 Mbp, about 4 Mbp to about 6 Mbp, about 4 Mbp to about 7 Mbp, about 4 Mbp to about 8 Mbp, about 4 Mbp to about 9 Mbp, about 4 Mbp to about 10 Mbp, about 5 Mbp to about 6 Mbp, about 5 Mbp to about 7 Mbp, about 5 Mbp to about 8 Mbp, about 5 Mbp to about 9 Mbp, about 5 Mbp to about 10 Mbp, about 6 Mbp to about 7 Mbp, about 6 Mbp to about 8 Mbp, about 6 Mbp to about 9 Mbp, about 6 Mbp to about 10 Mbp, about 7 Mbp to about 8 Mbp, about 7 Mbp to about 9 Mbp, about 7 Mbp to about 10 Mbp, about 8 Mbp to about 9 Mbp, about 8 Mbp to about 10 Mbp, or about 9 Mbp to about 10 Mbp. In some cases, the structural variant is about 1 Mbp, about 2 Mbp, about 3 Mbp, about 4 Mbp, about 5 Mbp, about 6 Mbp, about 7 Mbp, about 8 Mbp, about 9 Mbp, or about 10 Mbp.
[0056] As described elsewhere, the one or more target genomic variants may comprise one or more structural variants. In some cases, the one or more target genomic variants may comprise at least one structural variant. In some cases, the sample comprises about 1 target genomic variant to about 100 target genomic variants.
[0057] In some embodiments, the sample comprises RNA transcripts. In some embodiments, the sample comprises genomic DNA (gDNA). In some embodiments, the sample comprises gDNA and RNA transcripts.
[0058] In some embodiments, the sample comprises one or more target genomic variants. In some cases, the sample comprises about 1 target genomic variant to about 2 target genomic variants, about 1 target genomic variant to about 4 target genomic variants, about 1 target genomic variant to about 6 target genomic variants, about 1 target genomic variant to about 8 target genomic variants, about 1 target genomic variant to about 10 target genomic variants, about 1 target genomic variant to about 20 target genomic variants, about 1 target genomic variant to about 30 target genomic variants, about 1 target genomic variant to about 40 target genomic variants, about 1 target genomic variant to about 50 target genomic variants, about 1 target genomic variant to about 75 target genomic variants, about 1 target genomic variant to about 100 target genomic variants, about 2 target genomic variants to about 4 target genomic variants, about 2 target genomic variants to about 6 target genomic variants, about 2 target genomic variants to about 8 target genomic variants, about 2 target genomic variants to about 10 target genomic variants, about 2 target genomic variants to about 20 target genomic variants, about 2 target genomic variants to about 30 target genomic variants, about 2 target genomic variants to about 40 target genomic variants, about 2 target genomic variants to about 50 target genomic variants, about 2 target genomic variants to about 75 target genomic variants, about 2 target genomic variants to about 100 target genomic variants, about 4 target genomic variants to about 6 target genomic variants, about 4 target genomic variants to about 8 target genomic variants, about 4 target genomic variants to about 10 target genomic variants, about 4 target genomic variants to about 20 target genomic variants, about 4 target genomic variants to about 30 target genomic variants, about 4 target genomic variants to about 40 target genomic variants, about 4 target genomic variants to about 50 target genomic variants, about 4 target genomic variants to about 75 target genomic variants, about 4 target genomic variants to about 100 target genomic variants, about 6 target genomic variants to about 8 target genomic variants, about 6 target genomic variants to about 10 target genomic variants, about 6 target genomic variants to about 20 target genomic variants, about 6 target genomic variants to about 30 target genomic variants, about 6 target genomic variants to about 40 target genomic variants, about 6 target genomic variants to about 50 target genomic variants, about 6 target genomic variants to about 75 target genomic variants, about 6 target genomic variants to about 100 target genomic variants, about 8 target genomic variants to about 10 target genomic variants, about 8 target genomic variants to about 20 target genomic variants, about 8 target genomic variants to about 30 target genomic variants, about 8 target genomic variants to about 40 target genomic variants, about 8 target genomic variants to about 50 target genomic variants, about 8 target genomic variants to about 75 target genomic variants, about 8 target genomic variants to about 100 target genomic variants, about 10 target genomic variants to about 20 target genomic variants, about 10 target genomic variants to about 30 target genomic variants, about 10 target genomic variants to about 40 target genomic variants, about 10 target genomic variants to about 50 target genomic variants, about 10 target genomic variants to about 75 target genomic variants, about 10 target genomic variants to about 100 target genomic variants, about 20 target genomic variants to about 30 target genomic variants, about 20 target genomic variants to about 40 target genomic variants, about 20 target genomic variants to about 50 target genomic variants, about 20 target genomic variants to about 75 target genomic variants, about 20 target genomic variants to about 100 target genomic variants, about 30 target genomic variants to about 40 target genomic variants, about 30 target genomic variants to about 50 target genomic variants, about 30 target genomic variants to about 75 target genomic variants, about 30 target genomic variants to about 100 target genomic variants, about 40 target genomic variants to about 50 target genomic variants, about 40 target genomic variants to about 75 target genomic variants, about 40 target genomic variants to about 100 target genomic variants, about 50 target genomic variants to about 75 target genomic variants, about 50 target genomic variants to about 100 target genomic variants, or about 75 target genomic variants to about 100 target genomic variants. In some cases, the sample comprises about 1 target genomic variant, about 2 target genomic variants, about 4 target genomic variants, about 6 target genomic variants, about 8 target genomic variants, about 10 target genomic variants, about 20 target genomic variants, about 30 target genomic variants, about 40 target genomic variants, about 50 target genomic variants, about 75 target genomic variants, or about 100 target genomic variants. In some cases, the sample comprises at least about 1 target genomic variant, about 2 target genomic variants, about 4 target genomic variants, about 6 target genomic variants, about 8 target genomic variants, about 10 target genomic variants, about 20 target genomic variants, about 30 target genomic variants, about 40 target genomic variants, about 50 target genomic variants, or about 75 target genomic variants. In some cases, the sample comprises at most about 2 target genomic variants, about 4 target genomic variants, about 6 target genomic variants, about 8 target genomic variants, about 10 target genomic variants, about 20 target genomic variants, about 30 target genomic variants, about 40 target genomic variants, about 50 target genomic variants, about 75 target genomic variants, or about 100 target genomic variants.
Target Enrichment Sample Preparation for Sequencing
[0059] In some aspect, the target enrichment sample preparation approach describe herein may comprise one or more genome editing technologies. In some cases, the genome editing technology is an endonuclease-based genome editing technology. In some cases, the endonuclease-based genome editing technology comprises zinc-finger nucleases (ZFNs), homing nucleases, transcription activator-like effector nucleases (TALENs), and/or clustered regularly interspersed short palindromic repeats (CRISPR)-Cas systems. In some cases, the target enrichment sample preparation approach may further comprise DNA amplification. In some cases, the target enrichment sample preparation approach may not comprise DNA amplification.
[0060] In some embodiments, the target enrichment sample preparation approach comprises preparing a sample for sequencing using a non-amplification-based, gene-editing based approach. In some case, the sample preparation comprises Cas-mediated PCR-free enrichment of said sample as shown in
[0061] In some embodiments, a two RNP (ribonucleoprotein complex comprising Cas9-crRNA-tracrRNA) complexes, designed to excise a ROI, bind to sequences on the (+) and (−) strands, upstream and downstream of the ROI, respectively. The crRNAs confer specificity and ‘program’ the RNPs to bind to the specific sequences. Background DNA has been dephosphorylated (i.e. carries 5′-hydroxyl groups). Upon RNP binding, the duplex DNA is locally melted. crRNA hybridizes to the non-target DNA strand, which is complementary to the crRNA. Cas9 cleaves both of the DNA strands within the target site, 3 bp upstream of the PAM. Cleavage by Cas9 reveals 5′ phosphates at each end of the ROI. Existing ends of the same molecule, which carry 5′ hydroxyl groups, are considered non-target. The PAM-distal side is protected from ligation by Cas9 and/or the bound crRNA, whereas the PAM-proximal side is released for each RNP targeting the ROI. Because the RNPs here target the (+) strand and the (−) strand upstream and downstream of the ROI, the ROI is excised and both ends of the ROI are freed for dA-tailing and adapter ligation. Adapter ligation to the ROI results in directionality of the expected reads.
[0062] In some cases, an alternative to Cas9 may be used in the CRISPR-Cas system, wherein the alternative to Cas9 may be Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas10, Cas10d, Cas13a, Cas13b, Cas13c, Cse1, Cse2, Csy1, Csy2, Csy3, Csm2, Cmr5, Csx10, Csx10, Csf1, Csn2, Cpf1, C2c1, or C2c3.
[0063] In some cases, the target enrichment sample preparation comprises preparing a sample for sequencing using the PacBio® sequencing system. In such cases, genomic DNA (gDNA) is dephosphorylated and then subjected to Cas-mediated PCR-free enrichment as described herein. Following the cleavage reaction, SMRTbell® adapters are ligated to the blunt template ends, forming SMRTbell® templates. In the final step, unligated DNA is eliminated by exonuclease digestion and then prepared for sequencing by annealing to the Sequencing Primers and binding to the polymerase.
[0064] In some cases, the target enrichment sample preparation comprises preparing a sample for sequencing using Illumina® sequencing system. In such embodiments, gDNA is dephosphorylated and then filled in using biotinylated nucleotides. The gDNA is then subjected to Cas-mediated PCR-free enrichment as described herein. After the cleavage reaction, non-target gDNA is removed using streptavidin beads. The target gDNA is then fragmented to the appropriate size, end-repaired, and dA-tailed. Illumina® adapters are ligated to the end-repaired, dA-tailed target gDNA, and is then ready for sequencing.
crRNA Probes
[0065] In one aspect, provided herein are systems and methods to design crRNAs for use with the systems and methods described herein. In some embodiments as shown in
[0066] The preliminary crRNA probes are filtered using one or more approaches as shown in
[0067] In some cases, two or more of the approaches are used. In some cases, three or more of the approaches are used. In some cases, four approaches are used. In some cases, the following approaches are used in the following order: GC content, self-complementarity score, efficiency score and mismatches. After filtering the preliminary crRNA probes using one or more of the filter approaches, the on-target and off-target performance of candidate crRNA probes are confirmed using a guide RNA check tool. Examples of guide RNA check tools include IDT CRISPR-Cas9 gRNA checker, Cas-OFFinder, Dharmacon's CRISPR specificity analysis tool, Synthego's CRISPR specificity analysis tool, or a combination thereof.
[0068] Candidate crRNA probes obtained using the methods provided herein are more likely to cut the target genomic region of interest than crRNA probes obtained using other methods. In some cases, the probability that a candidate crRNA probe will cut a target is about 60% to about 99.9%. In some cases, the probability that a candidate crRNA probe will cut a target is at least about 60%. In some cases, the probability that a candidate crRNA probe will cut a target is at most about 99.9%. In some cases, the probability that a candidate crRNA probe will cut a target is about 60% to about 65%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 99.9%, about 65% to about 70%, about 65% to about 75%, about 65% to about 80%, about 65% to about 85%, about 65% to about 90%, about 65% to about 95%, about 65% to about 99.9%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 99.9%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 75% to about 99.9%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 99.9%, about 85% to about 90%, about 85% to about 95%, about 85% to about 99.9%, about 90% to about 95%, about 90% to about 99.9%, or about 95% to about 99.9%. In some cases, the probability that a candidate crRNA probe will cut a target is about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99.9%.
[0069] Provided herein are exemplary target genomic sequences (e.g., a protospacer) to which crRNA probes may be hybridizable for use with the systems and methods described herein. A guide RNA can target a nucleic acid sequence of or of about 20 nucleotides. A target nucleic acid can be less than or less than about 20 nucleotides. A target nucleic acid can be at least or at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. A target nucleic acid can be at most or at most about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. A target nucleic acid sequence can be or can be about 20 bases immediately 5′ of the first nucleotide of the PAM. A guide RNA can target the nucleic acid sequence. A guiding polynucleic acid, such as a guide RNA, can bind to a genomic sequence with at least or at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or up to about 100% sequence identity and/or sequence similarity to any of the sequences of the tables below.
TABLE-US-00001 TABLE 1 Target sequences for ABCD1 gene SEQ ID NOS Target sequence 1 TCAGCAACAACGTGACCCAGTGG 2 GTCATGACGAAGCAGAACCCTGG 3 TCATGACGAAGCAGAACCCTGGG 4 GTTCTGTTGCAAAACCCACAAGG 5 TTGGAGGCCATTAGTTAGTGCGG 6 GAGGCCATTAGTTAGTGCGGAGG 7 AGGCCATTAGTTAGTGCGGAGGG 8 CAGGTCTCCTGATTTACCTCGGG 9 CTTGCCCCATCTCGCATACCCGG 10 TGAGGGGTAACCACCTGTGCCGG 11 CCAGAAACCCGAGGTAAATCAGG 12 AAGTGTTACAAAGGGTCTCCAGG 13 CGGGTATGCGAGATGGGGCAAGG 14 TCATGGGGCCCCTGCGCGCAGGG 15 CGCAGGGCCACATATGCTCAGGG 16 GCGCAGGGCCACATATGCTCAGG 17 TTACCCCTCACCGCTCGCAGCGG 18 CTGAGGTAAGCTAAAGACCACGG 19 TCCAGGTAGACAGCTGTTCAAGG 20 GGGGCACTAAAGTGTTACAAAGG 21 CGGGGTTTATGATCAAGCATGGG 22 ACGGGGTTTATGATCAAGCATGG
TABLE-US-00002 TABLE 2 Target sequences for ACADM gene SEQ ID NOS Target sequence 23 AAGGGGTTACAATAGGCATATGG 24 CGTGTTGTAATCATCATAGAAGG 25 TGTATGGAGGGATTGAACACAGG 26 GATTACAACACGTGACCTCAGGG 27 CTTAATCACATGGTCCTCGGGGG 28 GTTCAATCCCTCCATACAAGTGG 29 ACTTAATCACATGGTCCTCGGGG 30 TGATTACAACACGTGACCTCAGG 31 AACTTAATCACATGGTCCTCGGG 32 TAGAATGAGGCCCAGCAACCAGG 33 ACAGTCATTTATTGCTACTAGGG 34 GAGGGATGCCAAAATCTATCAGG 35 GATAGGACTCTAATCTCACAGGG
TABLE-US-00003 TABLE 3 Target sequences for ACADVL gene SEQ ID NOS Target sequence 36 GGGTCTTGCCAAACGGCCAG 37 GGGGTCTTGCCAAACGGCCA 38 AGCACACCCCGATTCTCAGG 39 CACACCCCGATTCTCAGGAG 40 GGGAGCACACCCCGATTCTC 41 GAGGCCCGCAAGTATGCCAG 42 TCCCGCACTAGGTCCTGCAC 43 GACGTCCACCCATGTGCTGC
TABLE-US-00004 TABLE 4 Target sequences for AFF2 gene SEQ ID NOS Target sequence 44 GTGATACCATGTATGCCACGTGG 45 GATGCTAAGTGTACACCACGAGG 46 CTAACGAAAGACACCAACTG 47 CCTTCCCTAAGTGAACCAAGGGG 48 TCGTATCTAACACTCCCCTGGGG 49 CTAGTATTATCGATACCCAGAGG 50 GTAGGTTTCATACCACAATGTGG 51 GGTCTCTATCAAGTTCAAGGTGG 52 TCTGCCACTAAAGCAACCAGCGG 53 GTTCTCATGATCTCGCAGAGGGG 54 TGAGCACAACTTCAACTGGGGGG 55 GTCATAATCACAGTACATCGTGG 56 ACCATACACCAAATGCCACGTGG 57 GTGCTACTGCCACCTCACGGTGG 58 TTACGCCAGCACAAAAACGTGGG 59 TGCCTGCGATAATTACAGAGTGG 60 GAACTGTAAATATAGATACGTGG 61 GAACTCATATGCAAACCTCGTGG 62 TCTAACTAAGGATCAGCACAGGG 63 CGACACATTGGATGAAACGTGGG 64 TATCAAAAATACCCACAGCGGGG 65 TGTTACCTGGAGTACTACGATGG 66 TAGATGTCCACCATACTCAGAGG 67 CAACTTGTTCAGTATGACGAGGG 68 CAAGTTCCCTCAGATCGCTGAGG 69 ACCACAGTCCCTAATTACCGTGG 70 AGTGGCTTGGTACAACAATGAGG 71 GCTGGAGTATATAATCCCCGGGG 72 ATATGTGATATACTACCCTGTGG 73 TGGGCGTAAGAAACTAATTGAGG 74 GCTGGTGACGAGGTTAGACGTGG 75 CGTCTTCGCAGTAATTCTGGAGG 76 CCTTACAATGTATGTCCCAGTGG 77 GATAATGCTCATATGTGACAGGG 78 TGGGACCTTTGCAATACACAGGG 79 CTTTTAGTGATACTTCCACGTGG 80 ACCATACTGTCACAACAAGTGGG 81 GGCTATGTAGATACCTGTGGAGG 82 GATCACTAATCGCCCACCCAGGG 83 GAAAGTCCTTAAAGACCCCGTGG 84 GAGCATTGTTAGTGAACTGGGGG 85 ACTGACATAAATGCCGTGGGTGG 86 GGTATTATAGTTCATCAGGGTGG 87 CTCTATTGTGACATGCAAGGTGG 88 TTTTCATCGAGTGTGCGTCGTGG 89 ATAGCGAAATCTATCTCACGAGG 90 GGCACTTGTACATTTAACGTGGG 91 ATGAAAAACTCAGCTTACGGTGG 92 GGTACCAAGATCTAAAATGGGGG 93 ATCGTTTATATAATACCCAGAGG 94 CCACCTTGAAGAAATACCGGGGG 95 TATGTGATATACTACCCTGTGGG 96 GCTTGTTATAAATTACACCAAGG 97 GGATTCACTCTGGTAAAGCAGGG 98 TGTGCGCAGATTCTCCGATGAGG 99 GGGAGTTACATACCCGTACAGGG 100 CTCAAGATACCCCATGACAGAGG 101 GATAAGAAGCAATCCACTGGGGG 102 TACGTAGAGAGTAAGTCCAGAGG 103 GAGACAACGTTACAAAAGCGCGG 104 ATATCTTATCTCCTACACCAAGG 105 GCTCCCCTGATGGTAAGACGTGG 106 CAGTTCCCAGCAAATAAACGAGG 107 TGACCACGTCTTACCATCAGGGG 108 ACGGGCAACTGAGGTAATGGGGG 109 TCTGAACACTAATGTCACAAGGG 110 TAAGTTTGACAGCTAACCAGTGG 111 AATAGTCGGTTTCTTCAATGTGG 112 GACACACTTAAATAAGCACGTGG 113 TAGGCACTTGTCCAGAAACGAGG 114 CACGCAATGAGATAATTCTG 115 TCATAGGAAATGGCTCGCTGCGG 116 GGTATAATAGGCCAGTCTTGCGG 117 GGTGCTTATCCACTATTCAGGGG 118 ATAACTCAGTAGATCAACCTGGG 119 GTCCTTGGAGAACCCTTCGGAGG 120 ATGCTCTATTGTGACATGCAAGG 121 CCGTTAGGCTTCATAAACCATGG 122 ATGTTTCTGATGAAGTGCCGTGG 123 CAGGGTATTATAGTTCATCAGGG 124 TATCTCAAGAAGGTAACTCGGGG 125 AGGTTCTCATGATCTCGCAGAGG 126 GGGAATGAACACCATACCGAAGG 127 TCTAACCTAAATGGTCTGTGTGG 128 ACCTGTCCTATGGAGTATGGTGG 129 TCACACCCTTGTTAGACCAGTGG 130 CATAATCCTCTACTACGATG 131 CTTCTCTATAAATCTCAGGGAGG 132 TTACCAAAAAGGCTTATCCGTGG 133 AAATGCGGGGATAATTCCAAAGG 134 GGAGTTATAACAATTGTCCG 135 GAGTTGTCAAACCATTATCGTGG 136 TAGTGCCATCGCTTTAAGGGTGG 137 GCTAAAACACTGGTAACTCAGGG 138 GATGTGCTACTTCTACCTGAAGG 139 TATGAATAACAATGGTACGAAGG 140 ATTCATAGAGTGGTTGTCAGGGG 141 ATGATGATAGAACCTTAAGCCGG 142 GCTTGACATTAGATAGACCATGG 143 TCTGGAAGTTGGAACTCCGTAGG
TABLE-US-00005 TABLE 5 Target sequences for ALPL gene SEQ ID NOS Target sequence 144 TGATACCATCTTAAGTCTCCTGG 145 CACTTAGGTGATTAAGGGCTTGG 146 CAACATGCACGTACTAGGCATGG 147 CTGCCCAAGGCTTAGCTAGGTGG 148 AGGCCACCTAGCTAAGCCTTGGG 149 AATTTCCCCATTGTGCGTCTTGG 150 TTTACAACCCTTTGACCACCAGG 151 TAGGTCCCTCTGCTAAACAGGGG 152 GTACCTGCAAGTCCTGTCACAGG 153 GCATGGAACCTGGTGGTCAAAGG
TABLE-US-00006 TABLE 6 Target sequences for APC gene SEQ ID NOS Target sequence 154 TATATCAGGCATTGTAACAC 155 TGTGTATGGCCCCACAAAGA 156 ATCTTTGTGGGGCCATACAC 157 TCGTTATAACACCAGTTCTG 158 CGTTATAACACCAGTTCTGT 159 AGATCTAGTTAGTTCTACAA 160 GTACAATCCAATGACATCTG 161 AGATGTCATTGGATTGTACC 162 CCAGTCATGTTTGATATACT 163 GTCTCCTGCACTACAAGACT 164 AAGTTCACAACTAACTGGTT 165 CTCTTGGAGGTTGTAAACTC 166 CCTAGTATATCAAACATGAC 167 ATTAGGGTTTAGTGTACTAA 168 TTAGTCCTCTACCTTACTGG 169 GCCTATTTTGTGATTGCCAA 170 CATGAGAATAACGACCTCAA 171 GGTGACCCCCAGTAAGGTAG 172 CGACCTCAAAGGATATCATG 173 TAAGTGTCCATCAACTAGGG 174 CCATGCAGTTAAGAGGTACC 175 AACCTTTGCTTACATGCCTA 176 GGTGGTACTTACCCTTCCAT 177 TTGTTAATAGAGCTTACTAC 178 TGTCAACTATATTACCCTAT 179 CATCCAGGCTTATAATCTCC 180 TTTGCATGGATGCACCATAT 181 TGCACCATATAGGTTCCATG 182 TTTACGATCAATGTCCATTT 183 TTGGCCTCATGGAACCTATA 184 GTAGATTAGTTAAGTGGCTC 185 AATGAGGGGGATTAGCCACA 186 CAGGTAGTATATTAGTCACC 187 CTGGTGACTAATATACTACC 188 AAGACATCCAGACTGTCGCA 189 AATGCTTGGTACTCATGATA 190 TCTAAACTCATTTGGCCCAC 191 GAATGCGTATCTAACAAGGG 192 ATGCGTATCTAACAAGGGTG 193 AATGCGTATCTAACAAGGGT 194 CTTAACTTAGACCTGGGATT 195 TTGTTAGATACGCATTCATC 196 CTCATTTGTAGCTATCAAGC 197 ATGGGTCATCTAATTAGAGT 198 TAGCTACAAATGAGGACCAC 199 ACCAGTGAGGGACGGGCAAT 200 TGGTTGGCACTCTTACTTAC 201 TGGTACAAATAGCCAAGGTC 202 CTCTAGGTCAGATACAACTC
TABLE-US-00007 TABLE 7 Target sequences for ASPA gene SEQ ID NOS Target sequence 203 CTATGTAAGTTCACATGATGTGG 204 AACCTGGCGTTACTAGTACATGG 205 CACTAACTACAGTTCTGAGTAGG 206 TTGGATCTGCCTTCTCAACCAGG 207 AAATTCTGAGTCCGTAATCCAGG 208 TTAGCTAAGTGACAGGTCTCAGG 209 ACTAAGTTCGCAGTCTCACATGG
TABLE-US-00008 TABLE 8 Target sequences for ATM gene SEQ ID NOS Target sequence 210 AATTGCGAGGACAACTGTCT 211 GAATTGCGAGGACAACTGTC 212 GATCACAACTGGGTAAGGGT 213 ATCACAACTGGGTAAGGGTA 214 CAGGTCCAATCTTCCTATGA 215 CTTCATAGGAAGATTGGACC 216 GATTCTGTGAGATTGAATCG 217 GTTAAACTGTCAGGTCACTT 218 CATCGTCAAGGAGTTGACAG 219 GCCATGATGAGTTGGTCCAA 220 AAAGGCTAGTATAAGCCCAA 221 ATGCATAAGTAGCTCCTAGA 222 AGTGATACTCTAGGGCAAAC 223 GATGCATAAGTAGCTCCTAG 224 GGGCAATACTCTCTTGGTAT 225 GCCTTTGGACCAACTCATCA 226 CCCTAGAGTATCACTTGTTA 227 GGAACTTTATTGGCTGGAAC 228 TAGTTAGGAACTTTATTGGC 229 ACCGAATTCACTCCTTTGAA 230 AGGAGTGAATTCGGTAGCCA 231 GTTCTAATTAGGGACTCACC 232 GACCCAACTTGCTACTCGCT 233 TTCAGGTTGAGTGGATAGTC 234 GCTCTACCTCCACATACACT 235 GGCTCTACCTCCACATACAC 236 CGAGTAGCAAGTTGGGTCCT 237 TGGCCTAGCGAGTAGCAAGT 238 GCGTAACACCCACATATTTA 239 TCCCATTAGGCATAACCTAA 240 GGACTCAACTAATTGGTGTT 241 ATATGTGGGTGTTACGCAAA 242 CCAAATCCCTAACAGAGTTA 243 CCTTAACTCTGTTAGGGATT 244 GCTTCAAGCTGACTTTAACC 245 CAGGTGATTTCTCCATCCCG 246 TAGATTTAGTGACCACGGGA 247 CACGGGATGGAGAAATCACC 248 TCAGCTTGAAGCTCTCGTGA 249 GTGTTTAGATTTAGTGACCA 250 CTATAATCTAGTAGGATCAC 251 ATCTAGTAGGATCACAGGAT 252 CTGTGATCCTACTAGATTAT 253 CATGCTTGAAGGCTCATTAT 254 ACAAGTGGACAAGTCAGATC 255 GCCAAGCTGTTTCTATCCAA 256 TACACGATTCCTGACATCAA 257 GCTACTTATGTGTAGAGCAC 258 CTTTGCAGTTACCATAGGAG 259 TAGAGTATCTAACCCAACGT 260 GGGCATGTAGAATACTTATT 261 GTGAATTTATATACCTACGT 262 ATCTTAATGAACCACTCATA 263 AGACAGTCACGGATATTATA 264 TGGAGTACAACCCATATGAG 265 AAAGATGCCTCGGTTCATAA 266 CGGTTCATAAAGGTGCACAC 267 GCCCCACCCTTATTGACCAC 268 AAGATGAGTAACAGTCCATC 269 CATTAAGCCTGTGGTCAATA 270 GGCATTAACCATTAAGCCTG 271 AAGCCTGTGGTCAATAAGGG 272 AATAGGTCCCAATAATACGT 273 GTGTGCACCTTTATGAACCG 274 GGAGGTTGGTTGCACACCAC 275 ACTCACCATTAGTAGTATAC
TABLE-US-00009 TABLE 9 Target sequences for ATM gene SEQ ID NOS Target sequence 276 AATGGGATCCCTTCCTAAGG 277 GTACCAAGACGTGGATATGG 278 GGTACCAAGACGTGGATATG 279 GATGTGTAGGTACCAAGACG 280 CTGGAACCTATGATCAGGCA 281 TAGGTACCAAGACGTGGATA 282 AGGTACCAAGACGTGGATAT 283 TGTCTCTGGAACCTATGATC 284 TGTCACAAGAGGTGCTTACA 285 ATGACTCTGAACTGCCCACC
TABLE-US-00010 TABLE 10 Target sequences for ATXN1 gene SEQ ID NOS Target sequence 286 GGCAACTCAGATACTCACGTGGG 287 CCCCAATAGAGATTGCCCTGTGG 288 ACATCAGAACATGAGCACCGGGG 289 CAGGTGAGCGTACTGCACGGGGG 290 CGGGTCAAACCCCATCACAGTGG 291 TAAGTTGTCGTTGATCACAGGGG 292 GAAACAGGTATGATGCATGGGGG 293 GTCCACTTTATAAATCCCAGAGG 294 CTAAAACTTCTCATGCAAGGCGG 295 TTGCGATCAGAAACACAAGGAGG 296 TATAAGTGTTAAGGGCACCGGGG 297 AAGGTTACTCGGGTTCACAGAGG 298 CGAGACCTGACCATACTGTGGGG 299 GTAGTTCGAACACCCAACCAGGG 300 CAGAGTTTCGTACAGCAGCGTGG 301 TTGTAAACCAAGCTCCACCGAGG 302 TGTCACTTTAGACCAACCCGAGG 303 CCTGATCCAGTAAGTCACGGAGG 304 TTTCTGATGAGAGATCGCGGGGG 305 GTGTTTATGAACTCGCCAGGAGG 306 GTAGAAGATAGAATTCATTGGGG 307 AGTCTCAGCACATGACAACGTGG 308 GGTATACGTTCCAACCTCAGAGG 309 TGTATCACTACAGTTAAACGGGG 310 TGTCGGTAAATATTGCAAAGTGG 311 CAACCCCACATATCAAACCGTGG 312 GGACTGGTGGAACAACCGGGAGG 313 TGATCGCTGTAAGACCAAAGAGG 314 ACCACGTTGCAATATCTGGGAGG 315 GCAATGTGATTCTACACCCGGGG 316 AATGATTTGTCACTTTACCGAGG 317 ACATATACCTTACCCCAGCGAGG 318 TGTAGTAGAGCACACCAAGGGGG 319 TGGCCGGTTCCTATTCCATGGGG 320 GTGAACGCACCTGATCCATGAGG 321 GATCAATTCCAGGAGTTACGGGG 322 TGGTACTCTTGAGGTAAACGGGG 323 GGGCGATGAGGTAATTTGAGCGG 324 TCCAGAGATAAACTCCTCGGGGG 325 ACTAAGATTCATCTACCACGTGG 326 CATCTGGGTAGAGTACGTGGTGG 327 GCTCATTGTATCAACCAGTGTGG 328 GTACACTTTAAGATGCCACATGG 329 CTGTGCGATTGCCCACAAGGAGG 330 TTAGAAAGCACGTCCCAACGTGG 331 AGGCTATTATCTCATAACCGGGG 332 GTATCACTACAGTTAAACGGGGG 333 TTGACCGCCAAAACCAACCAGGG 334 GCTCATCGTAACTAAACCAGTGG 335 GCCGCAAACCAAGACATGTGAGG 336 TCCACATTCACTATTCCGTGTGG 337 ATCCGTAATAGATTGCTGAGAGG 338 GCGCAGCACTGGAACCACGTAGG 339 ATTAAAGTAGACCCCCCCAAGGG 340 ACGTCCTCTGATGAAAAGGGCGG 341 ACCTCCCTCTTGACAAACGGGGG 342 GCATCCAGATGCGACCCCCGAGG 343 TACACACAGCAGAGATCACGGGG 344 TATATCCAGGAGTTTGTAGGGGG 345 GTGACATTGTGATACCCCAAAGG 346 ATCTCCGGGGTATAAGACATAGG 347 GAAACTCACAAATGGCTACGAGG 348 ACTATTCCGTGTGGTGACAGGGG 349 CTTCACTCTAATGAGATACGTGG 350 CGGTGCATAGACACTTAAGGTGG 351 GAGCGTGACAGGAACCCCAAGGG 352 AGTACATGCATCATCCCAAGAGG 353 GTGCACAGGTCGTCTCTCAGGGG 354 AAGTTCTACAATGACACAGGTGG 355 ATACGTGGAACAAATTACTGGGG 356 ATTGAGGAGACACCTACCAGTGG 357 TCTGTGACCCTAATCTACGTGGG 358 TTAGGAAGTTCGATCCAACAGGG 359 TCAGGAGGAGAATGGTCGCGGGG 360 GTTTAAGATGATGTAATCGAGGG 361 CAGGATACATAACCCACAGGAGG 362 CTGTATACCACGAGACATGGAGG 363 GATACTTTTGGTAAACAACGTGG 364 ACTAGTTAGGATAGATGACAAGG 365 GAACACCGTAATGATGGCAGTGG 366 GCGTATCAGCACCACCCCCGTGG 367 GGTTCTAAGCTCAACTCCAGAGG 368 CGTCAAGTATGAAACCGGTGGGG 369 GCCACGACCAGATATCAGCTGGG 370 ATGATGGCACCCGAAGACAGTGG 371 GGGTGAAACAATTCCTTACGGGG 372 TCGCAACTTCAGCATAACAACGG 373 CATAGTATGTCAAACTCACGAGG 374 TCATCATCTTTTTGTCAACGGGG 375 CTAAGATTCATCTACCACGTGGG 376 GAATGGCGGTGATAAGCTAGAGG 377 ATTCGTTTGAGGGTGTTGGGAGG 378 AACCTACTTCCTACCCAAGGAGG 379 TCTAAGACGTTGCAGCAGTGGGG 380 GCTACTACCATATGCCCGAGGGG 381 GAAACTCTATGATACCCCAAAGG 382 CAGGTCATATACAACATCCGTGG 383 GCTATTTGAAAATCCCACGTAGG 384 AAGTGACGTTTTGGTCCTGGAGG 385 GTAATTCCCAAGACGCATGGTGG
TABLE-US-00011 TABLE 11 Target sequences for ATXN2 gene SEQ ID NOS Target sequence 386 GGTAACTCCACAATTCTACGAGG 387 TATGTGGTTCTGTACTTACGTGG 388 ACGATGATCTGGTATCCTGGTGG 389 GATACGCACAAACCTAAGTGAGG 390 ACCGTGGGTAAAGTCCTCTGGGG 391 TACCCCTTTAACCGGCACGGGGG 392 GTCCAAGATAATGACCTGAGAGG 393 AGGCCAGGGATCTATCATCAGGG 394 TGATGACCACGTTCCCCCCGAGG 395 TGTATCCATCTTCACGAGGGTGG 396 AAGTTGTATAGGCACTGACTTGG 397 GAACTTGGTACAGAGACGCTGGG 398 GTAAGTATGAGGATCTCGAATGG 399 TGACGATCAGTTCAGCATCAGGG 400 CAGATCACGTGTATTTGAGAGGG 401 TGTTAAATAGTGCGCCAGTGAGG 402 GATGACCACGTTCCCCCCGAGGG 403 GTGCCGCCAGAGCTTACCAAGGG 404 CCAGATCACGTGTATTTGAGAGG 405 GGAATCATCAGGGTCTGTCGGGG 406 ATTGTTTTGAGATCGTGCCCAGG 407 TGCCCAGTACAAAGCTCGAGTGG 408 AGTTTAGGCCCAAAGCTCCACGG 409 GGGACTGAATATGCGTGCAAAGG 410 TATAGTTACTTATCAACTGGAGG 411 ATTGCGTGGAGTAAGCTGGTGGG 412 GTAGTGTTGTACGATATCATGGG 413 AATCAAACAGCGTGTAACAGAGG 414 ACGGGTAGACATAATAGTTGGGG 415 AATGTGCGAACTTTAGACCTTGG 416 ATATTCAACGATTCCAAGGTCGG 417 AACCCCTCCCAACACGCGTGGGG 418 TTTAGGTGTGAACGTTGGAGGGG 419 CGTCTGTGGAAACCCCGAGTCGG 420 CATATGTTTTAGTGGTATCGGGG 421 TTGCGTGGAGTAAGCTGGTGGGG 422 TCACAGCTCATATGACGTAAAGG 423 GTAAGGTAGATTCTTCACGTTGG 424 CGTGGAGTAAGCTGGTGGGGTGG 425 TATCGCATCGTCAGAACACATGG 426 TCTGACCCAGAATTTGACGATGG 427 AACTTGCGAGTATATTAACAAGG 428 ACCGTGAGTTTATTCTCCCAGGG 429 AGACAGAATTCACCGCGTATGGG 430 TCACAGGATTATATGTACCCTGG 431 AAGCATCCTAAGTGGTGTGTGGG 432 TAAATACCGGTAAACTTGCAAGG 433 CCACGTAAATGGTGTGCAGAGGG 434 TGGGGTGGGTTGGTATACGCCGG 435 GATATGACGTCTTCATGCCAGGG 436 GAACCCCTCCCAACACGCGTGGG 437 ATCTCGAGTGATTGAATCTGAGG 438 AGACGCATGCGATGTATGGTAGG 439 ACAGGCCCCGGTAGTCACTTCGG 440 TACTGAACCGCAATAAACAAAGG 441 ACATCATGTGCGTAACATTGTGG 442 CTAAGTATATCATATTGACCAGG 443 TAGGGTCTTAAGCACCACAAAGG 444 AGAACCCCTCCCAACACGCGTGG 445 CGTAATAAAAAGTTACCGCAAGG 446 AGTGCCGCCAGAGCTTACCAAGG 447 GTCGGCTCTGTCTCTACCGAGGG 448 GCAAGAGTAAACTTCCATAGAGG 449 TGTGGAACATCGGTGGGTGAGGG 450 AGGCCTGTCAAACTTCGTAAAGG 451 GCAATGGAGCAGGTCGTCAATGG 452 TTTTAGGGGAAGTTGTGCTAAGG 453 GGATCTATCATCAGGGCCGAAGG 454 GCGTCTGAACCAAAGATGTACGG 455 GTTACGCACATGATGTATAGAGG 456 CTACCCCTTTAACCGGCACGGGG 457 TATTGCGTGGAGTAAGCTGGTGG 458 GTCGGCAATATAAGTGAACGTGG 459 CTAACCTATAACCTCAGCATAGG 460 ATCCGGTCATATAATCATCTAGG 461 AGTGATCGTTTCCCCAAGTAGGG 462 GTAGGCGCTCCAGTGGCTCGGGG 463 GTTGATGACCCACCATAGATGGG 464 GTTAGGGGGATGGCCGATGTTGG 465 TATACAACCGTTCCTCTCAAAGG 466 ACGATTAACCTCTAACTGCCAGG 467 GAAAACCTAACAACCAAGCTTGG 468 TGATGGTGCTGCAAAGCGACAGG 469 GTTCACACCTAAACCGGGAGTGG 470 GATGAAACTGTTCCACCGGCCGG 471 TCGAGTCAAACCCAGTTAGCCGG 472 GATAACCTATAGTCAGGGCATGG 473 TACGCCGGCTGAACGTGAGAAGG 474 GACACGCAAAGTCAGCTACATGG 475 GTGATTTCGAGGATGTCGCTGGG 476 ATACCACCTGTGTAAACTGCAGG 477 GTGTTCAGTAACACGTTGCAAGG 478 GTCCTATTCTCATTAACCTACGG 479 TGCTTCACTACTTGATCTGAGGG 480 TGGTATGCCCCTATGGATCAAGG 481 CTGTAGTGCACTTTGAGCGAGGG 482 ATACACGTGATCTGGCCCTAAGG 483 TACTTATTGACCTACTAAGCTGG 484 CGTTTAGGCATAGTAGAGACAGG 485 CAACAGGTAGGGGTCATAGAAGG
TABLE-US-00012 TABLE 12 Target sequences for BRCA1 gene SEQ ID NOS Target sequence 486 TCAGGTAGCACTCTTAACCTGGG 487 CCTGTCACCTGTCTATGGGTCGG 488 ACATAGACCCCTCTGTTGATGGG 489 GTAGTCAGACTAGTTCAATGAGG 490 GTAGTTGACCTGCACTCTACAGG 491 GTTTGATGTTTATCCAGACTTGG 492 TACTCCACTATGTAAGACAAAGG 493 GCTTTAACTTGTTAGATGCAAGG 494 AGTGCTAGATACTTTCACACAGG 495 TGTAATTTGGATTCCCGTCTCGG 496 ACGTCATATTTAAGGCATTCAGG 497 GCTAAGATCTGAACCCGAGACGG 498 GTATCTGAAGAACCGTTACCCGG 499 TTCCAAATATCCATACCTGCTGG 500 GTATCTTTACCATCTACCTCTGG 501 CTTTCAGGCAATCACTCCATTGG 502 GTCAACCCTGACATATTGGCAGG 503 ATATGTCAACCCTGACATATTGG 504 GCTGCAGATTAGACTACAAGTGG 505 CCATCGCCACCAATTGTGAAAGG 506 GTCAGGGTTGACATATAACATGG 507 TTATGCTAAGTAACTACCTATGG 508 CACGTAGAGGTTAGATGTGATGG 509 AACTACTCACGAGTACCACGTGG 510 TTTATGTAATGGCCACGTAGAGG 511 GTATTTGGCCACTTACCTGCTGG 512 TACTCACGAGTACCACGTGGTGG 513 TATTTGGCCACTTACCTGCTGGG 514 GGTTGCCAAAAAGTCCAGTGGGG 515 CATCACATCTAACCTCTACGTGG 516 TGGTTGCCAAAAAGTCCAGTGGG 517 GGTAAGTGGCCAAATACATTAGG 518 GTTGCCAAAAAGTCCAGTGGGGG 519 GCAATGCCATTGCCACCACGTGG 520 ATTACTGGTGGACTTACTTCTGG 521 AAGTAAGTCCACCAGTAATTAGG 522 GCTTTGCTACAATCCAATTCTGG 523 GGTACTTGAAGCATCTATATCGG 524 TAGAAAGTAGCCCAGCGCAATGG 525 AAGGTACTTATGTACAGTGGAGG 526 GGTTATATTGGATCCAGAATTGG 527 GTAGAGTGGTTAGCCCAAGGTGG 528 ATGTTGGTACAAGTTATCTCAGG 529 GAGATAACTTGTACCAACATTGG 530 AACTACGAGTGCGCAGACATGGG 531 CTCGGTCCCTCAGAACACGAAGG 532 AAAACGACCACCCCATTGACTGG
TABLE-US-00013 TABLE 13 Target sequences for BRCA2 gene SEQ ID NOS Target sequence 533 CACGTAAGCACTCTCCCACC 534 GTACTTTACCATCATGCAAG 535 AGGCCCCTGATTTACACTCT 536 GTCACTGGTTAAAACTAAGG 537 TCACTGGTTAAAACTAAGGT 538 GTCACAAATTTGTCTGTCAC 539 CCTTAGTACTACTCACAAGG 540 CTTCCTTAGTACTACTCACA 541 CCACCTTGTGAGTAGTACTA 542 GGTGTCTCTCTGTAATACAT 543 CGGTGTCTCTCTGTAATACA 544 GCAAATAACACCTCCCATGA 545 CTCTCAAAGATGGCACGTAC 546 ATATACTACCCATTAATGGC 547 AATCCAGTTCATTAAACCCC 548 ACACTTTGGGTTAGATATCC 549 AACTTCCCCTCATCTACTTG 550 ACTTCCCCTCATCTACTTGA 551 GGGACCCTCAAGTAGATGAG 552 TTGGGACCCTCAAGTAGATG 553 TGGGACCCTCAAGTAGATGA 554 AGGATTATCAAGTACACTCC 555 GATGTGCCAGACGAGTGTGG 556 TTGATAATCCTCTACCCTAA 557 CTTGATAATCCTCTACCCTA 558 TAATCCTCCACCCACACATA 559 TAGAGCCTGCCCATATGTGT 560 CAGCAAGCTGTCATATGATC 561 TCTTGGAACAGACGTGAGGT 562 TAATTCTTGGAACAGACGTG 563 AATTAGGCACCCCAGGATAT 564 TGCAAGAAATTAGGCACCCC
TABLE-US-00014 TABLE 14 Target sequences for C9orf72 gene SEQ ID NOS Target sequence 565 ACACTCCGATGATTATCCACTGG 566 TCTAACTCATCGGGGTCAAGTGG 567 TAGACAAGATCCCTATCCCATGG 568 TACTCTCAACTAAAAGTTAGAGG 569 CAGTAACAGCAAGGTGAGTCAGG 570 ACATGCAATGAGGTAGTGACTGG 571 TGTTACTGACGTGGTATCACCGG 572 GTTACTGACGTGGTATCACCGGG 573 CGTGGTATCACCGGGAATCATGG 574 GAATGTCCGCGCTCCACAGATGG
TABLE-US-00015 TABLE 15 Target sequences for CATSPER2 gene SEQ ID NOS Target sequence 575 GAGACTTCCGGTCCAGAAAC 576 ATTGCCAGGTGAGCTTGACT 577 CATAACTCTCATGTCAGATG 578 TGTCAGATGTGGGCCAAACT 579 GGGATGTCTAGTCTGTAGAC
TABLE-US-00016 TABLE 16 Target sequences for CDH1 gene SEQ ID NOS Target sequence 580 TAGGTTTGGGTGAACTCTAA 581 CCCATTTACAATCAACCTTA 582 GTCCAATCTGCCGTAACCTC 583 TCCAATCTGCCGTAACCTCA 584 GCCTGGTGCTAACACACAAC 585 GGACGGAACATACATGCCAA 586 GACGGAACATACATGCCAAT 587 ACATGAATAAATCGCTATCT 588 CCCTTAAGGTTGATTGTAAA 589 ACCCCAGGTACATGAGTCAA
TABLE-US-00017 TABLE 17 Target sequences for CDK4 gene SEQ ID NOS Target sequence 590 AATCTCTAGGGTACTTCCGG 591 CTAACCTTTGGGAGTGCCTA 592 CAAGTCCTCTTGTATGGCCT 593 gattaagggggtttgtctga 594 TCTAACCTTTGGGAGTGCCT 595 GGGAATGAACTGAAGGCCGT 596 AAGAGCTGTGCAAGTGTCGG 597 TGCAAGTGTCGGAGGTGTGA 598 AAGTTGACTAGGTGTGTGTC 599 GTGCAAGTGTCGGAGGTGTG 600 TAAATGACGCAGGTGTACCA 601 GGTGAGTGGTTAAATGACGC
TABLE-US-00018 TABLE 18 Target sequences for CDKN2A gene SEQ ID NOS Target sequence 602 CAGTTAGGAAGGTTGTATCGCGG 603 CAAGATATACTGGGTCTACAAGG 604 GCTAATTGAGAGGTACCCCGAGG 605 GGTGATTTCGATTCTCGGTGGGG 606 AGGTGATTTCGATTCTCGGTGGG 607 CAGGTGATTTCGATTCTCGGTGG 608 GTACAGGTGATTTCGATTCTCGG 609 AGCCGTTTTACACGCAGGAGGGG 610 CAGCCGTTTTACACGCAGGAGGG 611 GGCTTAACACAGCTGTACCTGGG
TABLE-US-00019 TABLE 19 Target sequences for CDKN2B gene SEQ ID NOS Target sequence 612 ATCTGAGTTATGTGCAACATTGG 613 TATCAGACGCTGCTTGTCAGGGG 614 TGCGGCAATTGACAGCATAGGGG 615 TTGCGGCAATTGACAGCATAGGG 616 CTAGTAAGCGCGAATGCCCCCGG 617 GCTCAAACTAAAGCGCCGCCGGG 618 CTCAAACTAAAGCGCCGCCGGGG 619 TCGCTTCATGGTGAGTGTCGAGG 620 CGCTTCATGGTGAGTGTCGAGGG 621 GGCTTTCCGCCGCTCCCCGTTGG
TABLE-US-00020 TABLE 20 Target sequences for CTFR gene SEQ ID NOS Target sequence 622 GTATGCTTTTGCCCACGGAA 623 GACCCTTGCCTTAGATGTGT 624 TTGCCGACACATCTAAGGCA 625 CTTTATTGCCGACACATCTA 626 GTGTCGGCAATAAAGTAATC 627 TGCCGACACATCTAAGGCAA 628 CACAATAAGGCCAAACAAGT 629 GGTCACACTATGCCACAATA 630 ATGTAGAGTGCCCACTTGTT 631 TCCAAGCACCCTAGACTGTA 632 GATAGAATAGAGCACACCAT 633 AAAGTGATGGCACACCCACC 634 TGGGATAGTATGCACCAGGT 635 ATACTGGGATAGTATGCACC 636 CTGAAGACCTTGCATGATCA 637 AAACACGCTTTCCCCTTCAA 638 GGATAATTAATACGCCATGA 639 GGAACTAGCAGCACCTTTGA 640 CAACTCCTACTGATAACCAA 641 ATTGGTGAGTAAAGGATCCT 642 TATTGGTGAGTAAAGGATCC 643 CATGGAGCTGTTACCATTCA 644 GACTATGTCCTCTTCGGTTG 645 CAGTACTCTATTGTCCCTAG 646 CCATTGTAGGCCAATAAGTG 647 GGAGGGTTGTCCAACCACTA 648 AATCACGATCTCTAAACTGG 649 ATCACGATCTCTAAACTGGA 650 CGGGTGTAGAGATCAAATAA 651 GTAGAGATCAAATAAGGGGC 652 TGGAGGGTTGTCCAACCACT 653 TGCCTTAATCCAACATTGGA 654 AATGTGCCTTAATCCAACAT 655 TTGGATTAAGGCACATTAGT 656 TGCCAGGTTAAGTTGTTCTT 657 GCCAGGTTAAGTTGTTCTTA 658 ACCCTAAGAACAACTTAACC 659 TGTATTAGCAAGTGGACTCC 660 GGGTCAATTGTATTAGCAAG 661 ACACTAACACCTACCCTACC 662 AGATCCTGAACTGTCTAGCC 663 CAGCCTACAAGTTCTTTGAC 664 CTGAGCTAGAGGTACCCTTA 665 GTAATTTAGATCTTAGGACC 666 AGACTTACTTACCAGGGAGC 667 CATGTACATTGGACCCTAAC 668 GGACCCTAACAGGAGTTCCA 669 TGTCTTAGATGATTCTAGTC 670 GAGTTTGGGGGCACACGAAA 671 GCTATTACTAAAGGTTTCTC 672 ATGGCCTTCAAAGTTGGCTC 673 CCCTTGAATAAGAGATATCC 674 ATGGCCTACACGACCCTACA 675 GGTCGTGTAGGCCATCTTAA 676 GTAGACAGCACGATGATTTC
TABLE-US-00021 TABLE 21 Target sequences for CHEK2 gene SEQ ID NOS Target sequence 677 AACGCACTGAGCTGTGTAGGAGG 678 TATTACTTGCAAGCTGAAACAGG 679 GTCATATGGGGAACTTCTGTTGG 680 ACCTCGACGTGTCTCTCGCCCGG 681 TGTTGACACAATACTTCAGCAGG 682 TTGGCAAATCGTATCTATGCAGG 683 CAACGTATGTATGTAGTAGTGGG 684 TCAACGTATGTATGTAGTAGTGG 685 GCAGATGTTCTAAGCTCTTGTGG 686 AAGGTGACCCTTATTAAAGTAGG
TABLE-US-00022 TABLE 22 Target sequences for CLN3 gene SEQ ID NOS Target sequence 687 GGGCATCGATTAGGGGTACGAGG 688 GCCAGAAGGGGCATCGATTAGGG 689 TGGGCGCCCCCCATCAGCTCAGG 690 CTTTCTCGTGCGGTTTTCCCAGG 691 GCTGTGAGGAGCTTCTCGAGAGG 692 AGTCCGACGAAAAGAGGGCCGGG 693 GAGTCCGACGAAAAGAGGGCCGG 694 GCATCATGCCAGGGTGCGCGAGG 695 GTTATCCCCGCCCAGTTCTGAGG 696 CTCCGCTTCTCTTCGGGTAAGGG
TABLE-US-00023 TABLE 23 Target sequences for CLN6 gene SEQ ID NOS Target sequence 697 CATGCACTCCTCAACTGTCGTGG 698 ACCCTTGAGATACGATCTACTGG 699 TTTGGTACGACCTGGATGAAGGG 700 TTTTGGTACGACCTGGATGAAGG 701 AGGAAGTTTTTGGTACGACCTGG 702 GAGTGAGCGGTCATCTTGGAGGG 703 GGTACACACACCTCGTCACTCGG 704 TTCCCGCGTTCCAGCGACCCGGG 705 CTTCCCGCGTTCCAGCGACCCGG 706 GAGCGCCCGCCCGAAGTTTGGGG
TABLE-US-00024 TABLE 24 Target sequences for CNBP gene SEQ ID NOS Target sequence 707 TTAATAGGGAGGGTAGTTCCAGG 708 TGGGGTGTTCGATGATTCAAAGG 709 GTTGGCAGGTATTGCTCAACTGG 710 GCTTCAGAAGCAAATACGAGAGG 711 AGTACTTGATTAGATTGGATTGG 712 CAGTGCAGTATACGTACATCAGG 713 ACCACCTGATTCACTGCGATAGG 714 GTTCCCACATGTTAACCATATGG 715 ATGCGGGTCTTTCGGCGCCACGG 716 AGCTGGGTCGCCGAGCATGCGGG
TABLE-US-00025 TABLE 25 Target sequences for COL3A1 gene SEQ ID NOS Target sequence 717 TTGGTGTGAAGAGTAATAACAGG 718 TCTATACTGCAGGTAAAGCAAGG 719 CTCTCAACTATGATACTTACAGG 720 GACTACCATTAATCCCAGGAGGG 721 GGACTACCATTAATCCCAGGAGG 722 AAACTTACGCGTTCACCACGTGG 723 TATTATGTCATCGCAGAGAACGG 724 TACCGTAATTGTTATACCTGAGG 725 GAAACATTTGTACGTACAGCTGG 726 GCCAGTTTTAGG1AACAATGAGG
TABLE-US-00026 TABLE 26 Target sequences for CRB1 gene SEQ ID NOS Target sequence 727 CCTGGAAACGAACAGCACCAAGG 728 CAGTAACCTAACTTACAGGGTGG 729 GACTCAAAGATAGTGCCGGGAGG 730 TCATCAGTAGAGATCCTGGGAGG 731 GATAAGCTCTGGTAACAGGGTGG 732 GACATGTGTATTCTATACGGTGG 733 ACCTCCAGCATAACACAAGGAGG 734 TAGGGGCTAAAACCGACATGCGG 735 TGAATTGCAGGAACTCATCGCGG 736 CTATAAGTAGAACGTCTGGGAGG
TABLE-US-00027 TABLE 27 Target sequences for CRX gene SEQ ID NOS Target sequence 737 ACACATCTGTGGAGGGTCTTGGG 738 GGCGTAGGTCATGGCATAGGGGG 739 GGGGCGTAGGTCATGGCATAGGG 740 CGGGGCGTAGGTCATGGCATAGG 741 ATCCCGGGATCTAAACTGCAGGG 742 CATCCCGGGATCTAAACTGCAGG 743 GCGGTCACAATCGTGCCAGACGG 744 GAGCTCGTGGTGTACTTCAGCGG 745 CTTACCAGTTACTCACCATGGGG 746 CACTTACCAGTTACTCACCATGG
TABLE-US-00028 TABLE 28 Target sequences for CTNS gene SEQ ID NOS Target sequence 747 GATGCCACGTACAGTTACCGAGG 748 GGTCTTAGAAAACCATCGTGGGG 749 TTATGCGCTTCCTTACACGAGGG 750 GAATCACAGGAGATCGCTAGCGG 751 ACGATCAGTCTCCAGCATGTGGG 752 ATTGGTTACTTACTTCATCGGGG 753 AGCGCAGAGGAGATTCACGATGG 754 AGAATGCTCACCCACGCAGGAGG 755 CTTGACGCCGCAATCCTCCAGGG 756 GCCGATGTTGAATACACTGTAGG
TABLE-US-00029 TABLE 29 Target sequences for CYPC1 gene SEQ ID NOS Target sequence 757 AATCTCTGATAGTATAAGATAGG 758 CTCATCATTAAACGTCACTACGG 759 GGGGGGGTCTCCCTACAGTAAGG 760 CATAAGTGTGGTGGTATCATGGG 761 GACTGTAACGCTTGTGCGATAGG 762 GTACCGAGGGTCAAAGATGGTGG 763 GCAATCAGGAAACCTCGTGTAGG 764 TGTCCAAACAAGTAACTACCAGG 765 CAAGTAAGCTCAGTGATCCAAGG 766 ACACCGATCTTTATCCCCCTGGG 767 GACACCGATCTTTATCCCCCTGG 768 TCTCGCTATTGAAACATTGTTGG 769 TACTCTTATACCCCAAAGTGAGG 770 GAGGGTCCAGATCAATCCATTGG 771 TGAACATTCGACCTCCATTACGG 772 GCAAGAGGCATAATGTGGGCAGG 773 CAATTCTGAATCATGACAACAGG 774 GATTCAATGGGTAATCCCCTTGG 775 TTAGTTATACTCTACACATAAGG 776 GTCCTGAGTACTTGGATACTTGG 777 GTACTGCCCTTCTTTGGAACGGG
TABLE-US-00030 TABLE 30 Target sequences for CYP2C19 gene SEQ ID NOS Target sequence 778 GGAGAACTATTAGTCATTGCTGG 779 TAGTAGGCTATATTAAATAGAGG 780 GTTAAGGGTCATCACTTTCAGGG 781 CTAACGTTTAAATCTTTGGCCGG 782 AGTATTGTAATCTATATGGGAGG 783 GTCCCCTCAATATTAGTATTTGG 784 GGGGCGCACGCATGTGTGACAGG 785 TTGTTCTGGCTACTCTTAAGTGG 786 GTACTAAATCAGTGACCTCAGGG 787 CTTATGTCAAGGGAATCCACTGG 788 AACTCCTCACTCACCTCTATAGG 789 TTGCTAAAATGCCCACAATCAGG 790 ACTGTTCGGTGAATCATAGGAGG 791 AGAACTGTTCGGTGAATCATAGG 792 CTACATATACTGCAGTATTGAGG 793 TGAATATCCCAATATAGATCAGG 794 GCGAATATAATACGTTTTTGTGG 795 CTTTAGTCTGGTGGCCACATTGG 796 AATAGACCTGCTGAATATGTTGG 797 AATGGCCCTATCACACCCCTAGG 798 ACGAGGAGTATGTACAAGGGAGG 799 CAGGTTTGTCATCGTACCCCAGG 800 TTATCCGATTTTACAGTGTGTGG 801 AAAGGAGCACCGGGCTGTATGGG 802 TAGTTACACCCCCATTGGAAGGG 803 ATAGTTACACCCCCATTGGAAGG 804 CTTTATAGTTACACCCCCATTGG 805 GGCTCCACTCCTCAATCTTAGGG 806 GGCTACTCACCCTTCAACTGGGG
TABLE-US-00031 TABLE 31 Target sequences for CYP2D6 gene SEQ ID NOS Target sequence 807 TCCGGTGTCGAAGTGGGGGGCGG 808 GAATCCGGTGTCGAAGTGGGGGG 809 CGGCCCGAAACCCAGGATCTGGG 810 GACGAGATCTCCAAATGCCCAGG 811 CCCTCTACAGGTGGATTGTATGG 812 GCCATACAATCCACCTGTAGAGG 813 CGGGGTTGATAAGTCCGCTGGGG 814 GGGGTTGATAAGTCCGCTGGGGG 815 ATAAGTCCGCTGGGGGTGACGGG 816 GGCACAGGATTCACTTATTGAGG 817 GCAGTCCGGTGGAGTGCTGTCGG 818 CTTTCCGACATACACGCAATGGG 819 AATTGTTCCAATCTGCTCTTGGG 820 CGGCTGGAACCTGCTGATCTCGG 821 GGGCGATAATGTGGCAACTCCGG 822 GAAGCGAAGTCTTTGCCGAGTGG 823 AAGCGAAGTCTTTGCCGAGTGGG 824 CCGGGCGTGGCTTCAGTGCTCGG 825 ACCTCCGGTTGCTTCCTGAGGGG 826 GTCAAGACAAGTTCTCACAGAGG 827 AAGGCGAGGTCGTTAAAGAAAGG 828 AGGCGAGGTCGTTAAAGAAAGGG 829 CAGCCTCGTCACCTCACCACAGG 830 ACGTACCCCTGTCTCAAATGCGG 831 GCCTGGCCGCATTTGAGACAGGG 832 TCGAAATCTCTGACGTGGATAGG
TABLE-US-00032 TABLE 32 Target sequences for CYP11B1 gene SEQ ID NOS Target sequence 833 CGCGTGTTCCTCTACTCTCTGGG 834 GCGCGTGTTCCTCTACTCTCTGG 835 ACAGTACACCAGCATCGTGGCGG 836 TCAACAGTACACCAGCATCGTGG 837 CATGACGTGATCCCTCTCGAAGG 838 CAAGGCTCTTGGATAAGATAAGG 839 GCGTACCAGATGACGAGAGTGGG 840 AGCGTACCAGATGACGAGAGTGG 841 GCTGCTAAACCGGGTCAGGTGGG 842 ATGGTGAGGAGCGTACCATCTGG 843 GAGCCGGTACTGGGAGAACCTGG 844 AACACGCGCACCAATGTCTGCGG 845 GACCCCCCGAGCTGCGACACTGG 846 ACGATGCTGGTGTACTGTTGAGG 847 TGACCCACAGGGCTTATCAGTGG 848 CAATGCAGGCACACCCCATTTGG 849 ACGCCGGGGCATGGCTTCAAAGG 850 CTTCGAGAGGGATCACGTCATGG 851 TTCGAGAGGGATCACGTCATGGG 852 GGGGGTGCATGAGCGTAGACAGG 853 GGATTATTCATCTCCTTGCAAGG 854 CTTAGAGATTTTCAAGTCCGTGG 855 TCATGCCCACTCTCGTCATCTGG 856 ACTCTCGTCATCTGGTACGCTGG 857 ATCACCAAATGTATTAGTGCAGG 858 GCACCGTTCCCCCTTGATACTGG 859 CTGTCAGTTCGAGGTGAATCTGG 860 AGTAGTGCATTCTGAACTGAGGG 861 GGTAAAAGGCTCTTTGGGGGAGG 862 GGTTATTAAGGATTGCCACAAGG 863 ACCGGTGAGTCATTCCAGTCTGG 864 CCGGTGAGTCATTCCAGTCTGGG 865 TGGTATATATGAGTGCTGTAGGG 866 GGCTGGGTACACTCTCAAACTGG 867 ATCCGGCCGGCCCAGAGTTCAGG 868 GTATGGCCACACGAGGAGCCTGG 869 TCGGGAGTTCCATTTGTGCTGGG 870 CGGGAGTTCCATTTGTGCTGGGG 871 GCAGAGACGTGATTAGTTGATGG
TABLE-US-00033 TABLE 33 Target sequences for CYP11B2 gene SEQ ID NOS Target sequence 872 TGTGAGAACCCGCCCTGAAGAGG 873 AACCGCCCTCAACACTACACAGG 874 CTTGTTGTAAGCGGCGAGTTGGG 875 TCTTGTTGTAAGCGGCGAGTTGG 876 ACGTATCGAGATTCCTCACATGG 877 CAGAAAAGCTCGTCTATGTCAGG 878 GGCTCTATGAATCTGAACTACGG 879 ACCTCTTCACTGCGTCAGCACGG 880 TGCGGCCAGACCTATGGGCAGGG 881 GTGCGGCCAGACCTATGGGCAGG 882 GGGGGGTGCGGCCAGACCTATGG 883 CCTTGGGCGACAGCACATCTGGG 884 ATCCCCACCTAAACACTGTCGGG 885 CCCCACCTAAACACTGTCGGGGG 886 CAGTGCAGACGCGACCCCACAGG 887 ACAGTAACCGCACCCCCGCCTGG 888 CCCAACCCGTGAACATTACAAGG 889 CCAACCCGTGAACATTACAAGGG 890 CTCACATGTGGCACGCTACACGG 891 GTACTTCCCGAATTACCAAATGG 892 CTGAGTTGAGGGCCGTTCTCAGG 893 ACCAGGCACCGAACCTTGCAGGG 894 AATCCCGAGATCTGCTCCGCTGG 895 AAGGCACTCACTCCAAGTTGAGG 896 AAGTTGAGGGGGGCGGCACCTGG 897 CAGAAAGGGCCGACCGCGGTGGG 898 CACCCCTCCGCATTCTCGTCAGG 899 ACCCCTCCGCATTCTCGTCAGGG 900 CAGAGCTTGCCGGCTAACTTGGG 901 AGAGCTTGCCGGCTAACTTGGGG 902 CGTTTCAGCGGGTGATTGCTCGG 903 TTTCAGCGGGTGATTGCTCGGGG 904 CCCGAGTCAAGTCCTTCCAACGG 905 CAACCAACATCCGCCCGCACAGG 906 TCCCGCTGTCATGTCAGAGCTGG 907 GCAACTGTCTTCGAATAGGCTGG 908 CAATAACATTGGCCAACCTCTGG 909 ATGGATCATACTGTTGTTCCAGG 910 ATTAACCATGGATTGTACCATGG 911 TTATGACCAAAAGGCCCCCATGG 912 TAACCACGCAACTTAGGCTCAGG 913 AGCTACACTAAGGCATGAACTGG 914 ACTTAGATGAAGGTGTTCGGGGG 915 TCCCGTGCCGAAGAGACACCTGG 916 GCCCAAGGCAGGTTCACGTAGGG 917 CATGGCTCCGTATCAACCAGAGG 918 TTACCAAAGTGTGACCTCGATGG 919 TAATCGCTCTGAAAGTGAGGAGG 920 TCTCCATGTATGAGCACTCCCGG 921 ACGCCGACCTCAACCAACCAAGG 922 CATTGCGACCCAGCGAGTAGAGG 923 GAGGTTACCGGTATGGAGCCAGG 924 CCTGTACCAATGTCTGCGGACGG
TABLE-US-00034 TABLE 34 Target sequences for DMD gene SEQ ID NOS Target sequence 925 CACTCATGCATCCTCTTAGATGG 926 GTGGTGGTTGACTATGGTAAGGG 927 AAATCCGAATCCCCAGGCCAGGG 928 GTGGCCAAATCCGAATCCCCAGG 929 TGAAACTCGCATTCATAAGGAGG 930 GCATTCATAAGGAGGCACACAGG 931 GTATATAGCAGTGCATGCCAGGG 932 ATTGTAATAGAGGAGGCCATAGG 933 TCTTGCCATATATGATCCTATGG 934 GTCCTCAGGAATACTGCCATTGG 935 AAGTACCATCTACACAGATCAGG 936 GCATCGAATCTCAAGAAATATGG 937 GATCTCAACATAACGTCTTCCGG 938 GGAAATGTAGTGAAGATCGGGGG 939 GGGAAATGTAGTGAAGATCGGGG 940 TGGCTAGCTTTCCCTACCAAAGG 941 GATCAAGTGCTTAATAGAGGTGG 942 TTGGTAGGGAAAGCTAGCCAGGG 943 AGGGTAAACAGGAACGCTTCAGG 944 ATGGATGGCCCTGAAGTCACAGG 945 GTATGGTGGGTCCTAAACAATGG 946 TATGGTGGGTCCTAAACAATGGG 947 CATACCTGCTACACGTATATAGG 948 GTCCAAATTGCTCTTTGAGCTGG 949 AATTCCTATATACGTGTAGCAGG 950 GGGATCTCAAGGATGTAGGCAGG 951 ATAGATTTCATGACGTACTAAGG 952 CCATAAGATTGCCTCAACTCAGG 953 GCCTCAACTCAGGTGTACCTCGG 954 CTGAGTTGAGGCAATCTTATGGG 955 TGAGTTGAGGCAATCTTATGGGG 956 ACGTCATGAAATCTATATAGTGG 957 CATTCCACTCAGGTACCTAAAGG 958 GATCTATCTGGCTTTCAATCAGG 959 TTTAAGGGGATCATTGCCACTGG 960 GTGCATACATACAAGTTCTATGG 961 TAGTAGGTGCTGGTATCACAAGG 962 GCTGTGGATTAGGCCTAGATTGG 963 CACGTCTTCTGACAATGAGATGG 964 ACGTCTTCTGACAATGAGATGGG 965 CGTGTTAAATATCCCTGTGTTGG 966 TAACACGTTGATTGCTGTTAAGG 967 TACTGCAAACCAGCCAACACAGG 968 ACTTGAATTGGAGCAATGCCTGG 969 GTTAAGGCTAAGATGTAGTTAGG 970 ATCTGGCTTTAGAGCTGAATGGG 971 TAACCACCACTCCTTCGTCACGG 972 GCACTATTTTGGTGGAATGCTGG 973 TAGTGGGATCACATCCCTGTGGG 974 TGGAAATTAGCCCGGTGGCATGG 975 TTACATGGAAATTAGCCCGGTGG 976 GAGATGGCATTACCCTTAGATGG 977 GAATGTTCTTGGAGAAGCGTTGG 978 AATGTTCTTGGAGAAGCGTTGGG 979 TCGGTGAGGTGAAAGATTAAAGG 980 GTGCATTTTAGAAATCGGTGAGG 981 ATGATACCCTTAAGGTACTTGGG 982 TCATAGACCCAAGTACCTTAAGG 983 CATAGACCCAAGTACCTTAAGGG 984 AACTATAGGTCCCACCCAACAGG 985 AGTTTGATGTGCTTTTCGAAAGG 986 ATGTGCTTTTCGAAAGGTTATGG 987 GTTCCTCAGAGCCTATGCCAGGG 988 TTAGGCCTCTTTCGGAGAGAAGG 989 AACAGTTTGTGTCGGTATAGAGG 990 AGATTTCAGGAGCCTAATAGAGG 991 GGCTATATTGTTGTCACAGCAGG 992 GAAGACCCAATCTTGACACCAGG 993 CTATACTGTGCCCTAAGATGAGG 994 CTGACCCTGGTGTCAAGATTGGG
TABLE-US-00035 TABLE 35 Target sequences for DMPK gene SEQ ID NOS Target sequence 995 GGGCACTCAGTCTTCCAACGGGG 996 CTGGTCATGGAGTATTACGTGGG 997 GATGGCGCGCTTCTACCTGGCGG 998 CGTCATTGGCTGCTTCCTAGCGG 999 GCGGTTGATCGACAAGACCAAGG 1000 TGGGCAGACGCCCTTCTACGCGG 1001 CAACTCCCCGAGTGGCACAGTGG 1002 ATAAATACCGAGGAATGTCGGGG 1003 GAAGTAACCTCGTCTCTCCGTGG 1004 AGTCCCCCACGTATATGGCAGGG 1005 CGAAGTTCTGGTTGTCCGTGCGG 1006 GACATTCTACATGAGAACGTGGG 1007 CCTTCTTATGAAACCCTTGGGGG 1008 CCCCTCTTCTCGACGCTCGGTGG 1009 GCCTGACGTAGTAAAGATCGGGG 1010 GGAGAGCGGTACCACTTGTGGGG 1011 GCTCCCGTTCACCAGGATGGAGG 1012 GTCTCAGTGCATCCAAAACGTGG 1013 AACCGCATCGTGAAGCAGGACGG 1014 TTGCGAACCAACGATAGGTGGGG 1015 GTGGGGTTCGCACTCTTACGAGG 1016 CAGCGTGCCCCCCTTTACACCGG 1017 GGACATTCTACATGAGAACGTGG 1018 GTCCTTCACCGAGGGCCGCGTGG 1019 TACATGGGAAGGTGGATCCGTGG 1020 TGCGAACCAACGATAGGTGGGGG 1021 CCAGGCCGTTGATGATGACGGGG 1022 GGGCCACACCCGTCACGATGGGG 1023 CAAATGCGCAGCTAAGCGGGTGG 1024 CCGGCCCACAACGCAAACCGCGG 1025 AAGAGGCATAGGGCGCGTGGAGG 1026 TCTAAAGTCGCAAAGACGTAGGG 1027 CCCAATAGAGGCTAAAACGGTGG 1028 GAAGCTCCCGTTCACCAGGATGG 1029 GTCATGGAGTATTACGTGGGCGG 1030 CACTTAGTCCCCGCGCCCCGCGG 1031 AGGTTCACGTTTCACAACAAAGG 1032 TCGAGCTTGCGTCCCAGGAGCGG 1033 GTCAACCTCACCCCCTGCGGTGG 1034 AAATATCCAAACCGCCGAAGCGG 1035 TAGGGTTCAGGGAGCGCGGGCGG 1036 ATGAAATGCGGGGTGTCGGAAGG 1037 GGCGCTTCTCGTCCGGCGTGGGG 1038 AAGATCCGCCCTCCTGCCGTGGG 1039 AAGCCTGACGTAGTAAAGATCGG 1040 AGCAAATTTCCCGAGTAAGCAGG 1041 CGGCCGGCCGCAGAGAGAAGTGG 1042 GCGAGGTCAACACCCGGCATGGG 1043 TGCGTCTTCAGCACCAATGTCGG 1044 GCAGCGGTTCAGAATCAAGCTGG
TABLE-US-00036 TABLE 36 Target sequences for EGFR gene SEQ ID NOS Target sequence 1045 GGGCACTCAGTCTTCCAACGGGG 1046 CTGGTCATGGAGTATTACGTGGG 1047 GATGGCGCGCTTCTACCTGGCGG 1048 CGTCATTGGCTGCTTCCTAGCGG 1049 GCGGTTGATCGACAAGACCAAGG 1050 TGGGCAGACGCCCTTCTACGCGG 1051 CAACTCCCCGAGTGGCACAGTGG 1052 ATAAATACCGAGGAATGTCGGGG 1053 GAAGTAACCTCGTCTCTCCGTGG 1054 AGTCCCCCACGTATATGGCAGGG
TABLE-US-00037 TABLE 37 Target sequences for EPCAM gene SEQ ID NOS Target sequence 1055 AGCAAATGATTCAACACCGGGGG 1056 AGGCTTTATATATGCCCCTCTGG 1057 GAGGTCTCTAAATCTATCAAAGG 1058 AACGGCAGCAGCGAACCATTTGG 1059 TCTAGCTGCCATCCCACTGAGGG 1060 TTAGGGTACTTGGGATACGAAGG 1061 CAGTCCCCCTCGCTACCCATTGG 1062 AAGATGAAGTTCTCCCGATTAGG 1063 AAAGATCCCTAACGCCGCCATGG 1064 CGCCATGGAGACGAAGCACCTGG 1065 CAACGAGCACCAGCGGCCAGAGG 1066 GCGAGCGAGCACCTTCGACGCGG 1067 CGAGCACCTTCGACGCGGTCCGG 1068 GAGCACCTTCGACGCGGTCCGGG 1069 AGCACCTTCGACGCGGTCCGGGG 1070 CCCCGCAGGTCCTCGCGTTCGGG 1071 GTTCGGGCTTCTGCTTGCCGCGG 1072 GCTTCTGCTTGCCGCGGCGACGG 1073 GCCCTCCGCGCGGTAGGAAACGG 1074 GTTTCCTGCGGCCACCGAACCGG 1075 CCCTGGCGCACCCACGTCCTCGG 1076 GCGCACCCACGTCCTCGGTTCGG 1077 GCACCCACGTCCTCGGTTCGGGG 1078 CCCACGTCCTCGGTTCGGGGTGG 1079 GGCCGCTATGCACCTGCGCGCGG 1080 GCTATGCACCTGCGCGCGGCAGG 1081 TATAATATTGCCCCAGCAGGTGG 1082 ATAATATTGCCCCAGCAGGTGGG 1083 TGTGTAATACTGATGTTCCCAGG 1084 GATCACAACGCGTTATCAACTGG 1085 ACAGTAGTAGGAAAGGCGTTGGG 1086 TGTTGATACAAGCTGTGCACAGG 1087 ATATTCTTGCGTGAGTTCCATGG 1088 CCATTCTGTAGTAGGTCATCTGG
TABLE-US-00038 TABLE 38 Target sequences for ERG gene SEQ ID NOS Target sequence 1089 CGGCACTGAATACATCCCAGAGG 1090 GTATTACATTGAGAACCATGTGG 1091 GGAATCTGACGATATCCCTGTGG 1092 AAAGCTGGTTCGATGCAGTGGGG 1093 ATCAGAGTCTACTTACAGCGAGG 1094 ATAACGTGATCACAGCGTGGCGG 1095 TTAATAACGTGATCACAGCGTGG 1096 GCTCACGAACACCATCACATGGG 1097 GATGCACAGAACACGCACAAGGG 1098 CGGGGCACAGGAGTACACCAAGG
TABLE-US-00039 TABLE 39 Target sequences for EVC gene SEQ ID NOS Target sequence 1099 TAGGTGGAAGATCTGAACCAGGG 1100 CCACCACACTCTCAATACGGAGG 1101 ATGCCTGAATAAACCCACCGGGG 1102 GCGATGCCCTGTGAGCAACACGG 1103 ATTTGAGAGATCCATCCGTGTGG 1104 GTGTCATCCCAATAACAGCGGGG 1105 TGTGGCTTAGATACCCTGGTAGG 1106 GCGCCCAAACCGAATCAGAGCGG 1107 GTGATGTGAGATCGTCAGGGAGG 1108 AGAGCGAAACCAGAGCTCGGTGG 1109 ATAATACAAGCATACCATGGAGG
TABLE-US-00040 TABLE 40 Target sequences for EVC2 gene SEQ ID NOS Target sequence 1110 GTATAGAAGACGAACCCCAGAGG 1111 ACCTACAATGTACCGCACAGTGG 1112 CGTAAGTGAACCCACCACAGGGG 1113 GCCGAAGCGTTAGTGCACAGTGG 1114 GGCGTAATCAGCAAACAGCGGGG 1115 ACAGGCTATATAGTCCAGAGGGG 1116 CCACCACACTCTCAATACGGAGG 1117 ATTGCGAAAGAATGGCCCAGAGG 1118 TAATATCTTTGAGTGCTACGGGG 1119 GCGCCCAAACCGAATCAGAGCGG
TABLE-US-00041 TABLE 41 Target sequences for F8 gene SEQ ID NOS Target sequence 1120 ACTGTAGTAAGAACACAACGTGG 1121 GTACACAGAATGACGCCACGAGG 1122 GTTGTGGGAGTGGAACTACGTGG 1123 TTATGGGCAGACAACCACACAGG 1124 CCGATCTGAGATACCCATGAAGG 1125 TACGATGGTAGACACAAAGGAGG 1126 GGACACACCCCACTAAACGATGG 1127 TGTATCGAGCAATAATTGGAGGG 1128 TGTATGCACTACTTCTGGAGGGG 1129 TGTTACGATGGTAGACACAAAGG
TABLE-US-00042 TABLE 42 Target sequences for FBN1 gene SEQ ID NOS Target sequence 1130 GAAGTCCAAGTACTACACAGTGG 1131 ATAACAGAGTGATACCCACGAGG 1132 CATATGTTTAGTCCACATGGGGG 1133 ACACTCGTCATTCAGCACCAGGG 1134 GGTACATACAAACACCTCTGGGG 1135 TGCTCATACGAAGACAACCGAGG 1136 GAGTGTATCAGATCACCTAGAGG 1137 TTTCTCCTTACCGATACACGCGG 1138 TACCAATACACTCCCCACGGAGG 1139 ACATACCATCAGGTTCCGTGGGG
TABLE-US-00043 TABLE 43 Target sequences for FGFRI gene SEQ ID NOS Target sequence 1140 CATGTGTTAACAGTGCATTGCGG 1141 GAACACGCTTGATACACATGTGG 1142 TACTGATCCAACATACAGGGTGG 1143 CTAAATTACAGTGACGAGGTGGG 1144 TGAGGAATGATCCCATTCGGGGG 1145 GTTGCCCGCCAACAAAACAGTGG 1146 GCACTGTCAAGGCTACGTGGGGG 1147 GTGAGGAATGATCCCATTCGGGG 1148 CCTCGACGTCCATCCAACTGAGG 1149 ATGAGTCCAGAAGTTGCGGGGGG
TABLE-US-00044 TABLE 44 Target sequences for FGFR2 gene SEQ ID NOS Target sequence 1150 TGACCAAACGTATCCCCCTGCGG 1151 GTGCGTTGCTTGGATCAATGGGG 1152 CAACTGTTACCTCCCACCCGGGG 1153 AACCAGTGCACTAAACACGTGGG 1154 TCCAGGAGTACTATCCACCTGGG 1155 AGACCAATGAGATTCCACGTGGG 1156 GTTGCGTTGACGTAATGACAGGG 1157 ACTTTAAAGTCCCCGCCATGTGG 1158 ATGACGTTAACACCCAGCAGAGG 1159 GAGGCCCTTAGAGCGTTCCGAGG
TABLE-US-00045 TABLE 45 Target sequences for FGFR3 gene SEQ ID NOS Target sequence 1160 ATCGTGAACGTATTGCCAAGTGG 1161 GAATTGCCGCTCACACCACAGGG 1162 GAGATCGCATGGCTCCCAGGGGG 1163 TTTCCGTCATGACCGCCGTGTGG 1164 AGAAGCTCCGTACCCCCGGGAGG 1165 CATCGTGGCACAGACATGGGGGG 1166 GACCCCCAAGGTACAGATCGAGG 1167 GTTAGAATATACCTCGTGTGAGG 1168 GTGCGTAGTGGGCAGAACGGCGG 1169 CGTGCAGGTGAGGGTCATCGTGG
TABLE-US-00046 TABLE 46 Target sequences for FMR1 gene SEQ ID NOS Target sequence 1170 TACACTAACCATCATAGTAG 1171 GGCATACTCGGTAGCAAACTAGG 1172 AACAATCTGCTATCAGTAAC 1173 CTGGGTTTGAGCACATCAAT 1174 GTATGTTTGCAATACAACACTGG 1175 AAACTGCTGGAGTACCCCAA 1176 AAGAGGACTATAACGGCAAG 1177 GCTTAAATTAGAGTGGCCCTTGG 1178 GATTGGATATGTCTCATTGCCGG 1179 CTTAAATTAGAGTGGCCCTTGGG 1180 TGCCAGACTTGGAGTGCCAAAGG 1181 CAACTATTCTAATGGCACTTAGG 1182 GACTGCATCAACTATTCTAA 1183 TAATGGCACTTAGGTGCTGAGGG
TABLE-US-00047 TABLE 47 Target sequences for FXN gene SEQ ID NOS Target sequence 1184 GTAATCCAGATACACCCAAGAGG 1185 GGCCTAAAGTAAGACACCAGGGG 1186 CTGCTGTAAACCCATACCGGCGG 1187 ACTTAGGGCAAGGTTACACAGGG 1188 TATCAGAGTATAGGGCCAAGGGG 1189 TACCCTGAGAGGATCGCATGTGG 1190 TATCTGACCCAGTTACGCCACGG 1191 TTTCAGAGTTCGAACCAACGTGG 1192 GGGCCTAAAGTAAGACACCAGGG 1193 GAAGTCAAGAGGTACCCCAAAGG
TABLE-US-00048 TABLE 48 Target sequences for G6PD gene SEQ ID NOS Target sequence 1194 GGTTCTGCATCACGTCCCTGGGG 1195 GCCGTGAGTTGATGTGACATGGG 1196 GGGGATTCGGGAGCACTACGCGG 1197 CAATGACAATATGCGTGGAGCGG 1198 AAAAAACCCGGTAAATTGCGGGG 1199 GUTTTTGAAACGAGGGCCCAGGG 1200 ATAATGGGAGAGGATTGCGAGGG 1201 GCTTCATCTCAAATTACACGTGG 1202 CTCGGTAATGATAAGCACGCCGG 1203 AGTAGGCGCCCAGAGCTGAAGGG
TABLE-US-00049 TABLE 49 Target sequences for GAA gene SEQ ID NOS Target sequence 1204 AATAGCAACGAGACCTGAGGGGG 1205 AGTTGGCATCAGTTCCAACGAGG 1206 CCGCAGGCTGAACACGACGGTGG 1207 ACCGTCCCCACTCTACAGCGTGG 1208 CTTAACGCACGCCAGAAACGCGG 1209 TCCAGCTAACAGGCGCTACGAGG 1210 TTGATTATATTCTCTCACGTGGG 1211 CTCCTTGATAACCTACACTGCGG 1212 GGTCGTACCATGTGCCCAAGGGG 1213 GCGGTCATTATAAATCTGCGTGG 1214 AACGCGGTGCTGCTTCAACACGG
TABLE-US-00050 TABLE 50 Target sequences for GALC gene SEQ ID NOS Target sequence 1215 TACTATACACCCACAATTAAGGG 1216 ACGCCGCTTTCATGATGTTCTGG 1217 ATGGGGCGCTGTTTTCTATCAGG 1218 TACTACTCAAACCACTCCTAAGG 1219 AATACGAATGCTGGTCTGTCTGG 1220 ATGTATGGCCCACTACTTAGTGG 1221 GTCTTGGAAGTATAACGTAATGG 1222 CCTCCCTGGTTAGAGAATCAGGG 1223 TACAGAGTATATGGGTCTTGTGG
TABLE-US-00051 TABLE 51 Target sequences for GALT gene SEQ ID NOS Target sequence 1224 GAATGAGCTCAATACCCCCGAGG 1225 AGGCAGACCTTATCACCCTGGGG 1226 GCTTGTATCAACATTCCCCAAGG 1227 GATCCGCTGGAAAATCTGCAGGG 1228 GAAGTCGTTGTCAAACAGGAAGG 1229 TGGGGATTCACCTACCGACAAGG 1230 ATGTCTGCCAGCGTGAGAGTGGG 1231 gtataagcgctcgtgacagaggg 1232 CTAGGCAGACCTTATCACCCTGG 1233 GACAATTCACTAAGAACCCTGGG
TABLE-US-00052 TABLE 52 Target sequences for GATA6 gene SEQ ID NOS Target sequence 1234 GCCGAAATAAATCAACCCTGGGG 1235 TTTTTLLGCGAAGTGCACGGGGG 1236 GCCAATATAGGAGAACGCGGCGG 1237 ACCCGAGTTAAAGTTCCCAAAGG 1238 CCGGGGGAGACACTTTAGGGCGG 1239 TACTCCAAACAGTCCTACCCCGG 1240 CTlTTTATTCACCAGCAGCGCGG 1241 CTTATTGATCTCCACGCCCGGGG 1242 TCGAATCGCGAATAGTGGTGTGG 1243 TCGCGAATAGTGGTGTGGCGCGG
TABLE-US-00053 TABLE 53 Target sequences for GBA Gene Cluster SEQ ID NOS Target sequence 1244 AAGCCATGGACGTTAGTAGT 1245 TAGAAAAGAGGGCTTACGGT 1246 AGCCATGGACGTTAGTAGTA 1247 AGGGCTTACGGTGGGCAATG 1248 AAAGATGGTACTTAAAGCCA 1249 GTAGAAAAGAGGGCTTACGG 1250 ACCAGATATGCTGAGTTGGA 1251 AGTTGGATGGCGCTCAAGAG 1252 TCCAACCAGATATGCTGAGT 1253 CGCTCAAGAGAGGTCAAGGC 1254 ACCTCCACTCTTTCTATAGG 1255 CCTGCTGAACTGCTTAACAT 1256 AAACTTTCCAGTGACCACAG 1257 TTGAATTTGTCCCTTTGAAA 1258 CTGCATCTCACTTGACCTCG 1259 CTGAACTGCTTAACATTGGA 1260 GCTGAACTGCTTAACATTGG 1261 CTCCTCCTTTTCACAGCAAT 1262 GCTTAACATTGGAGGGCCCC
TABLE-US-00054 TABLE 54 Target sequences for GCH1 gene SEQ ID NOS Target sequence 1263 CGCGATAGATCCTGTGGTATTGG 1264 GGGGTTACTTCGTACTATAATGG 1265 TAGTCTAAAGTCAACTTGATTGG 1266 CTACTAAGCATTAAGACAACAGG 1267 ATGGCGATTGAGCTGGGCGCAGG 1268 CACTACACCACTTTTATTGGAGG 1269 ATTGATGAGGTCGAGGAGCCGGG 1270 GTTTGGCTAAATGTTCGCACTGG 1271 GAACTTGGCCAATCAATCTTCGG 1272 GTTCAGGTGCGTGGAAGCTATGG 1273 ACTAACTGGAAGTTTTGCCCTGG 1274 TGGCGATTGAGCTGGGCGCAGGG 1275 CACCATTATGACGTTACTAAAGG 1276 TCTGTGCTCGTTCAGGTGCGTGG 1277 AGTGCATTTTCACAGATCGTTGG 1278 TGTAAGGCGCTCCTGAACTGTGG 1279 ATACGCTTTGGTTAAAACGTTGG 1280 GGTCCCTGATAGAACCAGAATGG 1281 AGGCAACGCGATAGATCCTGTGG 1282 GTTACCAAGCACCTCCATGGAGG
TABLE-US-00055 TABLE 55 Target sequences for GJB2 gene SEQ ID NOS Target sequence 1283 GCACTGATGGAACCGTCCTGAGG 1284 CCAAGTACAGGAGAACCGTGAGG 1285 GGCTACGTGATATTGCATGTAGG 1286 ATTTAGAGCATTLTTLCCGGCGG 1287 ACGCTGCAGACGATCCTGGGGGG 1288 TTGTCAAAGACCAACCCGTGGGG 1289 GACATAGAAGACGTACATGAAGG 1290 GTTCGCGAAGAGGTGGTGTGCGG 1291 GTCTTCTATGTCATGTACGACGG 1292 GCTCACAGGAGATTATCCACTGG
TABLE-US-00056 TABLE 56 Target sequences for GJB6 gene SEQ ID NOS Target sequence 1293 ACCCACTCATCATACCACGAGGG 1294 ACACGCAGCAAATGAAACGGGGG 1295 ACCGAGTCTTGGAATCACAATGG 1296 GTACCAATCTATAAAAACCAAGG 1297 TATCTCTTGACACTTGCGAGGGG 1298 TATGGCATAAAGTCTACTTGAGG 1299 AAACCAGCGCAATGGATTGGGGG 1300 ACGCTGCACACTTTCATCGGGGG 1301 ACCCTCGTGGTATGATGAGTGGG 1302 TCGCAGAAGGATAGACCCAATGG
TABLE-US-00057 TABLE 57 Target sequences for GLA gene SEQ ID NOS Target sequence 1303 ACCGAGATCTCACATGACGTAGG 1304 ACGGCCATAAAACTACACTGAGG 1305 ACGAAACGTTGAAAGCTGCGGGG 1306 ATAGCCATGAGCTTTCGAGGGGG 1307 GCCACACATACTGTACCACAGGG 1308 AGTGGGTTCGAACTTCAGCTCGG 1309 TCAATAAGGAGGGTATAAGGGGG 1310 CGATGGCAGAGTTACCGGTGAGG 1311 ACTGCGATGGTATAAGAGCGAGG 1312 TTAAGGAATAGAGCGGTGCAGGG
TABLE-US-00058 TABLE 58 Target sequences for HBA Gene Cluster SEQ ID NOS Target sequence 1313 AGAGTTTCACTGCATTAGCG 1314 TCCCGAGTAGCTGAGTAGCT 1315 ACATCTACAACTACTGCCAC 1316 CTGCCATAGGTGTTTACCAA 1317 GGGAAGGACATCACAAACGC 1318 ACAGTTGATACTGTACCCAC 1319 GGAGAAGGGACCTTCTAGCC 1320 GCCTGATCTTGACAGCCCCA 1321 CCAGCCTCAGGGGAGCTGAG 1322 CTCTCCAGTCGCAATGGGAC 1323 GTTTACCAAGGGTGATTCAT 1324 TGTTTACCAAGGGTGATTCA 1325 CTGCCATAGGTGTTTACCAA 1326 CTCTCCTCTCCAGTCGCAAT 1327 TTCCTATCAGTTGAGGGCCA 1328 AACCCTCCCTCTGATACCCC 1329 TGAGCATTCTGGGGTGACCT 1330 GTCTGGTGTGTGAGCATTCT 1331 AAGATATTCCTATCAGTTGA 1332 TCTGGTGTGTGAGCATTCTG 1333 GTGAGCATTCTGGGGTGACC 1334 TGTCTGGTGTGTGAGCATTC 1335 ATTCCTATCAGTTGAGGGCC
TABLE-US-00059 TABLE 59 Target sequences for HBB gene SEQ ID NOS Target sequence 1336 TTGGTTCTTCTATGGCTATCTGG 1337 CGGTTTGTTTCTATGGGTTCTGG 1338 GTAGACCTTATGATCTTGATAGG 1339 TACCTGTCTCAACCCTCATCAGG 1340 TTGTCTCTCCACATGGGTATGGG 1341 TAGACCTTATGATCTTGATAGGG 1342 AACCATCTCGCCGTAAAACATGG 1343 ATATCCCCCAGTTTAGTAGTTGG 1344 TCACACTAAGTAACTACCATTGG 1345 CCTAATTGTGTAATCGATTGTGG 1346 GATTACTGGTGGTCTACCCTTGG 1347 TTACCTCTATAATCATACATAGG 1348 CTTTCCTTACTAAACCGACATGG 1349 GGAGTAGATTGGCCAACCCTAGG 1350 GGCCAAGAGATATATCTTAGAGG 1351 GCGAGCTTAGTGATACTTGTGGG 1352 TGGTTATCAGGAAACAGTCCAGG 1353 CGTAAATACACTTGCAAAGGAGG 1354 GGGTTGGCCAATCTACTCCCAGG 1355 GAGTAGATTGGCCAACCCTAGGG 1356 ATCTCGCCGTAAAACATGGAAGG 1357 GCTGGCCCGCAACTTTGGCAAGG 1358 TGTATGATTATAGAGGTAAGAGG 1359 ACCGACATGGGTTTCCAGGTAGG 1360 AGCGAGCTTAGTGATACTTGTGG 1361 CTATCTTACTTACACATGAGTGG 1362 ACTATCAATGGGGTAATCAGTGG 1363 ACCACCAGTAATCTGAGGGTAGG 1364 GCATTTATGAGGTCAGCGTAGGG 1365 GACGAATGATTGCATCAGTGTGG 1366 AAGTCCAACTACTAAACTGGGGG 1367 TAAGTCCAACTACTAAACTGGGG
TABLE-US-00060 TABLE 60 Target sequences for HEXA gene SEQ ID NOS Target sequence 1368 TGGTTGACCCCACCTACAGGAGG 1369 ATTTACCACAGGCCCGCGTGCGG 1370 AGGAGGTCATTGAATACGCACGG 1371 TCCTTCTACATCCAGACGTGAGG 1372 TAGAAGGAAATGTCTCGTCGTGG 1373 AACCTGACCAATCTCCTTAGGGG 1374 GTCTGTATTTGGTGTCCGAGAGG 1375 CATGAGCTTTAAGTACGTAATGG 1376 GTAACATGAAAGTTATGACCAGG 1377 ATTACCCAGAAGCTTGTAGGAGG
TABLE-US-00061 TABLE 61 Target sequences for HLA-A gene SEQ ID NOS Target sequence 1378 TCCCTTGTCCGTTGTGTGAGCGG 1379 CTCACCTTTACAAGCTGTGAGGG
TABLE-US-00062 TABLE 62 Target sequences for HLA-C gene SEQ ID NOS Target sequence 1380 AGGCTGAAAACTACACATCCCGG 1381 GTAAGCGATGACACTCTGAACGG 1382 CGAGGCTGATGCAGACATGTGGG 1383 CTATATGTGGAGGTGGCATCTGG 1384 CATGTGGGATCCTGGTGTTCTGG 1385 TTGGAGTGGCATTGTGTGCTTGG 1386 GATGCAGACATGTGGGATCCTGG 1387 TAAGAGGTCACACCACATAAAGG 1388 CACGGATGTACTCACCAGTTGGG 1389 ACCGCACAGCAGGTCACTAGTGG 1390 GCACGTCTGTTTATAGGCTCTGG
TABLE-US-00063 TABLE 63 Target sequences for HTT gene SEQ ID NOS Target sequence 1391 ATATACAGTACGTTAATACGTGG 1392 TAATTGCCGAGGGATGAATGAGG 1393 TTATTCCAACCCATCCAGGGAGG 1394 TTTTGCAGTGATACGTCTGGGGG 1395 TGTAATCGTTGATATACGTGAGG 1396 AGTAAAGTGGTGAACTTACGTGG 1397 CCTGTCCTGAATTCACCGAGGGG 1398 CTTAGAAATCTTTCACCGAGGGG 1399 AGTAGTGGTATTCCAGATGGGGG 1400 TGTATCGTCACACGTTCTGTGGG
TABLE-US-00064 TABLE 64 Target sequences for IKBKG gene SEQ ID NOS Target sequence 1401 AAGACGAGGAGGGTTAAACGAGG 1402 CGAGTCACTTACAAACAAAGTGG 1403 CGGGGGCTCATGAGTCACCGGGG 1404 GCCGTGAGTTGATGTGACATGGG 1405 GGGGATTCGGGAGCACTACGCGG 1406 AAGTGTACGACCGTTTCCGGGGG 1407 CCTAAGTGTCCACCCCATCGTGG 1408 AACCGAGTAAAATCCTTGTGGGG 1409 GCTTTTGAAACGAGGGCCCAGGG 1410 ATAATGGGAGAGGATTGCGAGGG
TABLE-US-00065 TABLE 65 Target sequences for IKZF1 gene SEQ ID NOS Target sequence 1411 GGGTGTCGTAAACAAAACAGAGG 1412 GGTTTAGAGAGACGTACCAGCGG 1413 TGACTTGAGCGTCAAACCTGCGG 1414 CTACGCAAAACTCAGCACAAAGG 1415 GGTTAACGAAGAATTCATCAAGG 1416 ATTGAACCCCGATATCAGTGAGG 1417 TGGCACTCACCAACCAACCGAGG 1418 GCGTCACCCCAAAGTTTGCGGGG 1419 GTAGTGCTAAAGGATTTCTGTGG 1420 CGGAAGCATAAACACTCTGGTGG
TABLE-US-00066 TABLE 66 Target sequences for JAK2 gene SEQ ID NOS Target sequence 1421 GTGCACTTACTCACATCACATGG 1422 GCGCCATCTCACACTTACTGAGG 1423 TTGCTTATACTTTCCCTACGTGG 1424 TGTGTAACGTATGTACAGACTGG 1425 AGACAGTTGAGCGTATATTGTGG 1426 TCGATAACTTATAAATCTGAGGG 1427 GCCCGGTCTCCTGCCATTCGGGG 1428 GGCAGCACAATAATTGGTAGGGG 1429 GGTTTGCTTTTCAGTGACGGAGG 1430 GGGGCAGCACAATAATTGGTAGG
TABLE-US-00067 TABLE 67 Target sequences for KCNH2 gene SEQ ID NOS Target sequence 1431 GGGGTATAAAGTCTCCACGGGGG 1432 CCCTCCACTGAAAAACGACGGGG 1433 GGCTCCATCGAGATCCTGCGGGG 1434 TCGCCCGGGATACCTGACAGGGG 1435 CCGATGCGTGAGTCCATGTGTGG 1436 AGTCAACAAACCCACCTCCGAGG 1437 GTCAACAAACCCACCTCCGAGGG 1438 ACTGGCACATTTGCTGACGTGGG 1439 CTCTAACTCCGTACTGCCGGGGG 1440 ACCATCGTGACATGGTTTGGGGG
TABLE-US-00068 TABLE 68 Target sequences for KCNQ1 gene SEQ ID NOS Target sequence 1441 GTGCTGTAGATGGAGACGCGCGG 1442 AGTTATCTTACTGCACCCAAGGG 1443 CGGGATAGATGACACGAGCGGGG 1444 GCTCGAGGAAGTTGTAGACGCGG 1445 TTTGGCTCCACACCTCCGGGAGG 1446 GGGTGCGTGTTAATCAACAATGG 1447 ACGAGCGGGGCTAAGCAGGTGGG 1448 CAGGCGGGGTAAATGCACACTGG 1449 CGGTCTTTATGAGCATGCGGGGG 1450 GAACCTTTGCATATAACGTGCGG
TABLE-US-00069 TABLE 69 Target sequences for KLF5 gene SEQ ID NOS Target sequence 1451 GAAGTTGTGTACAAACTGCGCGG 1452 ACCCGTACCTACATAAGACGGGG 1453 CCCCAAGGTTTCATACCCGGTGG 1454 TTTACTCTCAGCGAAACGCGGGG 1455 GTTTCGCTGAGAGTAAATGGGGG 1456 TGCGTCGTTTCTCCAAATCGGGG 1457 GTCAAGTGTCAGTAGTCGCGGGG 1458 TTAAGGTCTCGTGCATTACGTGG 1459 TGGTACTGATAACTTCACATTGG 1460 AATGGTACAGCACTACTAAGCGG
TABLE-US-00070 TABLE 70 Target sequences for KRAS gene SEQ ID NOS Target sequence 1461 GATTAGGTCAAATCCCTTTATGG 1462 AATACGCATCGTGTTATCTCTGG 1463 GCTTACTATTCAACTCTAACAGG 1464 AACTTTTTCGTTCCACGTACTGG 1465 CCTACTGTCGCTAATGGATTGGG 1466 TCCTACTGTCGCTAATGGATTGG 1467 TAGTTACTACTCAGTTGAACAGG 1468 TATACTTACGTAAAATCCATTGG 1469 GCAATGTCATGAGTGAATACTGG 1470 CACCTATCCTACCCACGAATTGG 1471 ATACGCATCGTGTTATCTCTGGG 1472 ACTTTTTCGTTCCACGTACTGGG 1473 ACGCACCCTGAAATTGGAAGTGG 1474 TGCCAATTCGTGGGTAGGATAGG 1475 CGCCGAATGGTGACAGCAAGAGG 1476 GGGCAATGTTCATGAGTGCTGGG 1477 AAGGCTGCCAATTCGTGGGTAGG 1478 ATGACTTAGGTTTGCCAATGTGG 1479 ATCTCTGGGTCGTATACCAAAGG 1480 AGTATTCCATATCCATTTCGGGG 1481 GTTTAAAGTGACCCCAACACAGG 1482 AGTAGAGTGTGTGCGCCGAATGG 1483 GCTTTTTAGATCTGTATACGTGG 1484 TATAACTATATCCCAGTACGTGG
TABLE-US-00071 TABLE 71 Target sequences for LCA5 gene SEQ ID NOS Target sequence 1485 GGGTCACTGGGAAACTTATAAGG 1486 gaataacttcagaccgagtttgg 1487 CAATGAGCAGGTGCAGTATATGG 1488 TACGGTAGTTTGATGTGATATGG 1489 TCCCCTAATGAGTTCGCATTTGG 1490 TATCGTCTGCATGTTTTAATCGG 1491 AGAACTCCATGTCGTAAAACAGG 1492 GCGAACTCATTAGGGGAGGCTGG 1493 ACCCCTGGCCCTATCCATAAAGG 1494 ACGGGTTCAGTGACATAAGAAGG 1495 CGAGTAGTACTTTAGAATAGTGG 1496 CTCTATGGAATACCTCCGTATGG 1497 CCGATACGTTGTTTTCTTTGGGG 1498 TTGATGACCTTGGATCATGCTGG 1499 GAAAACGTTAGTTACTGTACAGG 1500 TATTCTAAAGTACTACTCGTTGG 1501 CGGGGATTCCTTAACTACCATGG 1502 TATGGCAAGTTTAACTGCACTGG 1503 GTAGCCCTCTTGTGTATGGTTGG 1504 TTCTAAAGTACTACTCGTTGGGG 1505 GCGTGGAGAGTAAACCAGACAGG 1506 AGGTCTGTTCTATACGAAGTGGG 1507 ATGCAACCCAAAAGTTCCGTGGG 1508 TATGCAACCCAAAAGTTCCGTGG 1509 TGCTTTACACATTATGACCGGGG 1510 CAGGTCTGTTCTATACGAAGTGG 1511 TGATGACCTTGGATCATGCTGGG 1512 AAAACGTTAGTTACTGTACAGGG 1513 AAATGCGAACTCATTAGGGGAGG 1514 GGTTACCCATGAGATTACACAGG 1515 ACTCCATGTCGTAAAACAGGAGG
TABLE-US-00072 TABLE 72 Target sequences for LRRK2 gene SEQ ID NOS Target sequence 1516 CAACGTCTGTTCAGCTTACGTGG 1517 CGTCTGTTCAGCTTACGTGGAGG 1518 CTGTTCAGCTTACGTGGAGGAGG 1519 TCATCCGTTCTTATACAATCTGG 1520 CTATAGAACTATACTTGACATGG 1521 TTCATCTCCGGTTTGAAATCAGG 1522 ACATGCCAATTGTCTAAATAAGG 1523 GGTACAATGCAAAGCTTAATGGG 1524 GCTTGATACACCCAGATATAAGG 1525 GGCTCCCCGCTTTCATCCTAGGG 1526 GCTCCCCGCTTTCATCCTAGGGG 1527 ACCCAATATCCAGGTTGAGTAGG 1528 CCAGTTCCCAGACCTTCCGTGGG 1529 TTCTGCGCGGCCCGTCGCCTCGG 1530 GGCCCCTGAGCTCGTTTTTGGGG 1531 GGCCCCAAAAACGAGCTCAGGGG 1532 TCCTCATAAACAGGCGGGCGTGG 1533 GTGTTCACGTACTCCGAGCGCGG 1534 TTTCAAGTGATTACCGCGCTCGG 1535 GAGTCCAAGACGATCAACAGAGG 1536 GAGAGTCGCGAGTGTGCAGCAGG 1537 CGCGAGTGTGCAGCAGGTAAAGG 1538 ACAAGGTATACTACAACTAAAGG 1539 GGGTAGGCGTTTTGGTCTGCAGG 1540 AGTGCTATACTTGACAACCCAGG 1541 GTGCTATACTTGACAACCCAGGG
TABLE-US-00073 TABLE 73 Target sequences for MDM4 gene SEQ ID NOS Target sequence 1542 GGGATATTATCGTTAAATATAGG 1543 ACTCCAACAACTTACTCATTGGG 1544 ATAGGGCCAGTTAGGGAGCGTGG 1545 AGTTAGGGAGCGTGGTTCATTGG 1546 AGATAGGGAATACAAGCGGTTGG 1547 GATAGGGAATACAAGCGGTTGGG 1548 TATAGGAACCTTAAGTCAGCGGG 1549 ATAGGAACCTTAAGTCAGCGGGG 1550 GGTGCATCCGTTACTATTATGGG 1551 CTCGTGTGAGGCCGTGTGGGAGG 1552 CGGCCGTACCGCCAGTTGTGCGG 1553 GGCCGTACCGCCAGTTGTGCGGG 1554 GTGAAGTAACTTTGGCCAACAGG 1555 AAACCTAAAGTCGACGTAGTTGG 1556 CACGTCAATGTCATTCTACCCGG 1557 ACCTACAGACAGTATCGAGATGG 1558 CCTACAGACAGTATCGAGATGGG 1559 CTACAGACAGTATCGAGATGGGG 1560 CGGGTGTTGCTTTTAAACTGTGG 1561 GTAACTTGCAGTTAGTAGGTAGG
TABLE-US-00074 TABLE 74 Target sequences for MET gene SEQ ID NOS Target sequence 1562 ATAACTGTTTGATAAGACCGTGG 1563 CCATAACATTCTCCTAACAGTGG 1564 CAATTTTTTGACAACCTACGAGG 1565 AACTCTTCATCAGCTAACCAAGG 1566 AGGTCGTTTTGGTATCAGAAAGG 1567 CAAATCTCTCTAAACCCGGGTGG 1568 CAAAGCTCGCGCCCTTCCCGGGG 1569 TGTCAGTTCCTATTGGCACGTGG 1570 CTATTATGTAGATCTGCAGAAGG 1571 GGTAGAGTATCATATGTGCTAGG
TABLE-US-00075 TABLE 75 Target sequences for MLH1 gene SEQ ID NOS Target sequence 1572 TCTTGTACTACAAAGCCTTA 1573 CAGTTTGGACGGCTGGTACT 1574 TTGTGATCAGTTTGGACGGC 1575 AGTTGTGGCAACCCGAAACA 1576 CAGTTGTGGCAACCCGAAAC 1577 ACCGGGCTCCATTTCAGTTG 1578 CTCACAAGGTCATCCCAACC 1579 TCTCACAAGGTCATCCCAAC 1580 TGGCAACCCGAAACAGGGCT 1581 TTAATTGTGATCAGTTTGGA 1582 TACCTATAAGAATACTCATC 1583 ACCTTAAACAAGGCCAGACG 1584 TGGGTAGAAAGATATCCAAC 1585 CTAGATAGGACTATATTTAC 1586 GTAGCCATTAAAACCTAGAT 1587 ACTCATCAGGACCTTAAACA 1588 TAGATAGGACTATATTTACT 1589 TCAGGAGTTCAAGACCAGCC 1590 GTCCTGATGAGTATTCTTAT 1591 CAGTAAATATAGTCCTATCT 1592 CTTGGCCTTGCAAAGTGCTG 1593 ACCACGTCTGGCCTTGTTTA 1594 ATGGTGATTTTTACATGCAG 1595 TGCAGAGGGGAGCAACTATG 1596 TGGTGATTTTTACATGCAGA 1597 CAACACGAATCTAGTCTTTA 1598 ATCCATATACCTCCCATATA 1599 TATAAAGTCCTGAGACCGCT 1600 AGACCGCTAGGAATCTATGA 1601 TGATTCACGCCACAGAATCT 1602 CACAAAGCCTGGAATATGAG 1603 AACACGAATCTAGTCTTTAA 1604 CGAATCTAGTCTTTAAGGGC 1605 CTAACTTCTAGCACAAAGCC 1606 GAGACCCTTCCATATATGGG 1607 TACTGAGACCCTTCCATATA 1608 GTGAATCATGTGTTCTTTCA 1609 GGGGAAAAGTGCTTGCATTA 1610 TTTCCATCATAGATTCCTAG 1611 ACTGAGACCCTTCCATATAT 1612 TTAATTGCCTCTCATATTCC 1613 CAGGCTTTGTGCTAGAAGTT 1614 AGGCTTTGTGCTAGAAGTTA 1615 CCAACCCCTTGGACCTCAAC 1616 ATGATCTCTGGCCAACCCCT 1617 CAACCCCTTGGACCTCAACT 1618 CCATTCTGATATTGCAACCA 1619 TACTCAACTATTAGTGAATG 1620 CATACTCAACTATTAGTGAA 1621 GAGCAAATGGCAATCACTCT 1622 TCCATTCTGATATTGCAACC 1623 ATACTCAACTATTAGTGAAT 1624 CACTTCTCCCAAATCACTGT 1625 AGAGCAAATGGCAATCACTC 1626 CTGAGAGGTTCTTCTCCCCA 1627 TCCATCTTCATTTCACACTT 1628 TATGTAGATTTGCTAGGACC 1629 GTTAGGGCAAGTGGCGGTGA 1630 TCTGTCCCAGTTGAGGTCCA 1631 CCAGTTGAGGTCCAAGGGGT 1632 CTGTCCCAGTTGAGGTCCAA 1633 CAAATAGAGAGGTTTTCATC 1634 CTACAGTGATTTGGGAGAAG 1635 TGTCCCAGTTGAGGTCCAAG 1636 AAGTGGCGGTGATGGAGTTG 1637 AAGGGGTTGGCCAGAGATCA 1638 GTTGAGTATGTAGATTTGCT 1639 CATTTGCTCTGTCCCAGTTG 1640 GGATCTGTTAGGGCAAGTGG 1641 GTCTGAGTATGGATCTGTTA 1642 CCCTGGTTGCAATATCAGAA 1643 GTTGCAATATCAGAATGGAT 1644 TATGGATCTGTTAGGGCAAG 1645 GGTCTGAGTATGGATCTGTT 1646 TGGTGAGGGACTTGAGACAC 1647 GATGGAAGCCTACAGTGATT 1648 CATGGTGTTCTTTAAGGCAG 1649 TGAAATTAAGTGTGGATATC 1650 CTGGAGGCAAAAAACGTTAA 1651 TCACTTCCTACTTCTGAGCT 1652 ACCCTGGCTTTCTGCTGAAC 1653 CCATGGTGTTCTTTAAGGCA 1654 TCTGGAGGCAAAAAACGTTA 1655 ACCATGGTGTTCTTTAAGGC 1656 TGTCACCTAGTGACAAACCA 1657 GGCCAGTTCAGCAGAAAGCC 1658 TGCTCTTTGGTGAACAGTCC 1659 GCAAAGGCACTGGCATACAG 1660 TATATCCATGGTTTGTCACT 1661 GCTCTTTGGTGAACAGTCCT 1662 GGTCTGAATGTATATATCCA 1663 AAGGGTGTCTTGATCATCTC 1664 ATGGTTTGTCACTAGGTGAC 1665 AATAATCAAAAGTAGACCTA 1666 GAGAAGTAATCCCTGAAACA 1667 ATGTTCTGTCCCTACCTGTC 1668 TTCCCAACGTCTTCAACCAG 1669 GATTCCCAACGTCTTCAACC 1670 ATTCCCAACGTCTTCAACCA 1671 TGCTTGAGATACAACCAGTT 1672 CTGGCCAGCCTCTAACAGAC 1673 TCTTCAACCAGGGGTCTGAC 1674 TAGAGCACTAAGACCAAGTC 1675 TGTGAATGGTTTTCCAGTAA 1676 GAATAAGTCAGCTACTCAAT 1677 GAAGTCTTTAAGCAAGTCTA 1678 TGAATGGTTTTCCAGTAAGG 1679 GTTTAAGGGAATGACCTCCA 1680 CGGGTTCAGAGTTCAATATC 1681 GTGAATGGTTTTCCAGTAAG 1682 AGTCTTTAAGCAAGTCTATG 1683 CACCAAAATGCAGACATAGA 1684 ATGTGAATGGTTTTCCAGTA 1685 CAGCTGTTAATAAATGTGAA 1686 GGGGAAAATCTAGTGACTAA 1687 TCACCCAGGCTGGAGTGCAG 1688 TCTTCTGCCTCAGCCTCCCG 1689 TTTCGCTCAGTCACCCAGGC 1690 GGAGTGCAGTGGCACGATCT 1691 AATAAAACTAGACTTTAAAA
TABLE-US-00076 TABLE 76 Target sequences for MSH2 gene SEQ ID NOS Target sequence 1692 AAGACCCATTATGTGTGGGC 1693 GGTATTTCAACGTTTGGCCT 1694 GTCTGTGGTATTTCAACGTT 1695 ACACTCAAGCTATAGGTCAT 1696 GGTAAACTAACAATCGAAGG 1697 TAATTTAACGACCCACTACT 1698 TGAGTCATCTGTAATGCCTA 1699 GCAAGGTGTGACCCAGTAGT 1700 TGCAAGGTGTGACCCAGTAG 1701 TTAGGGAGTTCCTAATGACC 1702 TAGGGAGTTCCTAATGACCA 1703 GGTCATTAGGAACTCCCTAA 1704 GTTGAATTTTAGGTGTACCC 1705 TTAGGTGTACCCTGGTCATT 1706 GCCATGGCAATTTGTTCCCG 1707 TCCACGGGAACAAATTGCCA 1708 TACCTACAGTATACTTACCT 1709 TGCCTAGGTAAGTATACTGT 1710 CCAAGACATTAGTACGTTGT 1711 GTTACAGTAGGACACATAAC 1712 CTACAACGTACTAATGTCTT 1713 CCTACAACGTACTAATGTCT 1714 GATTCCACTTGGATATACGT 1715 CACTTGGATATACGTTGGAG 1716 CGTTGGAGTGGAATTGTCTG
TABLE-US-00077 TABLE 77 Target sequences for MSH6 gene SEQ ID NOS Target sequence 1717 TAGTTCAACCTAGTATAAGG 1718 ACATAGTTCAACCTAGTATA 1719 GGGTGGTTGTAAACCAGACA 1720 GTAAACCAGACAAGGCCACC 1721 GTTTACAACCACCCCTTTGA 1722 TTGTATAGGTGCTACTAATT 1723 TCGAGCCTTTTCATGGTCAA 1724 GGACTTATTACTCCCAAAGC 1725 CGTGTTTAAGACTGTAACTG 1726 ATCCCATGCATGATTTCTAC
TABLE-US-00078 TABLE 78 Target sequences for MUTYH gene SEQ ID NOS Target sequence 1727 CAACTCCGGACGATCAGCCC 1728 TGAGCCGGACTCCCCAACTC 1729 TAAACCGAACTTTGGCCAGA 1730 TCACAGGTATTGTGTACCTC 1731 GCTGAACTCAAGAAGCCGCA 1732 ATTTCCTCACCATTTCCGGA
TABLE-US-00079 TABLE 79 Target sequences for MYC gene SEQ ID NOS Target sequence 1733 CTTCGGGGAGACAACGACGGCGG 1734 GCCGTATTTCTACTGCGACGAGG 1735 ACCCCTCCATAAATACAAGGGGG 1736 TCCGTATTGAGTGCGAAGGGAGG 1737 TAAGTGATCAGACACCGTCAGGG 1738 GCGCGCGTAGTTAATTCATGCGG 1739 GGCGGGTTGGAATCGCCGCGGGG 1740 TGCGTAGTTGTGCTGATGTGTGG 1741 GTCAAACAGTACTGCTACGGAGG 1742 CGAGGGGTCGATGCACTCTGAGG
TABLE-US-00080 TABLE 80 Target sequences for MYCN gene SEQ ID NOS Target sequence 1743 AAGCGAGTTAAACAACCCTGTGG 1744 ACAACACGCAGTCAAAGCGGGGG 1745 ACATACGAGCACTAACAAAGGGG 1746 GCTCCCCAACTGGTACAACGAGG 1747 TCGCACACCCTTGAGATACGAGG 1748 CTCCCCAACTGGTACAACGAGGG 1749 AGAAATCGACGTGGTCACTGTGG 1750 CTTTCTGCTCAGTCTCCGCGAGG 1751 TCCATGACAGCGCTAAACGTTGG 1752 TCACCAACCTCGTATCTCAAGGG
TABLE-US-00081 TABLE 81 Target sequences for MYH11 gene SEQ ID NOS Target sequence 1753 TTGTGTTGCACTAACCCAAGCGG 1754 TTATACGTGTTAATCCAAGGTGG 1755 GATGCTCAAATTCAGCGCAGAGG 1756 GATTTCCTACTTCCTACAAGCGG 1757 TGTTGCACTAACCCAAGCGGAGG 1758 TACACTCAAGATGATTCCCGAGG 1759 GTGGGATTTCCAACGCACCATGG 1760 AACTTTGAGACCTTTACACGTGG 1761 TGTGACGAAGAGAGCTGTGTGGG 1762 CCTTGTCAAAGACGTGAACGTGG
TABLE-US-00082 TABLE 82 Target sequences for NPC1 gene SEQ ID NOS Target sequence 1763 GTGGTAGGTCATGAAGTACGTGG 1764 CGTCCGTTCTGTCCACGATGTGG 1765 TGCCGAGCAGAGTTATGCGATGG 1766 AGCGAAACCAGCGTTTGCGAGGG 1767 TTATGCTCTGGAACTCACCGAGG 1768 GAGAAATATTAATCCGTGAGTGG 1769 CCTGTAAGGAAATACTCGGTAGG 1770 GTACAGTAAGATTGGTGTGATGG 1771 CCAACCGCACATCACACGCTGGG 1772 GGGTTATCCGAAAGGAACATGGG
TABLE-US-00083 TABLE 83 Target sequences for NPC2 gene SEQ ID NOS Target sequence 1773 AGCTGCCAGGAAACGCATCGCGG 1774 AACCCCGACGACAGGCAAGGAGG 1775 CAGATGCACCGAACTCAATGAGG 1776 TACCACTTAACACTGAACAGAGG 1777 TGCGCGGTCGGGTTTCATGGAGG 1778 GGCTTTTGGAAATCACCGAAGGG 1779 ATCAACCCCGACGACAGGCAAGG 1780 GGGTTCCCTAAATCTTAAGGAGG 1781 TAGTCGGTAGAAAGTCAGGCCGG 1782 CGGTCACAAGACAAACCTGTCGG
TABLE-US-00084 TABLE 84 Target sequences for OTOA gene SEQ ID NOS Target sequence 1783 AAGTTGGCAATTCCAGTAGAGGG 1784 AACTGGGTATCCCTGATATGAGG 1785 GTACCCATTGGTGTTATCTTAGG 1786 GTACAAAGTCCTAACACCCCTGG 1787 TCAACTGAAGCTCCCACGTGTGG 1788 AGCACAAGCGTGTTGATAGGTGG 1789 AGCGTGTTGATAGGTGGCAAAGG 1790 CAGTCATGATACTACCCACAAGG 1791 AATGGGGGAATCGGGCTGGCTGG 1792 CCTAAAAGGGGATGTGCGCCCGG 1793 TTAAATGTTGGCGGCTAATGAGG 1794 TCTCCCAACACCCCAAATACAGG 1795 CTCATACGACACAGTGATGCTGG 1796 ATGTATCAGCTACCCTAATCAGG 1797 CGTGATTCAGAACAGGTGACTGG 1798 TACTATGATCGATAAGAAATAGG 1799 CTATGATCGATAAGAAATAGGGG 1800 TCGATAAGAAATAGGGGTCTTGG 1801 CGATAAGAAATAGGGGTCTTGGG
TABLE-US-00085 TABLE 85 Target sequences for PAH gene SEQ ID NOS Target sequence 1802 GCAGCTTATAGGTTCACCAGAGG 1803 CTGTGATGTAGAAGGAATCGGGG 1804 TCCGTTTTGATATGCAACCTGGG 1805 ATCCGTTTTGATATGCAACCTGG 1806 TAAGTAATTTACACCTTACGAGG 1807 GCTACGACCCATACACCCAAAGG 1808 TATTATGGCCCTTGTGACCATGG 1809 TGATTTACCCCTACCCTACTAGG 1810 TCATTTTAGGCCACACCAAGTGG 1811 AAGTATTACAGACGCACTGGTGG 1812 ACTTGGTGGTTGCGTTGAACAGG 1813 CTTGGTGGTTGCGTTGAACAGGG 1814 AACTCTCTGCCACGTAATAGAGG 1815 ACTCCGTGACAGTGTAATTTTGG 1816 CGTGACAGTGTAATTTTGGATGG 1817 AGCTCATTAGGCACAACAGTGGG 1818 TCAGTACTGGCAACAAATGTGGG 1819 GTTCTACTCCAATATATGGCAGG 1820 ATATGGCAGGGTGGGTCTTAGGG 1821 AGGGTGCATACACACTTTACTGG 1822 CCCAGCTGGCATATATAAGCAGG 1823 TAACACCCCATCAGTGGATCAGG 1824 TCGATTACTGAGAAACCGAGTGG 1825 ACCTCAATCCTTTGGGTGTATGG
TABLE-US-00086 TABLE 86 Target sequences for PCCA gene SEQ ID NOS Target sequence 1826 GTTACCTAATGAGACCATGGGGG 1827 CTTATCGACATGGAAGTGAGTGG 1828 TTTGTGTCCAATTCAGCGTGCGG 1829 AGTACCACATCGAACTGGAAAGG 1830 CGAGTCTGTCGTTAATTCTGGGG 1831 TGTTCCTCGCGGGGATCCTGCGG 1832 GAAGTTCACTATCACTCTAGGGG 1833 TCCCCTTTCCGCAAGTTAGGGGG 1834 CGTTGCAGCTGTTCCTCGCGGGG 1835 GCCAGTAGTTGTACTAACAAGGG
TABLE-US-00087 TABLE 87 Target sequences for PCCB gene SEQ ID NOS Target sequence 1836 CGTGCCCCATGAAAGAGTGATGG 1837 ACATGCGTACTCAGGTGCGCCGG 1838 TGTGCGCGTGCAGGAACTTGTGG 1839 GTACTCAGGTGCGCCGGTAGGGG 1840 GCGACCTATCACTGCGTGCCCGG 1841 AACGCATCGAAAACAAGCGCCGG 1842 CGCATTTGACAAGGGTCCAAAGG 1843 AAGGTCAAGAGTACCCATTACGG 1844 GCGTACTCAGGTGCGCCGGTAGG 1845 GTCACAGATACCAGGATACTGGG 1846 CGGCACAGCAAAAATGGCGGCGG 1847 GTACTTGCATTGAGATCAACGGG 1848 TCCGTAGATTTTCCCAGAAGAGG 1849 TTGCCCAGTGTGTCCGTGACTGG 1850 TGAATGACCCTGTGTTATCCAGG 1851 TGGTATTAAAGGGCAATTACTGG 1852 GGCTACTCTCGATGTTTGGCTGG 1853 CGTACTCAGGTGCGCCGGTAGGG 1854 TACTTCTAACCTACTCTGTTAGG 1855 TGGAGCATAGTGGTATTAAAGGG 1856 CGCAGGCTACTCTCGATGTTTGG 1857 CGGGGCAAGGCTCAGCGTTCTGG
TABLE-US-00088 TABLE 88 Target sequences for PHEX gene SEQ ID NOS Target sequence 1858 TGGGTGTAAGTGGCTTCGAGTGG 1859 ATCGGTTGAAAGATTCTCCGCGG 1860 TATCTTGCGTATGTTTCCGAGGG 1861 CCTGTCGGTAAGTGATGGGTAGG 1862 AGGGTCGTCGTCTCTTCAAGGGG 1863 TATATCGTTAGTGAAAGGCCTGG 1864 CTAAACCATCCATACAGATACGG 1865 AATTCCTGTCGGTAAGTGATGGG 1866 TTTATCTAACGATGAGCAGAAGG 1867 TTTCCGTGTTACTTTAAGTGTGG
TABLE-US-00089 TABLE 89 Target sequences for PIK3CA gene SEQ ID NOS Target sequence 1868 AGCAAGCACATCCACAGCGTAGG 1869 GTAAAGGGAGCGCAACAAGAGGG 1870 AACTGTACATAAACTTCGGGCGG 1871 CCCCGAGCGTGAGTAGAGCGCGG 1872 GTAAACACCAGACGTTCAGCCGG 1873 AAGGTATAGGTACTCAGGAGAGG 1874 GGGTGTCATGTATAATACAGAGG 1875 GTGTCATGCATTCAAGTACCAGG 1876 CGATCACGAATCAGAAAACACGG 1877 CGAGTATTATGAGATTACCTGGG
TABLE-US-00090 TABLE 90 Target sequences for PKD1 gene SEQ ID NOS Target sequence 1878 GCTGCCGTCAGAAATCCCCGCGG 1879 CGGCAGAAAGTAATACTGAGCGG 1880 GACCGGGCATATCAGCATGGTGG 1881 ACGCAACACTCACGCCCGGGGGG 1882 CGGCGGTGTTAAGAGGGCAAAGG 1883 CCGATATCTACCCCTCCAAGTGG 1884 CACGCAACACTCACGCCCGGGGG 1885 CCGAAGCACTGTCCGAGCAAGGG 1886 GGCAGCGAAGACACGTTGAGGGG 1887 GGGCGTACCGAGGTGAGCAGAGG
TABLE-US-00091 TABLE 91: Target sequences for PLP1 gene SEQ ID NOS Target sequence 1888 ACTTAAATCTAAATGCACCGGGG 1889 GTGCACACTATGAGGAATCGGGG 1890 CAATGGTGCTCATTTCATGGGGG 1891 CGAATTGATTCATTAACCAGGGG 1892 GCACAGTTCGAGGTCCCAGAGGG 1893 GCACGATTGAGGATGCACATTGG 1894 TCCATAGATGACATACTGGAAGG 1895 GGTTATCCATGCTTTGAGTGAGG 1896 AACAAGGCTTCTTTGTCCGGGGG 1897 CGTAGAATCTGTGTAGACGAAGG
TABLE-US-00092 TABLE 92 Target sequences for PMP22 gene SEQ ID NOS Target sequence 1898 GCGCGTAAAGCTTCACACAGAGG 1899 CAGGATGTAGGCGAAACCGTAGG 1900 TGTCAGGAGCGAAATCATTGCGG 1901 TATAAATCCAGTATGCCGTGTGG 1902 CTTCTTTAAGGCTCAACACGAGG 1903 GCCAGGTTTTCCCAAAACGTGGG 1904 TCCGACCGTAAGAAAAATGTGGG 1905 ACACACAACAAAAGGTCGACGGG 1906 ACAGACAGCGTCCCCCCACAAGG 1907 TGTCACACGATAAGGGAACCAGG
TABLE-US-00093 TABLE 93 Target sequences for PMS2 gene SEQ ID NOS Target sequence 1908 CTCCTGTGTCTACGGTGAGC 1909 ACTAGTAAAAACTGGACCTT 1910 AAGGTCCAGTTTTTACTAGT 1911 TCTTTTTGACGAGCATAGAT 1912 CTATGCTCGTCAAAAAGACG 1913 TCGTCAAAAAGACGTGGATG 1914 GTGGTGCATTGGTTGACTGT 1915 TTAGACTTCATTGACAAACC 1916 TGAGATATAAGCGTCCTACC 1917 GACGCTTATATCTCATGTCT 1918 AGGATCACTATTGCAGTTCA 1919 GGATCACTATTGCAGTTCAC 1920 ACAGTCAACCAATGCACCAC 1921 GAGACCCACCCCAGGGATAC 1922 AGGATGGTCAAAGTGCAACG 1923 CCAATAAAGAGAACGGGGAC 1924 GTCCTCAAGTTAGAGAAGTC
TABLE-US-00094 TABLE 94 Target sequences for PRSS1 gene SEQ ID NOS Target sequence 1925 TAGTAAGTTATGTGCTATATAGG 1926 GCCCITTCCCGCAAGGATGCTGG 1927 AACGCCCTGCAGGCTTGTTAAGG 1928 CGGGGTTGGCACATGACATATGG 1929 TGACCTTGCCCGACACTGACTGG 1930 CCATAAACTAATCGACAGTCAGG 1931 CACGGTTCCACGTGAGTACATGG 1932 GTATCTACAGTTGTTAGAGCAGG 1933 AGAGGCACGTCATCACCAACAGG 1934 TCTTCCTGTCGTATTGGGGGTGG 1935 GAGTCTTCCTGTCGTATTGGGGG 1936 GGCGTTGATTACTGCACGTGAGG 1937 AAGAGTCTTAGTGGCCCAGGTGG 1938 TAGGAGCTTAGTGCATCTGGAGG
TABLE-US-00095 TABLE 95 Target sequences for PTCH1 gene SEQ ID NOS Target sequence 1939 ATTTCAAAAGCGTCTCTGCGCGG 1940 TTGAAAGAGCACTAATGACGGGG 1941 GGAGGTCTATAATTACCAAGAGG 1942 CGAGGAGCTTCGGCACTACGAGG 1943 CCCATGTGACCAATTCGCTGTGG 1944 TAAGAGATGCCGTAGACACGAGG 1945 AGTGCCGAAGCTCCTCGCTGAGG 1946 GAAGCACGTACCCTAAACACTGG 1947 GTCCAATTATGCATCTCAAGGGG 1948 TATTACTGCTACCCAAGATGGGG
TABLE-US-00096 TABLE 96 Target sequences for PTEN gene SEQ ID NOS Target sequence 1949 GTAGTCCCGGAGTTAGGTAA 1950 CCAGGTTTAATTAGTAGTCC 1951 CCTATGGAAGAACGTATATG 1952 AGGTTAGACTAACCTTAAAT 1953 CCACATATACGTTCTTCCAT 1954 CATATACGTTCTTCCATAGG 1955 ATTTAAGTTGCCCAACCAAC 1956 TAGCGAGAGCAAAACTGTAG 1957 GGTTATAGCTACCAATACTC 1958 ATTGGTAGCTATAACCACTT 1959 TTGGTAGCTATAACCACTTT 1960 GGTATGAGTACTAATCTGGC 1961 GTATGAGTACTAATCTGGCT 1962 GGTGAAGTTATTGCAATCTA 1963 TTTTGGTATGAGTACTAATC 1964 TGGTGTGCTAGTTTTTACGT 1965 CCCGATTAATATTTAGCCAG 1966 CAATGGTTGGTACTAACAGG 1967 GAACAATGGTTGGTACTAAC 1968 ACGTGATATCTTTTTGTAAC 1969 AGTTTAAACCATAGACGCAA 1970 GGGAACATACTACCACTGTT 1971 CTTTGTAGGAGAGGTTTATC 1972 TCCTACAAAGAGCCTTGTTG 1973 CGGATACCATAGTGTTTCTT 1974 AGGGTTAGACTATCAGAACT 1975 GGGTTAGACTATCAGAACTG 1976 CCATTAAACTGAGTCACTTC 1977 CCTATTTCACAACACCCTAC 1978 GGGATATTCCAACCTATGCA 1979 GATGAAATCGTAAGTCCTGT 1980 ATGAAATCGTAAGTCCTGTA 1981 CTATCACTCAATAACTCTTC 1982 GCCCTACCCACAACATAAAC 1983 AATTCATTTGTCATACGCTG 1984 TGGCACTTCTTAACCTCCTA 1985 GTAGTAGGTGTTTACTAAAC 1986 GCTCATATTACAACGTACAA
TABLE-US-00097 TABLE 97 Target sequences for REEP1 gene SEQ ID NOS Target sequence 1987 CATCTGGTCCAATCACCGTGAGG 1988 TCCCCATATAAGTCTCACAAGGG 1989 ATTGGCGTTTTCTGACGACGAGG 1990 GGCGTTTTCTGACGACGAGGAGG 1991 TGATCTGTGTATCCCATGGAAGG 1992 GTTGGCTCATCTCACTCACGTGG 1993 AGGCAGATTACTATAAAGGTGGG 1994 CACTTAACATCTAACACACCAGG 1995 CAGATGTTAATTAAGCTGGATGG 1996 GGTTTTAGAAGATTGCGAGTTGG
TABLE-US-00098 TABLE 98 Target sequences for RPGR gene SEQ ID NOS Target sequence 1997 TCGCTTGTCAGAGATCCCAGAGG 1998 ATATTGACCCTACGACAACAAGG 1999 AGGTTTCTCTCAGAACATCGTGG 2000 TGCCAACTCAGTAAACCGAAGGG 2001 AATGGCACCAAGTAACCAGTGGG 2002 GGTAGCAACTAATAATGACCAGG 2003 GTTCTTAACGAGCAAACCAGAGG 2004 AATACAGGTATGATGCGTGATGG 2005 GGACTCTATCAGCACGTATGCGG 2006 TAAACTAATTCGTACCAGAAAGG
TABLE-US-00099 TABLE 99 Target sequences for SBDS gene SEQ ID NOS Target sequence 2007 TGGCGAAAGTAAATACGCCAAGG 2008 GCTGTATCAAATGGTGCACATGG 2009 GCGGTACCAGTGCGAATCATCGG 2010 CGGTACCAGTGCGAATCATCGGG 2011 ATCCTGGTGGTATCTTGTCGTGG 2012 TGCGAATCATCGGGCTATCCAGG 2013 CACTCGGTACGCCGCTAACGCGG
TABLE-US-00100 TABLE 100 Target sequences for SCNIA gene SEQ ID NOS Target sequence 2014 TCCCGATGCAACTCAGTTCATGG 2015 GACCCTAATAAAGTTAACCCTGG 2016 AAACTTGTACCTATACTGTTGGG 2017 CATTTTGICACGCATCAATCTGG 2018 TGATATGTGTTGCATACCTCTGG 2019 ATGGTTGCCAAGTAATATCAGGG 2020 ACTCACTAAGCATAAGGTCTTGG 2021 TTCGCCACTCACTAAGCATAAGG 2022 GCTTAGTGAGTGGCGAAATTTGG 2023 ATCAGAAGTTATCCCATTATAGG 2024 TGAACAATTGAATTGCTCCGTGG 2025 TTTATATAGTTCGAGTGTCTGGG 2026 GGTAGTATAAAAAGTCTGCAGGG 2027 GTCAGTCCATTGTACAAGGATGG 2028 CCAATTGGGAGCTCAAAGGTAGG 2029 TCCAGTGACATATCTACCCTAGG 2030 ACCTTCAATTCAGTTAGTGCAGG 2031 TGGGGGGTATGGCAACCACATGG 2032 TTTGTCACCCGGTCATAGGAAGG 2033 GTCACCCGGTCATAGGAAGGTGG
TABLE-US-00101 TABLE 101 Target sequences for SDHB gene SEQ ID NOS Target sequence 2034 GCGTCTCTGGGAAGAAACCGGGG 2035 TGAAATTTCCAGTCCCACGTGGG 2036 TTTGGTACAGGAACACACGTTGG 2037 GTGTGTATAATTAAGCACCCGGG 2038 GGCCGATCATGAAACTGGAAGGG 2039 CTCTCGGTGTGTGGTCATCGAGG 2040 GCCACTGCCAATCCTGACGGAGG 2041 GGTCCCCACAGGGTCAGTAAGGG 2042 CTTCCAATCCCGCGGCTGAGGGG 2043 CGAGTTAATCACATGACCATAGG
TABLE-US-00102 TABLE 102 Target sequences for SDHC gene SEQ ID NOS Target sequence 2044 TATTATCACTGGTCTCCCCGAGG 2045 ATACATTCACCACATCGCGGTGG 2046 TAGTATCTCACCTTGGAACGGGG 2047 GTGGTTCCATCAATATCCTGAGG 2048 AAACAACGCACTTCACAACGTGG 2049 ATTGTGGGGTCTAATCGAGGTGG 2050 ATTGTCTGACCAACGCTGGGGGG 2051 TGGTCCGCAAGGTCTTCTCGAGG 2052 GTGCGCTCCGTAGGGCTTCGGGG 2053 TTGATGGATATGTACGACAGTGG
TABLE-US-00103 TABLE 103 Target sequences for SDHD gene SEQ ID NOS Target sequence 2054 CTGAGCACTACCGGTCACCAGGG 2055 AAAACTCTGAATCGGTCGAGGGG 2056 TAGGTGGGTTAATAAGCTAGAGG 2057 GCGTTAGAACCATGTCCGAAGGG 2058 TAGCATTACTACAGTACCTGAGG 2059 TATTCCCAGCAGAACCACGAGGG 2060 GCTGGATCCAATAGTGACCTGGG 2061 GAGATTCCTTGAACATGCCAGGG 2062 GTTCGAAAATCATTTAACCTGGG 2063 GCGATGGAGAGAACATACAATGG
TABLE-US-00104 TABLE 104 Target sequences for SHOX gene SEQ ID NOS Target sequence 2064 TTGGCAACGAAAAACGTGTGGGG 2065 CCCAAGATCGTGCGTCCCCGGGG 2066 AATCAATAAACAGCGTCGGAGGG 2067 CGTCAATCAATAAACAGCGTCGG 2068 AACCCCTGCGCTCACCCGCGGGG 2069 TTGCAGCTCCCGTCTCGCCAGGG 2070 ATTGGCAACGAAAAACGTGTGGG 2071 CGGCGCATCTTCCTCCCCGGCGG 2072 GCTTTTTCTCCGAGGCCGAGGGG 2073 CCAAATCACCTGGCCACACGGGG
TABLE-US-00105 TABLE 105 Target sequences for SLC6A4 gene SEQ ID NOS Target sequence 2074 GTAGTAAAAAGGGGCAAACGTGG 2075 CTACTGCACCCATAAATATGAGG 2076 TAAGGGGAGTTGCTTTACAGTGG 2077 GAACGTATTTGTGAACCGATAGG 2078 AGGAGCTCGTAGAATTGTCATGG 2079 TGAAAGTTCTGCCCCCGAGAGGG 2080 CTTTCCAGCAACAGCACGAGCGG 2081 GTGCAGGCCACGAGACCCGAAGG 2082 CACGCTGCAAGGTAAGATGTTGG 2083 TGAGAGCGCTATAAAGGCAGCGG
TABLE-US-00106 TABLE 106 Target sequences for SLC6A5 gene SEQ ID NOS Target sequence 2084 CTTGCTTAACCTCCGCACTGCGG 2085 GTTTAGGCAGAAACACTCGTAGG 2086 GCTACCCCCATACAACCGAGTGG 2087 TACCTCTTCTGTACCCACCGAGG 2088 ATTCAGACCGAATGGCTGCGCGG 2089 CGGTCCGGTTGAGAAGATGTGGG 2090 ACGCGCGGCAGTCTCCACGCCGG 2091 CGAGTTGCTCTGGGTCCTAGAGG 2092 AGGCTTGAGTGCATAACCAGAGG 2093 GGGATCTGCGAAGAGCGGCGGGG
TABLE-US-00107 TABLE 107 Target sequences for SLC6A8 gene SEQ ID NOS Target sequence 2094 ACTCTCCAAGCACATTACAGGGG 2095 ATAGGTCTATGTGGTCCGGGTGG 2096 GGTCTGATCAGGTCTTGAAGGGG 2097 GATGAGGCGCTTCACCCCCGTGG 2098 AGGCAAGCGAGTCCTCTACCCGG 2099 CGCGTCCAGGTCTCGCGCGGCGG 2100 GGTCATCCTGCAAACCTTCGGGG 2101 ACCGCAGCATTCTGGTCCGTAGG 2102 GCAGACAAACGAGGCGCCCAGGG 2103 CCGCAGCATTCTGGTCCGTAGGG
TABLE-US-00108 TABLE 108 Target sequences for SLC22A5 gene SEQ ID NOS Target sequence 2104 GCTAATTCCCCAGTACCCCAGGG 2105 TGGCTTGGGAACGCTTCACGAGG 2106 GCATCCAACCCCTAATCAGGAGG 2107 GGGCCATAGAGCATCGCCCAGGG 2108 GAGTTGTCAAGGGCGGTCAGTGG 2109 GTCCCTCTTATAAGATTAGGCGG 2110 GTATTATAGAAGGGTTTTCGGGG 2111 TGAGGTAAGGGATGTGCTCGGGG 2112 GTGGGTAAGTATCCCTGCAGTGG 2113 TTACATAGGGCGCACGACCAGGG
TABLE-US-00109 TABLE 109 Target sequences for SMAD4 gene SEQ ID NOS Target sequence 2114 CTTCGGGAAGAAACAGACGCTGG 2115 TCTTATAACCACCTACCACTAGG 2116 TAGTAGAATCATTACATGCGAGG 2117 GTCATACCAAAAGGCCACATTGG 2118 TGAGTGGCGAAGGCGTACGGTGG 2119 ATTCTCCCACGAGCTGCAAGCGG 2120 GTTTGAGGGAGTGGTCGCCGGGG 2121 GCAATTCAACCATGTGAGGGTGG 2122 TTAATGGGGTAAGCTAAGCCAGG 2123 TTTTGCACCGTAGTTTAAGGTGG
TABLE-US-00110 TABLE 110 Target sequences for SMARCE1 gene SEQ ID NOS Target sequence 2124 GTAGTGCTATGGATTAAACGAGG 2125 GTGTAGGAATCATATCACCTGGG 2126 CAGAACCATGACGACCTTAGGGG 2127 AGCTATTGTCCCAGAATACGTGG 2128 GCATCGTTGCAAGAAGTGGGAGG 2129 AAGTAACTACTCTAACTATGGGG 2130 CAGTGAGGGCCATAGTTCGTTGG 2131 GTGCCTATACCACAAATCCCAGG 2132 GGTAGATTTAGGCATGGTGTAGG 2133 AGTCCTCTCCATATAGGCACAGG
TABLE-US-00111 TABLE 111 Target sequences for SMN1 gene SEQ ID NOS Target sequence 2134 CCGGGTGTAAGGGGGCCATTAGG 2135 TTCAAATAATGTCGGGGTGGTGG 2136 CTTCATATCACTGTACCTACTGG 2137 GCCGAGTTCCGGGTGTAAGGGGG 2138 ACACACTGGAGTTCGAGACGAGG 2139 GAAGGATGGCCAGCTCTTATTGG 2140 AAGGATGGCCAGCTCTTATTGGG 2141 TACATGAGTGGCTATCATACTGG 2142 GTTGTTGCGCAATAGATCTTCGG 2143 CATATCTTATACAGGTGACATGG 2144 TCATCTCGTTTTGATCAGTGGGG 2145 GGTGTAGATTAGTAATGAAGTGG 2146 GCATGGCAGCGCACTGTTAAAGG 2147 GCAGTCCTGGTGGTCCGTTCTGG 2148 CACATCTATGATACGTGAATGGG 2149 TCATACACAATCTTGCTGTCTGG 2150 AAACCCGCGGGTGCGCAGCGTGG 2151 ACGAATCTGCCAAAACTTAGTGG 2152 CTTCTCACGCTTTCTACGAGTGG 2153 GCGTTTGGAGCATATTGTGTAGG 2154 AGTTTCAAATAATGTCGGGGTGG 2155 CGCACGAAAACTGCCCAGCACGG 2156 CGTGCTGGGCAGTTTTCGTGCGG 2157 TGCCGCACCCAGCTGTAAACTGG 2158 CTATAGGGTAGAGTTGGATTTGG 2159 CAGGAAACTTACCTGGTTAGAGG 2160 TTCCCTGGTCATATCTTGGTTGG 2161 ATCATCTCGTTTTGATCAGTGGG 2162 AAGTTGGTGTCTATGCCATAAGG 2163 ATATCTTATACAGGTGACATGGG 2164 GTGTAGATTAGTAATGAAGTGGG 2165 AGAGCTCAATTCATTAAGCGTGG 2166 ACATCGGTAGGCATATTTCAAGG 2167 GATTCGTGGTCATGAGTTGAAGG 2168 CGTCACTCTTAAGAAGGGACGGG 2169 GCTATGGCGATGAGCAGCGGCGG 2170 GAGCCCAAACTGCTCGAGGAAGG 2171 GATTCCGTGCTGTTCCGGCGCGG 2172 CCGCTATTCACAACAAATATGGG 2173 TCTACTCATGGTATGTGGATAGG 2174 TAGGCATTCCCAATAAGAGCTGG 2175 GATTGAAATGGGGCTCGATGTGG 2176 CAGAAGTAATGAAACCGTTGGGG 2177 CACGTTACTAAGAGCAACTCTGG 2178 AACCCGCGGGTGCGCAGCGTGGG 2179 GGCCGAGTTCCGGGTGTAAGGGG 2180 GTTACTACAAGCGGTCCTCCCGG 2181 GTTTTCGTGCGGCTGTCTCGTGG 2182 CCCGCTATTCACAACAAATATGG 2183 GAAGCGTTATAGAAGATAACTGG 2184 CGTGAGCTTAGAGCATAGACTGG 2185 TAGGCCGAGTTCCGGGTGTAAGG 2186 GAACTGCGATGTAAACATTAAGG 2187 TGTCTTTATATAGATCAAGCAGG 2188 CGATAGTTAGACAGAGTCCTCGG 2189 TCAGATAGATTCGATAACGGAGG 2190 CTTAAGGTTACATTCGCACTTGG 2191 ATAGCAATGTAGGGCCCCAACGG 2192 AATAAGGTATAAGCGGGCTCAGG 2193 AGGCCGAGTTCCGGGTGTAAGGG 2194 CATCAAGTCGATCCGCTCACTGG 2195 CGATCCGCTCACTGGAGTTGTGG 2196 AGGTTACATTCGCACTTGGAAGG 2197 GGTTACATTCGCACTTGGAAGGG 2198 GTTGTCAGTTTGATCCACCGAGG
TABLE-US-00112 TABLE 112 Target sequences for SMN2 gene SEQ ID NOS Target sequence 2199 TCATCTCGTTTTGATCAGTGGGG 2200 GTTGTCAGTTTGATCCACCGAGG 2201 GATTCCGTGCTGTTCCGGCGCGG 2202 ATATCTTATACAGGTGACATGGG 2203 ACACACTGGAGTTCGAGACGAGG 2204 CAGAAGTAATGAAACCGTTGGGG 2205 CGTGCTGGGCAGTTTTCGTGCGG 2206 ATAGCAATGTAGGGCCCCAACGG 2207 AGAGCTCAATTCATTAAGCGTGG 2208 TCAGATAGATTCGATAACGGAGG 2209 CGCACGAAAACTGCCCAGCACGG 2210 AGTTTCAAATAATGTCGGGGTGG 2211 GTGTAGATTAGTAATGAAGTGGG 2212 GAGCCCAAACTGCTCGAGGAAGG 2213 GCCGAGTTCCGGGTGTAAGGGGG 2214 GGTTACATTCGCACTTGGAAGGG 2215 GGTGTAGATTAGTAATGAAGTGG 2216 GCGTTTGGAGCATATTGTGTAGG 2217 CGATAGTTAGACAGAGTCCTCGG 2218 GTTTTCGTGCGGCTGTCTCGTGG 2219 GATTGAAATGGGGCTCGATGTGG 2220 TGTCTTTATATAGATCAAGCAGG 2221 GCTATGGCGATGAGCAGCGGCGG 2222 CGATCCGCTCACTGGAGTTGTGG 2223 TAGGCATTCCCAATAAGAGCTGG 2224 GTTACTACAAGCGGTCCTCCCGG 2225 AGGTTACATTCGCACTTGGAAGG 2226 CATATCTTATACAGGTGACATGG 2227 CGTCACTCTTAAGAAGGGACGGG 2228 CATCAAGTCGATCCGCTCACTGG 2229 CTTCTCACGCTTTCTACGAGTGG 2230 CGTGAGCTTAGAGCATAGACTGG 2231 AAGTTGGTGTCTATGCCATAAGG 2232 CTTCATATCACTGTACCTACTGG 2233 GTTGTTGCGCAATAGATCTTCGG 2234 ATCATCTCGTTTTGATCAGTGGG 2235 GATTCGTGGTCATGAGTTGAAGG 2236 ACGAATCTGCCAAAACTTAGTGG 2237 GGCCGAGTTCCGGGTGTAAGGGG 2238 TACATGAGTGGCTATCATACTGG 2239 CTTAAGGTTACATTCGCACTTGG 2240 AAACCCGCGGGTGCGCAGCGTGG 2241 ACATCGGTAGGCATATTTCAAGG 2242 TCATACACAATCTTGCTGTCTGG 2243 AATAAGGTATAAGCGGGCTCAGG 2244 TTCAAATAATGTCGGGGTGGTGG 2245 TCTACTCATGGTATGTGGATAGG 2246 GAACTGCGATGTAAACATTAAGG 2247 CACATCTATGATACGTGAATGGG 2248 TTCCCTGGTCATATCTTGGTTGG 2249 CAGGAAACTTACCTGGTTAGAGG 2250 AGGCCGAGTTCCGGGTGTAAGGG 2251 CTATAGGGTAGAGTTGGATTTGG 2252 GAAGCGTTATAGAAGATAACTGG 2253 TAGGCCGAGTTCCGGGTGTAAGG 2254 CACGTTACTAAGAGCAACTCTGG 2255 CCGCTATTCACAACAAATATGGG 2256 TGCCGCACCCAGCTGTAAACTGG 2257 CCGGGTGTAAGGGGGCCATTAGG 2258 GCAGTCCTGGTGGTCCGTTCTGG 2259 AAGGATGGCCAGCTCTTATTGGG 2260 AACCCGCGGGTGCGCAGCGTGGG 2261 GCATGGCAGCGCACTGTTAAAGG 2262 GAAGGATGGCCAGCTCTTATTGG 2263 CCCGCTATTCACAACAAATATGG
TABLE-US-00113 TABLE 113 Target sequences for STK11 gene SEQ ID NOS Target sequence 2264 GGACTCTTCTGTCAATTTCG 2265 ACACCCAGGCCTATTTGTCG 2266 GGGCACAAACAGAGGCCTCG 2267 GCGAAAATCCTCTTTACCAT 2268 CAGATGCTGGAACCCCATAA 2269 TGCTTGGACCTATGGTAAAG 2270 TTGGCAGATGCTTGGACCTA 2271 GTAGGTCTTTACATCCCAGG 2272 GATACCTGGACGCTCCTAAG 2273 ATACCTGGACGCTCCTAAGG 2274 GTGATACCTGGACGCTCCTA 2275 TGATACCTGGACGCTCCTAA 2276 TCCACTCCTGGGACATGCCG 2277 GAGCGTCCAGGTATCACCCA 2278 GGAGCGTCCAGGTATCACCC 2279 AAGCCCAGGGCCCACGTCGG 2280 CGGCTCCCACGTCCACTGGG 2281 CACGTCGGTGGGATGGGAAT
TABLE-US-00114 TABLE 114 Target sequences for TGFBR1 gene SEQ ID NOS Target sequence 2282 TGGGTTTTTAGTGACACCTCAGG 2283 TTTCCAACCTGGATCGGGAAGGG 2284 TCACAACGATCAGGTAAATTAGG 2285 ACACTATCTTCACAACGATCAGG 2286 GTCATGGTTGCTGATGTTACAGG 2287 GTGTCAGCTTTACTATCTCCTGG 2288 CTGAAGTCCTAGCTTGTATCTGG 2289 TTTTATTCGTAGGCCACCAAAGG 2290 GTAGTAGAAAGGTCCTAAACAGG 2291 GTAGGAGTCTAAACCAAATCAGG 2292 CATGTCTTAACCTTTCAGTCTGG 2293 TAAACCAAATCAGGTCCACCTGG 2294 GTCTTAACCTTTCAGTCTGGAGG 2295 AGTTGCGTAGGTTTCACTCGTGG 2296 CCTTCCCCACTTATCACATCAGG 2297 AGTGACCTGATGTGATAAGTGGG 2298 TCAATAAGTCAGCTCCATGGTGG 2299 AGTGATACCTCTAACACATGGGG 2300 TAGGTTAAATTAGATTGTCGTGG 2301 ACTATGTTCTGATACACTAAAGG 2302 AACACTGTAATAGGTCTCTCAGG 2303 GATGCTTCAGTGGTTACTCCAGG 2304 AAGAGTGTGCATTCTGTTCGTGG 2305 AGTGTGCATTCTGTTCGTGGTGG
TABLE-US-00115 TABLE 115 Target sequences for TGFBR2 gene SEQ ID NOS Target sequence 2306 CACCACTATCACTTCGTGATAGG 2307 TACCCCGTTTGCACATGAGAGGG 2308 TTCCATTGAGATCACAAGACAGG 2309 TTCCAACACCCATGCTATAATGG 2310 ACTACTTGTCCATTATAGCATGG 2311 GTCCATTATAGCATGGGTGTTGG 2312 CATGGGTGTTGGAAGACTAGAGG 2313 ACAGTCCTAATCAAGCCCACTGG 2314 GGATTCCATAGCAAGTCTTCTGG 2315 TGAGATACAGGCCACATAACAGG 2316 TTGTTAGAAACCAAGCGCCTTGG 2317 TCCCAAATATGGTAGTACTCTGG 2318 TATACAACTTATGCTGCTGAGGG 2319 ATAGAAATTCTTCTCCGTGCTGG 2320 AACCCAGACCTATAGTTAGTTGG 2321 TTTCCAACTAACTATAGGTCTGG 2322 TCACTATTCTCACGTTTCTAAGG 2323 TTCCAACTAACTATAGGTCTGGG 2324 CAATGCTAGTAAACATGCCTGGG 2325 TTGATAAATGGCCTGCAAGTTGG 2326 TCTCTGACAGTAGAATACCCAGG 2327 TCTAGTCAATTAACTGGTGGAGG 2328 CGGGCACACTTAGAATAACGAGG 2329 CTTGCCATCCCCCACGGACAGGG 2330 ACTGAGTGTTATCTAAGCTCAGG 2331 ACGGACAGGGAACTCCATGCTGG 2332 GTTGTACTGAATTGTTACCTAGG 2333 ATGGAGTTCCCTGTCCGTGGGGG 2334 AGCAACTTGACAATACACTAAGG 2335 ACGTGTCAGCTTCTATTCAAAGG 2336 GGGACAGCAATGGTATTCCTCGG 2337 GTGTTACTGTTCTACGAAAAAGG 2338 ACGGGTAGTCTGAAAGGTGCTGG 2339 GAGGTCACGGGTAGTCTGAAAGG 2340 GTTACATGAGGTCTCATCCTAGG 2341 AGGTTGAAATACCCTGGTGCAGG
TABLE-US-00116 TABLE 116 Target sequences for VHL gene SEQ ID NOS Target sequence 2342 ACCATAGGTGGTACATAGTAGGG 2343 CACCATAGGTGGTACATAGTAGG 2344 TATTGAAGTGCAGTGAAGGCAGG 2345 TCAACACTTATCACCATAGGTGG 2346 TAGTAATTTCACCTTGAAATGGG 2347 GGCCCCCTATGGACACCTCATGG 2348 ATTTCACCTTGAAATGGGCTGGG 2349 CAGTACAAGGAACGAACAAGAGG 2350 CTCAGGCGATCTACTGACGTTGG 2351 GTATAAAAGCAGAAGTCAGCAGG 2352 CACCATGAGGTGTCCATAGGGGG 2353 TCAAGGTGAAATTACTACAGAGG 2354 TCTAGCCCATGCCCTCACTGTGG 2355 GCCAATGACTAGCAGAGCGTGGG 2356 ACTAGCAGAGCGTGGGACTGAGG
TABLE-US-00117 TABLE 117 Target sequences for WT1 gene SEQ ID NOS Target sequence 2357 AATCTTGTCTAACATTCCCGAGG 2358 GTTCCCAACTTACTCAACAAGGG 2359 TGGTATGGTTTCTCACCTTGGGG 2360 TTGATCGTCCTAACTGTACAGGG 2361 TGTAGCGAGGATCTACAGGGTGG 2362 GAATGCTACTAACACTGGTGGGG 2363 GTCCTGAGCTCATAATTCGGTGG 2364 GTAGCGAGGATCTACAGGGTGGG 2365 TACTCCTTACAACTGCCCGTAGG 2366 CTCCTTACAACTGCCCGTAGGGG
Sequencing
[0070] Following target enrichment of the sample using the methods and systems described elsewhere, the highly fragmented gDNA samples can be sequenced to detect genomic variations. In some embodiments, short-read sequencing is used. In some embodiments, long-read sequencing. In some cases, the sample contains high fragmented RNA samples. In some case the sample contains full-length RNA transcripts.
[0071] In some embodiments, the long-read sequencing platform may be single molecule real time sequencing (SMRT) (e.g. Pacific Biosciences long-read sequencing technology), or a variation thereof. Single-molecule real-time sequencing (SMRT) is a parallelized single molecule DNA sequencing method. Single-molecule real-time sequencing utilizes a zero-mode waveguide (ZMW). A single DNA polymerase enzyme is affixed at the bottom of a ZMW with a single molecule of DNA as a template. The ZMW is a structure that creates an illuminated observation volume that is small enough to observe only a single nucleotide of DNA being incorporated by DNA polymerase. Each of the four DNA bases is attached to one of four different fluorescent dyes. When a nucleotide is incorporated by the DNA polymerase, the fluorescent tag is cleaved off and diffuses out of the observation area of the ZMW where its fluorescence is no longer observable. A detector detects the fluorescent signal of the nucleotide incorporation, and the base call is made according to the corresponding fluorescence of the dye.
[0072] In other embodiments, the long-read sequencing platform may be nanopore sequencing (e.g. Oxford Nanopore long-read sequencing technology), or a variation thereof. Nanopore sequencing uses electrophoresis to transport an unknown sample through an orifice of about 10.sup.−9 meters in diameter. A nanopore system can contains an electrolytic solution; when a constant electric field is applied, an electric current can be observed in the system. The magnitude of the electric current density across a nanopore surface depends on the nanopore's dimensions and the composition of DNA or RNA molecule that is occupying the nanopore. Sequencing is made possible because, while traversing through the nanopore, samples cause characteristic changes in electric current density across nanopore surfaces. The total charge flowing through a nanopore channel is equal to the surface integral of electric current density flux across the nanopore unit normal surfaces between times t.sub.1 and t.sub.2.
[0073] In some cases, long-read sequencing requires application of the sample. In other cases, long-read sequencing does not require application of the sample.
Clinical Applications
[0074] The systems and methods described herein can be used in clinical settings to detect and diagnose genetic diseases or disorders. In some embodiments, the systems and methods described herein can be used can be used in the detection, treatment and/or monitoring of hereditary breast cancer-related disorders by detecting genetic variations in relevant genes such as BRCA1, BRCA2, MLH1, MSH2, and STK11. In some embodiments, the systems and methods described herein can be used can be used in the detection, treatment and/or monitoring of hereditary colon cancer-related disorders by detecting genetic variations in relevant genes such as MLH1, MSH2, EPCAM, SMAD4, and STK11. In some embodiments, the systems and methods described herein can be used can be used in the detection, treatment and/or monitoring of hereditary neuroendocrine tumor disorders by detecting genetic variations in relevant genes such as SDHB, SHDC, SDHD, and VHL. In some embodiments, the systems and methods described herein can be used can be used in the detection, treatment and/or monitoring of Cowden Syndrome by detecting genetic variations in relevant genes such as PTEN.
[0075] In some embodiments, the systems and methods described herein can be used can be used in the detection, treatment and/or monitoring of neuromuscular disorders such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy by detecting genetic variations in relevant genes such as DMD, SMN1, and SMN2.
[0076] In some embodiments, the systems and methods described herein can be used can be used in the detection, treatment and/or monitoring of Fragile X Syndrome by detecting genetic variations in relevant genes such as FMR1.
[0077] In some embodiments, the systems and methods described herein can be used can be used in the detection, treatment and/or monitoring of cardiovascular disorders such as aortic dysfunction and dilation, and cardiac ion channelopathies, by detecting genetic variations in relevant genes such as TGFBR1, TFRBR2, MYH11, COL3A1, KCNH2 and KCNQ1.
[0078] In some embodiments, the systems and methods described herein can be used can be used in the detection, treatment and/or monitoring of movement disorders such as Parkinson Disease, Hereditary Ataxia, and Dystonia 5, by detecting genetic variations in relevant genes such as SCNA, PARK2, PARK7, PINK1, SCA1 (ATXN1), SCA10 (ATXN10), SCA17 (TBP), SCA2 (ATXN2), SCA3 (MJD/ATXN3), SCA6 (CACNA1A), SCAT (ATXN7), SCAB (ATXN8OS) and GCH1.
[0079] In some embodiments, the systems and methods described herein can be used can be used in the detection, treatment and/or monitoring of renal disorders (e.g. Alport Syndrome and Polycystic Kidney Disease) by detecting genetic variations in relevant genes such as COL4A5, PKD1 and PKD2.
[0080] In some embodiments, the systems and methods described herein can be used can be used in the detection, treatment and/or monitoring of adrenal disorders (e.g. Congenital Adrenal Hyperplasia) by detecting genetic variations in relevant genes such as CYP21A2.
[0081] In some embodiments, the systems and methods described herein can be used can be used in the detection, treatment and/or monitoring of neurodevelopmental disorders (e.g. Rett Syndrome) by detecting genetic variations in relevant genes such as FOXG1, and MeCP2.
[0082] In some embodiments, the systems and methods described herein can be used can be used in the detection, treatment and/or monitoring of cerebrovascular disorders (e.g. Cerebral Cavernous Malformations) by detecting genetic variations in relevant genes such as KRIT1 and PDCD10.
[0083] In some embodiments, the systems and methods described herein can be used can be used in the detection, treatment and/or monitoring of neuro-oncology (e.g. Neurofibromatosis Type 1 and Neurofibromatosis Type 2) by detecting genetic variations in relevant genes such as NF1 and NF2.
[0084] In some embodiments, the systems and methods described herein can be used can be used in the detection, treatment and/or monitoring of epilepsy (e.g. Unverricht-Lundborg disease) by detecting genetic variations in relevant genes such as CSTB.
[0085] In some embodiments, the systems and methods described herein can be used can be used in the detection, treatment and/or monitoring of peripheral neuropathy by detecting genetic variations in relevant genes such as GJB1 and PMP22.
[0086] Use systems and methods described herein with existing clinical sequencing methods. In some cases, a sample can be analyzed using short-read sequencing to detect SNVs and indels, and long-read sequencing to detect SVs.
Kits
[0087] In some embodiment, a kit Is described herein. The kit may comprise a plurality of crRNA probes disclosed herein. Further, the kit may comprise a plurality of tracerRNA molecules. The kit may comprise reagents that can be used to performing dA tailing and adapter ligation. Moreover, the kit may comprise any buffer that can be used in performing needed experiments. The kit may comprise instructions for performing any experiments and procedures described herein.
Computer Systems
[0088] The present disclosure provides computer systems that are programmed to implement methods of the disclosure.
[0089] The computer system 501 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 505, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 501 also includes memory or memory location 510 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 515 (e.g., hard disk), communication interface 520 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 525, such as cache, other memory, data storage and/or electronic display adapters. The memory 510, storage unit 515, interface 520 and peripheral devices 525 are in communication with the CPU 505 through a communication bus (solid lines), such as a motherboard. The storage unit 515 can be a data storage unit (or data repository) for storing data. The computer system 501 can be operatively coupled to a computer network (“network”) 530 with the aid of the communication interface 520. The network 530 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 530 in some cases is a telecommunication and/or data network. The network 530 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 530, in some cases with the aid of the computer system 501, can implement a peer-to-peer network, which may enable devices coupled to the computer system 501 to behave as a client or a server.
[0090] The CPU 505 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 510. The instructions can be directed to the CPU 505, which can subsequently program or otherwise configure the CPU 505 to implement methods of the present disclosure. Examples of operations performed by the CPU 505 can include fetch, decode, execute, and writeback.
[0091] The CPU 505 can be part of a circuit, such as an integrated circuit. One or more other components of the system 501 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).
[0092] The storage unit 515 can store files, such as drivers, libraries and saved programs. The storage unit 515 can store user data, e.g., user preferences and user programs. The computer system 501 in some cases can include one or more additional data storage units that are external to the computer system 501, such as located on a remote server that is in communication with the computer system 501 through an intranet or the Internet.
[0093] The computer system 501 can communicate with one or more remote computer systems through the network 530. For instance, the computer system 501 can communicate with a remote computer system of a user (e.g., operator). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 501 via the network 530.
[0094] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 501, such as, for example, on the memory 510 or electronic storage unit 515. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 505. In some cases, the code can be retrieved from the storage unit 515 and stored on the memory 510 for ready access by the processor 505. In some situations, the electronic storage unit 515 can be precluded, and machine-executable instructions are stored on memory 510.
[0095] The code can be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.
[0096] Aspects of the systems and methods provided herein, such as the computer system 501, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.
[0097] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.
[0098] The computer system 501 can include or be in communication with an electronic display 535 that comprises a user interface (UI) 540 for providing, for example, monitoring of sample preparation, monitoring of reactions and/or reaction conditions, monitoring of sequencing, results of sequencing, and permitting user inputs for sample preparation, reactions, sequencing and/or sequencing analysis. Examples of UIs include, without limitation, a graphical user interface (GUI) and web-based user interface.
[0099] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 505. The algorithm can, for example, implement sample preparation protocols, reaction protocols, sequencing protocols, data analysis protocols and system or device operation protocols.
[0100] Devices, systems, compositions and methods of the present disclosure may be used for various applications, such as, for example, processing a single analyte (e.g., RNA, DNA, or protein) or multiple analytes (e.g., DNA and RNA, DNA and protein, RNA and protein, or RNA, DNA and protein) from a cell. For example, a biological particle or analyte carrier (e.g., a cell or cell bead) is partitioned in a partition (e.g., droplet), and multiple analytes from the biological particle or analyte carrier are processed for subsequent processing. The multiple analytes may be from the cell. This may enable, for example, simultaneous proteomic, transcriptomic and genomic analysis of the cell.
EXAMPLES
Example 1: Target Enrichment Protocol
[0101] An exemplary target enrichment protocol begins with preparing the Cas9 ribonucleoprotein complexes (RNPs). Prior to guide RNA assembly, all crRNAs are pooled into an equimolar mix, with a total concentration of 50-100 μM. The crRNA mix and tracrRNA are then combined such that the tracrRNA concentration and the total crRNA concentration are both 5-10 μM. The gRNA duplexes are formed by denaturation at 95° C. and then cooling to room temperature. Ribonucleoprotein complexes (RNPs) are constructed by combining the gRNA duplexes with Cas9 nucleases and then incubating at room temperature.
[0102] The next stage comprises dephosphorylating the genomic DNA. Between one to four genomic DNA samples can be pooled into the dephosphorylation reaction, for a total of 1-5 μg of gDNA in each phosphorylation reaction. The input DNA is dephosphorylated using Calf Intestinal Phosphatase or Shrimp Alkaline Phosphatase.
[0103] The next stage comprises cleaving and dA-tailing target DNA. RNPs are added to the dephosphorylated gDNA along with dATP and Taq DNA polymerase. The sample is then incubated at 37° C. for Cas9 cleavage followed by 72° C. for dA-tailing. The reaction is then cleaned up using SPRI beads. Next is barcode ligation. Barcodes are ligated to the dA-tailed ends of the gDNA using ligase. The reaction is incubated at room temperature and then cleaned up using SPRI beads.
[0104] Next stage is sequencing adapter ligation and clean-up. All the barcoded DNA are pooled together at an equimolar amount. Sequencing adapters are ligated to the pool of barcoded DNA using ligase. The DNA is then cleaned up using SPRI beads, and then eluted in elution buffer.
[0105] The next stage is priming and loading the Flow Cell. Libraries were prepared for sequencing by adding the following to the eluate: Sequencing Buffer, Loading Beads, and Flush Tether. The sequencing libraries are then loaded onto the flow cell for sequencing.
Example 2: BRCA1 crRNA Probe Design
[0106] In the case of BRCA1, the CHOPCHOP design program yielded a total of 5567 possible crRNA probes along the entire length of the BRCA1 genomic locus. These crRNA sequences were then filtered using the filtering scheme described in [0041], reducing the number to 233 crRNA probes. The crRNA sequences were then checked using a second design checker tool, e.g. IDT CRISPR-Cas9 guide RNA design checker tool. The number of candidate crRNA probes was reduced to 86 probes. The final set of crRNA probes was chosen based upon the location of the target sites.
[0107] As shown in
[0108] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the inventions be limited by the specific examples provided within the specification. While the invention has been described with reference to aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the inventions are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.