Methods and systems for analyzing complex genomic regions - Patents.com

Abstract

Provided herein is an improved method for analyzing (e.g., sequencing, genotyping, structural analysis) complex genomic regions.In some cases, the method involves the use of CRISPR-associated endonucleases and pairs of outer guide RNAs and pairs of inner guide RNAs to excise genomic regions of interest from genomic DNA.The method further involves the use of long-read sequencing to sequence gene regions of interest.In some cases, the method is amplification-free.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 171,387, filed April 6, 2021, which is incorporated by reference herein in its entirety.

[0002] Sequence Listing This application has been submitted electronically in ASCII format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy was created on April 5, 2022, is named 57312-702_601_SL.txt, and is 109,652 bytes in size. [Background technology]

[0003] background Pharmacogenetics (PGx) is a component of precision medicine that allows for the determination of individualized drug responses, since genetic variations can affect response to drug application. Benefits of PGx include reduced cost and risk of adverse drug reactions (SADRs), as well as improved efficacy. Although numerous PGx genes are currently being tested, the diagnostic value of cytochrome P450 2D6 (CYP2D6) is enormous, since up to 25% of all drugs are activated or metabolized by CYP2D6. These drugs include cancer drugs, opioid agonists, and some antidepressants and anxiolytics. The CYP2D6 enzyme is encoded by the CYP2D6 gene, and genetic variations can lead to reduced or complete loss of enzyme function. CYP2D6 is primarily expressed in the liver and is a major contributor to hepatic drug metabolism and clearance. Problems with correctly diagnosing genetic variations in CYP2D6 can directly affect the risk of developing SADRs. The NIH Clinical Pharmacogenetics Implementation Consortium (CPIC) currently lists 58 drugs with associated evidence supporting CYP2D6 clinical trials, making CYP2D6 one of the top genes. CYP2D6 testing in the United States alone was a $522M market in 2019, with an estimated annual growth rate of 6-8%.

[0004] To date, over 100 pharmacogenetic relevant alterations (also referred to as star allele haplotypes) have been described for CYP2D6, including frequent copy number variations. In addition, variant calling is complicated by gene fusions and hybrids with nearby highly homologous (up to 94% identical) pseudogenes (CYP2D7 and CYP2D8). Approximately 13% of individuals in the United States have CYP2D6 structural variants, and these variants represent 7% of all mutations associated with the gene. These features complicate genetic analysis using current testing platforms, and many rare or complex haplotypes are not accurately analyzed. Studies by many groups have demonstrated that currently used commercial genotyping platforms are prone to mischaracterization of CYP2D6. This leads to erroneous assignments and, consequently, incorrect medication recommendations. Gene sequencing is similarly hampered whether based on short reads (NGS) or template length (Sanger sequencing). Although numerous methods have been developed that combine targeted amplification, copy number analysis, and long-range PCR to more precisely determine the overall structure, these methods are not suitable for routine clinical testing due to their complex workflow, time requirements, and overall cost. Summary of the Invention [Means for solving the problem]

[0005] overview There is an unmet need for improved methods and systems for accurately and cost-effectively analyzing complex genomic regions. The present disclosure meets this unmet need.

[0006] In one aspect of the present disclosure, a method for analyzing (e.g., sequencing, genotyping, structural analysis) a genomic region of interest is provided, comprising: a) contacting genomic DNA comprising the genomic region of interest with a pair of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease and a pair of outer guide RNA (gRNA), thereby generating a first excised fragment comprising the genomic region of interest; b) contacting the first excised fragment with a pair of CRISPR-associated endonuclease and a pair of inner gRNA, thereby generating a second excised fragment comprising the genomic region of interest; and c) analyzing the genomic region of interest contained in the second excised fragment. In some cases, the CRISPR-associated endonuclease and the pair of outer gRNA in a) associate with and block the 5'-end and 3'-end of the first excised fragment. In some cases, the method further comprises contacting the product of a) with one or more exonucleases prior to b), such that background genomic DNA is digested and the first excised fragment is not digested. In some cases, the one or more exonucleases are selected from the group consisting of exonuclease I, exonuclease II, exonuclease III, exonuclease IV, exonuclease V, exonuclease VI, exonuclease VII, exonuclease VIII, and any combination thereof. In some cases, the pair of outer gRNAs comprises a first outer gRNA and a second outer gRNA. In some cases, the first outer gRNA comprises a nucleotide sequence substantially complementary to a first nucleotide sequence present in the genomic DNA, and the second outer gRNA comprises a nucleotide sequence substantially complementary to a second nucleotide sequence present in the genomic DNA. In some cases, the first nucleotide sequence and the second nucleotide sequence are different. In some cases, the first nucleotide sequence and the second nucleotide sequence flank the genomic region of interest.In some cases, the first nucleotide sequence, the second nucleotide sequence, or both are present in the genomic DNA up to about 100 kilobases in length from the genomic region of interest. In some cases, the pair of inner gRNAs includes a first inner gRNA and a second inner gRNA. In some cases, the first inner gRNA includes a nucleotide sequence that is substantially complementary to a third nucleotide sequence present in the genomic DNA, and the second inner gRNA includes a nucleotide sequence that is substantially complementary to a fourth nucleotide sequence present in the genomic DNA. In some cases, the third nucleotide sequence and the fourth nucleotide sequence are different. In some cases, the third nucleotide sequence and the fourth nucleotide sequence flank the genomic region of interest. In some cases, the third nucleotide sequence and the fourth nucleotide sequence are present on the genomic DNA at a base length closer to the genomic region of interest than the first nucleotide sequence and the second nucleotide sequence. In some cases, the second excised fragment is shorter in base length than the first excised fragment. In some cases, the analyzing step comprises sequencing the genomic region of interest contained within the second excised fragment. In some cases, the genomic DNA is provided in an amount of about 10 μg or more. In some cases, the analyzing step comprises genotyping the genomic region of interest contained within the second excised fragment. In some cases, the analyzing step comprises performing a structural analysis on the genomic region of interest contained within the second excised fragment. In some cases, the method further comprises isolating the first excised fragment before b). In some cases, the method further comprises isolating the second excised fragment before c). In some cases, the method does not involve DNA amplification. In some cases, the method further comprises attaching one or more adaptors to the 5' end, 3' end, or both of the second excised fragment before c). In some cases, the CRISPR-associated endonuclease is a class 1 CRISPR-associated endonuclease or a class 2 CRISPR-associated endonuclease.In some cases, the class 1 CRISPR-associated endonuclease is selected from the group consisting of Cas3, Cas5, Cas8a, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Csx11, Csx10, and Csf1. In some cases, the class 2 CRISPR-associated endonuclease is selected from the group consisting of Cas9, Cas12a, Csn2, Cas4, Cas12b, Cas12c, Cas13a, Cas13b, Cas13c, and Cas13d. In some cases, the CRISPR-associated endonuclease comprises an amino acid sequence having at least 80% sequence identity to a wild-type CRISPR-associated endonuclease. In some cases, the CRISPR-associated endonuclease is Cas9 or a mutant thereof. In some cases, Cas9 is Streptococcus pyogenes Cas9 (spCas9). In some cases, the Cas9 mutant comprises one or more point mutations selected from the group consisting of R780A, K810A, K848A, K855A, H982A, K1003A, R1060A, D1135E, N497A, R661A, Q695A, Q926A, L169A, Y450A, M495A, M694A, and M698A compared to wild-type Streptococcus pyogenes Cas9 (spCas9). In some cases, the genomic DNA is not fragmented, digested, or sheared before a). In some cases, the genomic DNA is not subjected to restriction enzyme digestion before a). In some cases, the genomic region of interest is a complex genomic region. In some cases, the complex genomic region comprises a gene of interest and one or more of its pseudogenes. In some cases, the one or more pseudogenes comprise a nucleotide sequence with at least 75% sequence identity to the gene of interest. In some cases, the complex genomic region comprises one or more repeat regions, one or more duplications, one or more insertions, one or more inversions, one or more tandem repeats, one or more retrotransposons, or any combination thereof. In some cases, the genomic region of interest is a highly polymorphic locus.In some cases, the length of the first excised fragment is at least about 0.06 kilobases. In some cases, the length of the first excised fragment is up to about 200 kilobases. In some cases, the length of the second excised fragment is at least about 0.02 kilobases. In some cases, the length of the second excised fragment is up to about 199.98 kilobases. In some cases, the sequencing comprises long-read sequencing. In some cases, the long-read sequencing comprises single molecule real-time sequencing or nanopore sequencing. In some cases, the method does not involve either polymerase chain reaction (PCR) or isothermal amplification. In some cases, the method does not involve either multiple displacement amplification (MDA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification, rolling circle amplification (RCA), ligase chain reaction (LCR), helicase-dependent amplification, or ramification amplification method. In some cases, the genomic DNA is provided or obtained in a biological sample. In some cases, the biological sample includes a bodily fluid (e.g., blood (e.g., whole blood, plasma, serum), urine, saliva, bone marrow, spinal fluid, sputum, peritoneal fluid, lymphatic fluid, pleural fluid, amniotic fluid, semen, vaginal fluid, sweat, stool, glandular secretion, ocular fluid, breast milk) or a solid tissue sample. In some cases, the biological sample is a diagnostic sample. In some cases, the genomic region of interest is a locus including CYP2D6, CYP2D7, and CYP2D8. In some cases, the analyzing step includes identifying one or more genetic variations of CYP2D6. In some cases, the method further includes identifying the subject as having a reduced, lost, or increased CYP2D6 function based on the genetic variation. In some cases, the method further includes recommending a treatment or an alternative treatment to the subject based on the identifying step. In some cases, if the subject is identified as having reduced, lost, or increased CYP2D6 function, an alternative treatment is recommended for the subject. In some cases, the method further includes recommending a dosage of a therapeutic agent to the subject based on the identifying step.In some cases, if a subject is identified as having reduced, lost, or increased CYP2D6 function, the dosage of the therapeutic agent is altered. In some cases, the outer gRNA pair, the inner gRNA pair, or both are selected from any one of SEQ ID NOs: 1-418.

[0007] In another aspect, a kit for analyzing a genomic region of interest is provided, comprising: a) a clustered regularly interspaced short palindromic repeats (CRISPR) associated endonuclease; b) a pair of outer gRNAs comprising i) a first outer gRNA comprising a nucleotide sequence substantially complementary to a first nucleotide sequence present upstream of the genomic region of interest in genomic DNA, and ii) a second outer gRNA comprising a nucleotide sequence substantially complementary to a second nucleotide sequence present downstream of the genomic region of interest in genomic DNA; c) a pair of inner gRNAs comprising iii) a first inner gRNA comprising a nucleotide sequence substantially complementary to a third nucleotide sequence present upstream of the genomic region of interest in genomic DNA, and iv) a second inner gRNA comprising a nucleotide sequence substantially complementary to a fourth nucleotide sequence present downstream of the genomic region of interest in genomic DNA, wherein the third nucleotide sequence and the fourth nucleotide sequence are present on the genomic DNA at a base length closer to the genomic region of interest than the first nucleotide sequence and the second nucleotide sequence. In some cases, the kit further comprises one or more exonucleases.In some cases, the one or more exonucleases are selected from the group consisting of exonuclease I, exonuclease II, exonuclease III, exonuclease IV, exonuclease V, exonuclease VI, exonuclease VII, exonuclease VIII, and any combination thereof.In some cases, the CRISPR-associated endonuclease is class 1 or class 2 CRISPR-associated endonuclease. In some cases, the class 1 CRISPR-associated endonuclease is selected from the group consisting of Cas3, Cas5, Cas8a, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Csx11, Csx10, and Csf1. In some cases, the class 2 CRISPR-associated endonuclease is selected from the group consisting of Cas9, Cas12a, Csn2, Cas4, Cas12b, Cas12c, Cas13a, Cas13b, Cas13c, and Cas13d.In some cases, the CRISPR-associated endonuclease comprises an amino acid sequence having at least 80% sequence identity to the wild-type CRISPR-associated endonuclease. In some cases, the CRISPR-associated endonuclease is Cas9 or a mutant thereof. In some cases, the Cas9 is Streptococcus pyogenes Cas9 (spCas9). In some cases, the Cas9 mutant comprises one or more point mutations selected from the group consisting of R780A, K810A, K848A, K855A, H982A, K1003A, R1060A, D1135E, N497A, R661A, Q695A, Q926A, L169A, Y450A, M495A, M694A and M698A compared to the wild-type Streptococcus pyogenes Cas9 (spCas9). In some cases, the genomic region of interest is a genomic locus including CYP2D6, CYP2D7, and CYP2D8. In some cases, the first outer guide RNA, the first inner guide RNA, or both, comprises a nucleotide sequence of any one of SEQ ID NOs: 3-12, 17-26, 68-77, 82-214, and 344-418. In some cases, the second outer guide RNA, the second inner guide RNA, or both, comprises a nucleotide sequence of any one of SEQ ID NOs: 1, 2, 13-16, 27-67, 78-81, and 215-343. In some cases, the kit further comprises instructions for using the kit in a nested CRISPR reaction. In some cases, the kit further comprises instructions for using the kit to excise a genomic region of interest from genomic DNA.

[0008] In one aspect, a method of analyzing a genomic region of interest is provided, comprising: (a) contacting genomic DNA comprising the genomic region of interest with a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease and two or more gRNAs, thereby generating an excised genomic region of interest; (b) isolating the genomic DNA comprising the genomic region of interest; and (c) analyzing the excised genomic region of interest, without DNA amplification. In some cases, the analyzing step comprises sequencing the excised genomic region of interest. In some cases, the analyzing step comprises genotyping the excised genomic region of interest. In some cases, the analyzing step comprises performing a structural analysis on the excised region of interest. In some cases, the isolating step of (b) is performed before the contacting step of (a). In some cases, the isolating step of (b) is performed after the contacting step of (a). In some cases, two or more gRNAs each comprise a nucleotide sequence that is substantially complementary to a different nucleotide sequence present in genomic DNA.In some cases, the different nucleotide sequences flank the genomic region of interest.In some cases, the CRISPR-associated endonuclease cuts the genomic region of interest at the genomic site that flanks the genomic region of interest.In some cases, the CRISPR-associated endonuclease is a class 1 or class 2 CRISPR-associated endonuclease.In some cases, the class 1 CRISPR-associated endonuclease is selected from the group consisting of Cas3, Cas5, Cas8a, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Csx11, Csx10, and Csf1.In some cases, the class 2 CRISPR-associated endonuclease is selected from the group consisting of Cas9, Cas12a, Csn2, Cas4, Cas12b, Cas12c, Cas13a, Cas13b, Cas13c, and Cas13d. In some cases, the CRISPR-associated endonuclease comprises an amino acid sequence having at least 80% sequence identity to the wild-type CRISPR-associated endonuclease. In some cases, the CRISPR-associated endonuclease is Cas9 or a mutant thereof. In some cases, the Cas9 is Streptococcus pyogenes Cas9 (spCas9). In some cases, the Cas9 mutant comprises one or more point mutations selected from the group consisting of R780A, K810A, K848A, K855A, H982A, K1003A, R1060A, D1135E, N497A, R661A, Q695A, Q926A, L169A, Y450A, M495A, M694A and M698A compared to wild-type Streptococcus pyogenes Cas9 (spCas9). In some cases, the genomic DNA is not fragmented, digested or sheared prior to (a). In some cases, the genomic DNA is not subjected to restriction enzyme digestion prior to (a). In some cases, the genomic region of interest is a complex genomic region. In some cases, the complex genomic region comprises a gene and one or more pseudogenes thereof. In some cases, the one or more pseudogenes comprise a nucleotide sequence having at least 75% sequence identity to the gene. In some cases, the complex genomic region comprises one or more repeat regions, one or more duplications, one or more insertions, one or more inversions, one or more tandem repeats, one or more retrotransposons, or any combination thereof. In some cases, the genomic region of interest is a highly polymorphic locus. In some cases, the length of the excised genomic region of interest is at least 10 kilobases. In some cases, the length of the excised genomic region of interest is up to 250 kilobases. In some cases, the isolating step comprises isolating high molecular weight DNA. In some cases, the length of the high molecular weight DNA is at least 50 kilobases.In some cases, the sequencing comprises long-read sequencing. In some cases, the long-read sequencing comprises single molecule real-time sequencing or nanopore sequencing. In some cases, the method further comprises ligating one or more sequencing adaptors to one or both ends of the excised genomic region of interest. In some cases, the method further comprises dephosphorylating the genomic DNA prior to a). In some cases, the dephosphorylating comprises treating the genomic DNA with a phosphatase. In some cases, the phosphatase is shrimp alkaline phosphatase. In some cases, the method further comprises treating the genomic DNA with terminal transferase (TdT) after the dephosphorylating. In some cases, the method further comprises end-tailing the excised genomic region of interest. In some cases, the end-tailing comprises adding one or more adenosine nucleotides to the free 3' end of the excised genomic region of interest. In some cases, the method does not involve polymerase chain reaction (PCR) or isothermal amplification. In some cases, the method does not involve multiple displacement amplification (MDA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification, rolling circle amplification (RCA), ligase chain reaction (LCR), helicase-dependent amplification, or ramification amplification method. In some cases, the genomic DNA is provided in a biological sample. In some cases, the genomic DNA is provided in or obtained from a biological sample. In some cases, the biological sample comprises a bodily fluid (e.g., blood (e.g., whole blood, plasma, serum), urine, saliva, bone marrow, spinal fluid, sputum, peritoneal fluid, lymphatic fluid, pleural fluid, amniotic fluid, semen, vaginal fluid, sweat, stool, glandular secretion, ocular fluid, breast milk) or a solid tissue sample. In some cases, the biological sample is a diagnostic sample.

[0009] In another aspect, a method for analyzing a complex genomic region of interest that is at least 10 kilobases in length is provided, comprising: (a) providing genomic DNA comprising the complex genomic region of interest; (b) isolating high molecular weight DNA comprising the complex genomic region of interest; (c) contacting the genomic DNA with a clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease and two or more gRNAs to excise the complex genomic region of interest, wherein the two or more gRNAs each comprise a nucleotide sequence substantially complementary to a different nucleotide sequence present in the genomic DNA, and the different nucleotide sequences flank the complex genomic region of interest; and (d) analyzing the complex genomic region of interest, without DNA amplification. In some cases, the analyzing step comprises sequencing the complex genomic region of interest. In some cases, the sequencing comprises long-read sequencing. In some cases, the long-read sequencing comprises single-molecule real-time sequencing or nanopore sequencing. In some cases, the analyzing step includes genotyping the complex genomic region of interest. In some cases, the analyzing step includes performing structural analysis of the genomic region of interest. In some cases, the isolating step (b) is performed before the contacting step (c). In some cases, the isolating step (b) is performed after the contacting step (c). In some cases, the length of the high molecular weight DNA is at least 10 kilobases. In some cases, the complex genomic region of interest includes a target gene and one or more pseudogenes thereof. In some cases, the one or more pseudogenes have at least 75% sequence identity to the target gene. In some cases, the complex genomic region of interest includes CYP2D6, CYP2D7, and CYP2D8. In some cases, the complex genomic region of interest includes CYP2C8, CYP2C9, CYP2C18, and CYP2C19.In some cases, the complex genomic region of interest comprises one or more repeat regions, one or more duplications, one or more insertions, one or more inversions, one or more tandem repeats, one or more retrotransposons, or any combination thereof.In some cases, the complex genomic region of interest is a highly polymorphic locus.In some cases, the CRISPR-associated endonuclease is a class 1 or class 2 CRISPR-associated endonuclease.In some cases, the class 1 CRISPR-associated endonuclease is selected from the group consisting of Cas3, Cas5, Cas8a, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Csx11, Csx10, and Csf1. In some cases, the class 2 CRISPR-associated endonuclease is selected from the group consisting of Cas9, Cas12a, Csn2, Cas4, Cas12b, Cas12c, Cas13a, Cas13b, Cas13c, and Cas13d. In some cases, the CRISPR-associated endonuclease comprises an amino acid sequence having at least 80% sequence identity to the wild-type CRISPR-associated endonuclease. In some cases, the CRISPR-associated endonuclease is Cas9 or a mutant thereof. In some cases, the Cas9 is Streptococcus pyogenes Cas9 (spCas9). In some cases, the Cas9 mutant comprises one or more point mutations selected from the group consisting of R780A, K810A, K848A, K855A, H982A, K1003A, R1060A, D1135E, N497A, R661A, Q695A, Q926A, L169A, Y450A, M495A, M694A and M698A compared to wild-type Streptococcus pyogenes Cas9 (spCas9). In some cases, the genomic DNA is not fragmented or digested prior to a). In some cases, the genomic DNA is not subjected to restriction enzyme digestion prior to a). In some cases, the complex genomic region of interest is up to 250 kilobases in length.In some cases, the method further comprises ligating one or more sequencing adaptors to one or both ends of the excised genomic region of interest. In some cases, the method does not involve polymerase chain reaction (PCR) or isothermal amplification. In some cases, the method does not involve multiple displacement amplification (MDA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification, rolling circle amplification (RCA), ligase chain reaction (LCR), helicase-dependent amplification, or ramification amplification method. In some cases, the genomic DNA is provided or obtained in a biological sample. In some cases, the biological sample is a bodily fluid (e.g., blood (e.g., whole blood, plasma, serum), urine, saliva, bone marrow, spinal fluid, sputum, peritoneal fluid, lymphatic fluid, pleural fluid, amniotic fluid, semen, vaginal fluid, sweat, stool, glandular secretions, intraocular fluid, breast milk) or a solid tissue sample. In some cases, the biological sample is a diagnostic sample.

[0010] In another aspect, a method for analyzing a locus comprising CYP2D6, CYP2D7, and CYP2D8 is provided, the method comprising: (a) providing genomic DNA comprising the locus; (b) contacting the genomic DNA with a clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease and two or more gRNAs to excise the locus from the genomic DNA, the two or more gRNAs each comprising a nucleotide sequence substantially complementary to a different nucleotide sequence present in the genomic DNA, the different nucleotide sequence flanking the locus comprising CYP2D6, CYP2D7, and CYP2D8; and (c) analyzing the locus. In some cases, the analyzing step comprises sequencing the locus. In some cases, the sequencing comprises long-read sequencing. In some cases, the long-read sequencing comprises single-molecule real-time sequencing or nanopore sequencing. In some cases, the analyzing step includes genotyping the locus. In some cases, the analyzing step includes performing a structural analysis of the locus. In some cases, the method further includes isolating high molecular weight DNA comprising the locus prior to c). In some cases, the high molecular weight DNA is at least 10 kilobases in length. In some cases, the two or more gRNAs comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-418. In some cases, the locus is at least 40 kilobases in length. In some cases, the CRISPR-associated endonuclease is a class 1 or class 2 CRISPR-associated endonuclease. In some cases, the class 1 CRISPR-associated endonuclease is selected from the group consisting of Cas3, Cas5, Cas8a, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Csx11, Csx10, and Csf1.In some cases, the class 2 CRISPR-associated endonuclease is selected from the group consisting of Cas9, Cas12a, Csn2, Cas4, Cas12b, Cas12c, Cas13a, Cas13b, Cas13c, and Cas13d. In some cases, the CRISPR-associated endonuclease comprises an amino acid sequence having at least 80% sequence identity to the wild-type CRISPR-associated endonuclease. In some cases, the CRISPR-associated endonuclease is Cas9 or a mutant thereof. In some cases, the Cas9 is Streptococcus pyogenes Cas9 (spCas9). In some cases, the Cas9 mutant comprises one or more point mutations selected from the group consisting of R780A, K810A, K848A, K855A, H982A, K1003A, R1060A, D1135E, N497A, R661A, Q695A, Q926A, L169A, Y450A, M495A, M694A and M698A compared to wild-type Streptococcus pyogenes Cas9 (spCas9). In some cases, the genomic DNA is not fragmented, digested or sheared prior to a). In some cases, the genomic DNA is not subjected to restriction enzyme digestion prior to a). In some cases, the method further comprises ligating one or more sequencing adaptors to one or both ends of the excised locus. In some cases, the method does not involve DNA amplification. In some cases, the method does not involve polymerase chain reaction (PCR) or isothermal amplification. In some cases, the method does not involve multiple displacement amplification (MDA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification, rolling circle amplification (RCA), ligase chain reaction (LCR), helicase-dependent amplification, or ramification amplification method. In some cases, the genomic DNA is provided or obtained from a biological sample. In some cases, the genomic DNA is provided or obtained from a biological sample.In some cases, the biological sample comprises a bodily fluid (e.g., blood (e.g., whole blood, plasma, serum), urine, saliva, bone marrow, spinal fluid, sputum, peritoneal fluid, lymphatic fluid, pleural fluid, amniotic fluid, semen, vaginal fluid, sweat, stool, glandular secretion, ocular fluid, breast milk) or a solid tissue sample. In some cases, the biological sample is a diagnostic sample.

[0011] In yet another aspect, a method for identifying genetic variations of CYP2D6 in a subject is provided, the method comprising: (a) providing a biological sample comprising genomic DNA from a subject; (b) contacting the genomic DNA with a clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease and two or more gRNAs to excise a locus comprising CYP2D6, CYP2D7, and CYP2D8; (c) performing long-read sequencing of the locus; and (d) identifying one or more genetic variations of CYP2D6 in the subject. In some cases, the method further comprises identifying the subject as having reduced, lost, or increased CYP2D6 function based on the genetic variation. In some cases, the method further comprises recommending a treatment or alternative treatment to the subject based on the identifying step. In some cases, the method further comprises recommending an alternative treatment to the subject if the subject is identified as having reduced, lost, or increased CYP2D6 function. In some cases, the method further comprises recommending a dosage of a therapeutic agent to the subject based on the identifying step. In some cases, if the subject is identified as having a reduced, lost, or increased CYP2D6 function, the method further comprises modifying the dosage of the therapeutic agent. In some cases, the method further comprises isolating high molecular weight DNA comprising the locus of interest prior to c). In some cases, the high molecular weight DNA is at least 40 kilobases in length. In some cases, the two or more gRNAs each comprise a nucleotide sequence substantially complementary to a different nucleotide sequence present in the genomic DNA, the different nucleotide sequences flanking the locus comprising CYP2D6, CYP2D7, and CYP2D8. In some cases, the two or more gRNAs comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-418. In some cases, the locus is at least 40 kilobases in length. In some cases, the long-read sequencing comprises single molecule real-time sequencing or nanopore sequencing.In some cases, CRISPR-associated endonuclease is class 1 or class 2 CRISPR-associated endonuclease.In some cases, class 1 CRISPR-associated endonuclease is selected from the group consisting of Cas3, Cas5, Cas8a, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Csx11, Csx10 and Csf1.In some cases, class 2 CRISPR-associated endonuclease is selected from the group consisting of Cas9, Cas12a, Csn2, Cas4, Cas12b, Cas12c, Cas13a, Cas13b, Cas13c and Cas13d. In some cases, the CRISPR-associated endonuclease comprises an amino acid sequence having at least 80% sequence identity to the wild-type CRISPR-associated endonuclease. In some cases, the CRISPR-associated endonuclease is Cas9 or a mutant thereof. In some cases, the Cas9 is Streptococcus pyogenes Cas9 (spCas9). In some cases, the Cas9 mutant comprises one or more point mutations selected from the group consisting of R780A, K810A, K848A, K855A, H982A, K1003A, R1060A, D1135E, N497A, R661A, Q695A, Q926A, L169A, Y450A, M495A, M694A and M698A compared to the wild-type Streptococcus pyogenes Cas9 (spCas9). In some cases, the genomic DNA is not fragmented, digested, or sheared prior to (a). In some cases, the genomic DNA is not subjected to restriction enzyme digestion prior to (a). In some cases, the method further comprises ligating one or more sequencing adaptors to one or both ends of the excised genomic region of interest. In some cases, the method does not involve DNA amplification. In some cases, the method does not involve either polymerase chain reaction (PCR) or isothermal amplification.In some cases, the method does not involve multiple displacement amplification (MDA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification, rolling circle amplification (RCA), ligase chain reaction (LCR), helicase-dependent amplification, or ramification amplification methods. In some cases, the biological sample is a bodily fluid (e.g., blood (e.g., whole blood, plasma, serum), urine, saliva, bone marrow, spinal fluid, sputum, peritoneal fluid, lymphatic fluid, pleural fluid, amniotic fluid, semen, vaginal fluid, sweat, stool, glandular secretions, ocular fluid, breast milk) or a solid tissue sample.

[0012] In yet another aspect, a composition is provided that includes: (a) a clustered regularly interspaced short palindromic repeats (CRISPR) associated endonuclease; (b) a first guide RNA (gRNA) that includes a nucleotide sequence that is substantially complementary to a nucleotide sequence upstream of a locus that includes CYP2D6, CYP2D7, and CYP2D8 present in genomic DNA; and (c) a second guide RNA (gRNA) that includes a nucleotide sequence that is substantially complementary to a nucleotide sequence downstream of a locus that includes CYP2D6, CYP2D7, and CYP2D8 present in genomic DNA. In some cases, the first guide RNA includes a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, or 13-16. In some cases, the second guide RNA includes a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3-12 or 17-26. In some cases, the CRISPR associated endonuclease is a class 1 or class 2 CRISPR associated endonuclease. In some cases, the class 1 CRISPR-associated endonuclease is selected from the group consisting of Cas3, Cas5, Cas8a, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Csx11, Csx10, and Csf1. In some cases, the class 2 CRISPR-associated endonuclease is selected from the group consisting of Cas9, Cas12a, Csn2, Cas4, Cas12b, Cas12c, Cas13a, Cas13b, Cas13c, and Cas13d. In some cases, the CRISPR-associated endonuclease comprises an amino acid sequence having at least 80% sequence identity to a wild-type CRISPR-associated endonuclease. In some cases, the CRISPR-associated endonuclease is Cas9 or a variant thereof. In some cases, the Cas9 is Streptococcus pyogenes Cas9 (spCas9).In some cases, the Cas9 mutant comprises one or more point mutations selected from the group consisting of R780A, K810A, K848A, K855A, H982A, K1003A, R1060A, D1135E, N497A, R661A, Q695A, Q926A, L169A, Y450A, M495A, M694A and M698A compared to wild-type Streptococcus pyogenes Cas9 (spCas9).

[0013] In yet another aspect, a kit for genotyping CYP2D6 is provided, the kit comprising: (a) a clustered regularly interspaced short palindromic repeats (CRISPR) associated endonuclease; (b) a first guide RNA (gRNA) comprising a nucleotide sequence substantially complementary to a nucleotide sequence upstream of a locus comprising CYP2D6, CYP2D7, and CYP2D8 present in genomic DNA; and (c) a second guide RNA (gRNA) comprising a nucleotide sequence substantially complementary to a nucleotide sequence downstream of a locus comprising CYP2D6, CYP2D7, and CYP2D8 present in genomic DNA. In some cases, the first guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, or 13-16. In some cases, the second guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3-12 or 17-26. In some cases, the CRISPR associated endonuclease is a class 1 or class 2 CRISPR associated endonuclease. In some cases, the class 1 CRISPR-associated endonuclease is selected from the group consisting of Cas3, Cas5, Cas8a, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Csx11, Csx10, and Csf1. In some cases, the class 2 CRISPR-associated endonuclease is selected from the group consisting of Cas9, Cas12a, Csn2, Cas4, Cas12b, Cas12c, Cas13a, Cas13b, Cas13c, and Cas13d. In some cases, the CRISPR-associated endonuclease comprises an amino acid sequence having at least 80% sequence identity to a wild-type CRISPR-associated endonuclease. In some cases, the CRISPR-associated endonuclease is Cas9 or a variant thereof. In some cases, the Cas9 is Streptococcus pyogenes Cas9 (spCas9).In some cases, the Cas9 mutant comprises one or more point mutations selected from the group consisting of R780A, K810A, K848A, K855A, H982A, K1003A, R1060A, D1135E, N497A, R661A, Q695A, Q926A, L169A, Y450A, M495A, M694A and M698A compared to wild-type Streptococcus pyogenes Cas9 (spCas9).

[0014] In yet another aspect, a system for analyzing a complex genomic region of interest is provided, the system comprising: (a) at least one memory location configured to receive a data input comprising data generated by a method that includes: (i) isolating high molecular weight DNA from genomic DNA that includes the complex genomic region of interest; (ii) contacting the genomic DNA with a clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease and two or more gRNAs to excise the complex genomic region of interest, each of the two or more gRNAs comprising a nucleotide sequence substantially complementary to a different nucleotide sequence present in the genomic DNA, the different nucleotide sequences flanking the complex genomic region of interest; and (iii) analyzing the complex genomic region of interest to generate data, the method not involving DNA amplification; and (b) a computer processor operably coupled to the at least one memory location, the computer processor being programmed to generate an output based on the data. In some cases, the output is a report. In some cases, the output is a genotype of the complex genomic region of interest. In some cases, the output is a genetic sequence of the complex genomic region of interest. In some cases, the output is a structural analysis of the complex genomic region of interest. In some cases, the analyzing step includes genotyping the complex genomic region of interest. In some cases, the analyzing step includes performing a structural analysis of the complex genomic region of interest. In some cases, the analyzing step includes sequencing the complex genomic region of interest. In some cases, the sequencing step includes long-read sequencing. In some cases, the long-read sequencing includes single molecule real-time sequencing or nanopore sequencing. In some cases, the isolating step of (i) is performed before the contacting step of (ii). In some cases, the isolating step of (i) is performed after the contacting step of (ii). In some cases, the high molecular weight DNA is at least 10 kilobases in length.In some cases, the complex genomic region of interest comprises a target gene and one or more of its pseudogenes. In some cases, the one or more pseudogenes have at least 75% sequence identity to the target gene. In some cases, the complex genomic region of interest comprises CYP2D6, CYP2D7, and CYP2D8. In some cases, the complex genomic region of interest comprises CYP2C8, CYP2C9, CYP2C18, and CYP2C19. In some cases, the complex genomic region of interest comprises one or more repeat regions, one or more duplications, one or more insertions, one or more inversions, one or more tandem repeats, one or more retrotransposons, or any combination thereof. In some cases, the complex genomic region of interest is a highly polymorphic locus. In some cases, the CRISPR-associated endonuclease is a class 1 or class 2 CRISPR-associated endonuclease. In some cases, the class 1 CRISPR-associated endonuclease is selected from the group consisting of Cas3, Cas5, Cas8a, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Csx11, Csx10, and Csf1. In some cases, the class 2 CRISPR-associated endonuclease is selected from the group consisting of Cas9, Cas12a, Csn2, Cas4, Cas12b, Cas12c, Cas13a, Cas13b, Cas13c, and Cas13d. In some cases, the CRISPR-associated endonuclease comprises an amino acid sequence having at least 80% sequence identity to a wild-type CRISPR-associated endonuclease. In some cases, the CRISPR-associated endonuclease is Cas9 or a variant thereof. In some cases, the Cas9 is Streptococcus pyogenes Cas9 (spCas9).In some cases, the Cas9 mutant comprises one or more point mutations selected from the group consisting of R780A, K810A, K848A, K855A, H982A, K1003A, R1060A, D1135E, N497A, R661A, Q695A, Q926A, L169A, Y450A, M495A, M694A and M698A compared to wild-type Streptococcus pyogenes Cas9 (spCas9). In some cases, the genomic DNA is not fragmented, digested or sheared prior to a). In some cases, the genomic DNA is not subjected to restriction enzyme digestion prior to a). In some cases, the complex genomic region of interest is up to 250 kilobases in length. In some cases, the method further comprises ligating one or more sequencing adaptors to one or both ends of the excised genomic region of interest. In some cases, the method does not involve polymerase chain reaction (PCR) or isothermal amplification. In some cases, the method does not involve multiple displacement amplification (MDA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification, rolling circle amplification (RCA), ligase chain reaction (LCR), helicase-dependent amplification, or ramification amplification method. In some cases, the genomic DNA is provided or obtained in a biological sample. In some cases, the genomic DNA is provided or obtained in a biological sample. In some cases, the biological sample comprises a bodily fluid (e.g., blood (e.g., whole blood, plasma, serum), urine, saliva, bone marrow, spinal fluid, sputum, peritoneal fluid, lymphatic fluid, pleural fluid, amniotic fluid, semen, vaginal fluid, sweat, stool, glandular secretion, ocular fluid, breast milk) or a solid tissue sample. In some cases, the biological sample is a diagnostic sample.

[0015] In yet another aspect, a system for identifying genetic variation of CYP2D6 in a subject is provided, the system comprising: (a) at least one memory location configured to receive data input comprising sequencing data generated by a method comprising: (ii) contacting genomic DNA from a subject with a clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease and two or more gRNAs to excise a locus comprising CYP2D6, CYP2D7, and CYP2D8; and (iii) performing long-read sequencing of the locus to generate sequencing data; and (b) a computer processor operably coupled to the at least one memory location, the computer processor being programmed to generate output based on the sequencing data. In some cases, the output is a report. In some cases, the output identifies a genetic variation of CYP2D6. In some cases, the output identifies a reduction, loss, or increase in the function of CYP2D6. In some cases, the report recommends a treatment for the subject based on the genetic variation. In some cases, the report recommends a dosage of the therapeutic agent to the subject based on the genetic variation. In some cases, the report recommends modifying the dosage of the therapeutic agent based on the genetic variation. In some cases, the therapeutic agent is a therapeutic agent that is activated or metabolized by CYP2D6. In some cases, the method further comprises, prior to (ii), isolating high molecular weight DNA comprising the locus. In some cases, the high molecular weight DNA is at least 40 kilobases in length. In some cases, the two or more gRNAs each comprise a nucleotide sequence substantially complementary to a different nucleotide sequence present in the genomic DNA, the different nucleotide sequences flanking the locus comprising CYP2D6, CYP2D7, and CYP2D8. In some cases, the two or more gRNAs comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-26. In some cases, the locus is at least 40 kilobases in length.In some cases, the long-read sequencing comprises single molecule real-time sequencing or nanopore sequencing. In some cases, the CRISPR-associated endonuclease is a class 1 or class 2 CRISPR-associated endonuclease. In some cases, the class 1 CRISPR-associated endonuclease is selected from the group consisting of Cas3, Cas5, Cas8a, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Csx11, Csx10, and Csf1. In some cases, the class 2 CRISPR-associated endonuclease is selected from the group consisting of Cas9, Cas12a, Csn2, Cas4, Cas12b, Cas12c, Cas13a, Cas13b, Cas13c, and Cas13d. In some cases, the CRISPR-associated endonuclease comprises an amino acid sequence having at least 80% sequence identity to the wild-type CRISPR-associated endonuclease. In some cases, the CRISPR-associated endonuclease is Cas9 or a mutant thereof. In some cases, the Cas9 is Streptococcus pyogenes Cas9 (spCas9). In some cases, the Cas9 mutant comprises one or more point mutations selected from the group consisting of R780A, K810A, K848A, K855A, H982A, K1003A, R1060A, D1135E, N497A, R661A, Q695A, Q926A, L169A, Y450A, M495A, M694A and M698A compared to the wild-type Streptococcus pyogenes Cas9 (spCas9). In some cases, the genomic DNA is not fragmented, digested, or sheared prior to (a). In some cases, the genomic DNA is not subjected to restriction enzyme digestion prior to (a). In some cases, the method further comprises ligating one or more sequencing adaptors to one or both ends of the excised genomic region of interest. In some cases, the method does not involve DNA amplification. In some cases, the method does not involve either polymerase chain reaction (PCR) or isothermal amplification.In some cases, the method does not involve multiple displacement amplification (MDA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification, rolling circle amplification (RCA), ligase chain reaction (LCR), helicase-dependent amplification, or ramification amplification methods. In some cases, the biological sample comprises a bodily fluid (e.g., blood (e.g., whole blood, plasma, serum), urine, saliva, bone marrow, spinal fluid, sputum, ascites, lymphatic fluid, pleural fluid, amniotic fluid, semen, vaginal fluid, sweat, stool, glandular secretion, ocular fluid, breast milk) or a solid tissue sample.

[0016] In another aspect, a system for analyzing a genomic region of interest is provided, the system comprising: (a) at least one memory location configured to receive a data input comprising data generated by a method comprising: (i) contacting a genomic DNA comprising the genomic region of interest with a clustered regularly interspaced short palindromic repeats (CRISPR) associated endonuclease and a pair of outer guide RNAs (gRNAs), thereby generating a first excised fragment comprising the genomic region of interest; (ii) contacting the first excised fragment with a CRISPR associated endonuclease and a pair of inner gRNAs, thereby generating a second excised fragment comprising the genomic region of interest; and (iii) analyzing the genomic region of interest contained within the second excised fragment; and (b) a computer processor operably coupled to the at least one memory location, the computer processor being programmed to generate an output based on the data. In some cases, the output is a report. In some cases, the output is a genotype of the genomic region of interest. In some cases, the output is a gene sequence of the genomic region of interest. In some cases, the output is a structural analysis of the genomic region of interest. In some cases, the analyzing step includes genotyping the genomic region of interest. In some cases, the analyzing step includes performing a structural analysis of the genomic region of interest. In some cases, the analyzing step includes sequencing the genomic region of interest. In some cases, the sequencing step includes long-read sequencing. In some cases, the long-read sequencing includes single-molecule real-time sequencing or nanopore sequencing. In some cases, the CRISPR-associated endonuclease and the pair of outer gRNAs of (i) associate with and block the 5'-end and 3'-end of the first excised fragment. In some cases, the method further includes, prior to (ii), contacting the product of (i) with one or more exonucleases, such that background genomic DNA is digested and the first excised fragment is not digested.In some cases, the one or more exonucleases are selected from the group consisting of exonuclease I, exonuclease II, exonuclease III, exonuclease IV, exonuclease V, exonuclease VI, exonuclease VII, exonuclease VIII, and any combination thereof. In some cases, the pair of outer gRNAs comprises a first outer gRNA and a second outer gRNA. In some cases, the first outer gRNA comprises a nucleotide sequence substantially complementary to a first nucleotide sequence present in the genomic DNA, and the second outer gRNA comprises a nucleotide sequence substantially complementary to a second nucleotide sequence present in the genomic DNA. In some cases, the first nucleotide sequence and the second nucleotide sequence are different. In some cases, the first nucleotide sequence and the second nucleotide sequence flank the genomic region of interest. In some cases, the first nucleotide sequence, the second nucleotide sequence, or both are present in the genomic DNA up to about 100 kilobases in length from the genomic region of interest. In some cases, the pair of inner gRNAs includes a first inner gRNA and a second inner gRNA. In some cases, the first inner gRNA includes a nucleotide sequence that is substantially complementary to a third nucleotide sequence present in the genomic DNA, and the second inner gRNA includes a nucleotide sequence that is substantially complementary to a fourth nucleotide sequence present in the genomic DNA. In some cases, the third nucleotide sequence and the fourth nucleotide sequence are different. In some cases, the third nucleotide sequence and the fourth nucleotide sequence flank the genomic region of interest. In some cases, the third nucleotide sequence and the fourth nucleotide sequence are present on the genomic DNA at a base length closer to the genomic region of interest than the first nucleotide sequence and the second nucleotide sequence. In some cases, the second excised fragment is shorter in base length than the first excised fragment. In some cases, the analyzing step comprises sequencing the genomic region of interest contained within the second excised fragment. In some cases, the genomic DNA is provided in an amount of about 10 μg or more.In some cases, the analyzing step comprises genotyping the genomic region of interest contained within the second excised fragment. In some cases, the analyzing step comprises performing structural analysis on the genomic region of interest contained within the second excised fragment. In some cases, the method further comprises isolating the first excised fragment before (ii). In some cases, the method further comprises isolating the second excised fragment before (iii). In some cases, the method does not involve DNA amplification. In some cases, the method further comprises attaching one or more adaptors to the 5' end, 3' end, or both of the second excised fragment before (iii). In some cases, the CRISPR-associated endonuclease is a class 1 CRISPR-associated endonuclease or a class 2 CRISPR-associated endonuclease. In some cases, the class 1 CRISPR-associated endonuclease is selected from the group consisting of Cas3, Cas5, Cas8a, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Csx11, Csx10, and Csf1. In some cases, the class 2 CRISPR-associated endonuclease is selected from the group consisting of Cas9, Cas12a, Csn2, Cas4, Cas12b, Cas12c, Cas13a, Cas13b, Cas13c, and Cas13d. In some cases, the CRISPR-associated endonuclease comprises an amino acid sequence having at least 80% sequence identity to a wild-type CRISPR-associated endonuclease. In some cases, the CRISPR-associated endonuclease is Cas9 or a variant thereof. In some cases, the Cas9 is Streptococcus pyogenes Cas9 (spCas9).In some cases, the Cas9 mutant comprises one or more point mutations selected from the group consisting of R780A, K810A, K848A, K855A, H982A, K1003A, R1060A, D1135E, N497A, R661A, Q695A, Q926A, L169A, Y450A, M495A, M694A and M698A compared to wild-type Streptococcus pyogenes Cas9 (spCas9). In some cases, the genomic DNA is not fragmented, digested or sheared prior to (i). In some cases, the genomic DNA is not subjected to restriction enzyme digestion prior to (i). In some cases, the genomic region of interest is a complex genomic region. In some cases, the complex genomic region comprises a gene of interest and one or more pseudogenes thereof. In some cases, the one or more pseudogenes comprise a nucleotide sequence having at least 75% sequence identity to the gene of interest. In some cases, the complex genomic region comprises one or more repeat regions, one or more duplications, one or more insertions, one or more inversions, one or more tandem repeats, one or more retrotransposons, or any combination thereof. In some cases, the genomic region of interest is a highly polymorphic locus. In some cases, the length of the first excised fragment is at least about 0.06 kilobases. In some cases, the length of the first excised fragment is up to about 200 kilobases. In some cases, the length of the second excised fragment is at least about 0.02 kilobases. In some cases, the length of the second excised fragment is up to about 199.98 kilobases. In some cases, the method does not involve either polymerase chain reaction (PCR) or isothermal amplification. In some cases, the method does not involve multiple displacement amplification (MDA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification, rolling circle amplification (RCA), ligase chain reaction (LCR), helicase-dependent amplification, or ramification amplification methods. In some cases, the genomic DNA is provided or obtained from a biological sample.In some cases, the biological sample comprises a bodily fluid (e.g., blood (e.g., whole blood, plasma, serum), urine, saliva, bone marrow, spinal fluid, sputum, peritoneal fluid, lymphatic fluid, pleural fluid, amniotic fluid, semen, vaginal fluid, sweat, stool, glandular secretion, ocular fluid, breast milk) or a solid tissue sample. In some cases, the biological sample is a diagnostic sample. In some cases, the genomic region of interest is a locus comprising CYP2D6, CYP2D7, and CYP2D8. In some cases, the analyzing step comprises identifying one or more genetic variants of CYP2D6. In some cases, the output comprises an identification of the subject having a reduction, loss, or increase in CYP2D6 function based on the genetic variant. In some cases, the output comprises a recommendation of a treatment or alternative treatment for the subject based on the identification. In some cases, if the subject is identified as having reduced, lost, or increased CYP2D6 function, the output further comprises a recommendation of an alternative treatment for the subject. In some cases, the output further provides a recommendation of a dosage of a therapeutic agent for the subject based on the identification. In some cases, if the subject is identified as having reduced, lost, or increased CYP2D6 function, the output further comprises a recommendation to modify the dosage of the therapeutic agent. In some cases, the outer gRNA pair, the inner gRNA pair, or both comprise a gRNA selected from any one of SEQ ID NOs: 1-418.

[0017] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0018] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description and accompanying drawings that set forth illustrative embodiments, in which the principles of the disclosure are utilized. [Brief description of the drawings]

[0019] [Figure 1] Figure 1 shows the CYP2D6 locus according to the embodiment presented herein. Panel A shows the orientation of the reference locus containing a single copy of the CYP2D6 gene relative to CYP2D7 and CYP2D8. Representative examples of structural variants illustrating the complexity of CYP2D6 gene copy number variation, including complete CYP2D6 deletion (panel B), duplication (panel C), and the presence of CYPD6 / CYPD7 hybrid alleles at either 5' (panel D) or 3' (panel E). Duplicated genes in such an arrangement often have a CYP2D7-like downstream region that contains a 1.6 kb long spacer sequence. The 5'-3' orientation is shown relative to the reference sequence (NG_008376.3).

[0020] [Diagram 2] FIG. 2 depicts a non-limiting example of a flow chart showing a method for isolating and sequencing the CYP2D6 locus according to embodiments presented herein.

[0021] [Diagram 3] Figure 3 shows a non-limiting example of a comparison of genomic DNA extraction according to embodiments presented herein: Lane A is 50 ng of gDNA (>50 kb) extracted from lymphoblastoid cell line (LCL) cells using a modified high molecular weight protocol, lane B is 50 ng of gDNA (~10-48 kb) extracted using a Maxwell Rapid Sample Concentrator (RSC), lane C is 50 ng of gDNA control (Coriell; ~10 kb-50 kb), lane D is lambda phage DNA (~50 kDa; NEB), and lane E is a HINDIII lambda phage digest.

[0022] [Figure 4]Figure 4A and Figure 4B show non-limiting examples of design and validation of sgRNA targeting CYP2D6 locus according to embodiments presented herein. Figure 4A shows a schematic of CRISPR cut sites required to capture allele CYP2D6 and hybrid alleles. Figure 4B shows CRISPR Cut XL-PCR amplicon of target site. Sample A added Cas9 without sgRNA, sample B added Cas9 with sgRNA_1, and sample C added Cas9 with sgRNA_2.

[0023] [Diagram 5] Figures 5A and 5B show non-limiting examples of the efficiency of sgRNA targeting the CYP2D6 locus on genomic DNA according to embodiments of the present disclosure. Figure 5A shows a gel image of XL-PCR products containing sgRNA binding sites in the upstream and downstream regions of CYP2D6. Lane C is the control. Figure 5B shows the percentage of uncut gDNA normalized to the negative control. *=P value < 0.010.

[0024] [Figure 6] FIG. 6 shows a non-limiting example of an NGS alignment of an XL-PCR and NGS based analysis method according to an embodiment of the present disclosure.

[0025] [Figure 7-1] 7A-7C show non-limiting examples of problems with alternative CRISPR / Cas9 design approaches to the CYP2D6 locus according to embodiments of the present disclosure. Cut sites are indicated with scissors. X represents an allele where the design shown in the A allele results in an undesired cut in the B-E allele configuration. [Figure 7-2] Same as above. [Figure 7-3] Same as above.

[0026] [Figure 8]Figure 8 shows a non-limiting example of a global targeting design for the CYP2D6 locus. Cut sites are indicated with scissors. Checkmarks represent alleles where the design shown for the A allele results in on-target cuts only in the B-E allele configuration.

[0027] [Figure 9-1] Figures 9A-9C show non-limiting examples of design and validation of sgRNAs targeting the CYP2D6 locus. Figure 9A shows a schematic of the required cut sites on the target to capture the allele CYP2D6 and hybrid alleles. Figures 9B and 9C show the CRISPR Cut XL-PCR amplicons of the target sites. Sample A added Cas9 without sgRNA, sample B added Cas9 with sgRNA_1, and sample C added Cas9 with sgRNA_2. [Figure 9-2] Same as above.

[0028] [Figure 10] 10 shows a non-limiting example of isolated high molecular weight DNA according to embodiments of the present disclosure. 2% DNA agarose gel of 100 ng of high molecular weight genomic DNA extracted from LCL cell pellets compared to lambda control and pre-extracted DNA from the Coriell Institute.

[0029] [Figure 11-1] 11A and 11B show non-limiting examples of sequencing run coverage according to embodiments disclosed herein. [Figure 11-2] Same as above.

[0030] [Figure 12-1] 12A and 12B show non-limiting examples of sequence alignment sizes according to embodiments disclosed herein. [Figure 12-2] Same as above.

[0031] [Figure 13]Figure 13 shows a non-limiting example of an alignment plot according to embodiments disclosed herein. 121x coverage of the targeted capture region was achieved. Boxes outline CYP2D6 and CYP2D7.

[0032] [Figure 14] Figure 14 shows a non-limiting example of a Sashimi plot showing sgRNA specificity according to embodiments disclosed herein. The plot shows aligned regions for two sequencing runs. The top alignment shows sequence data from a run using an sgRNA designed to capture the region of interest (ROI) (chr22:42,122,115-41,161,320). The bottom alignment shows enrichment performed on the same DNA sample using an sgRNA targeting the opposite strand.

[0033] [Figure 15] Figure 15 shows a non-limiting example of a Sashimi plot showing sgRNA specificity for multiple complex structural arrangements according to embodiments disclosed herein. The plot shows aligned regions for four sequencing runs. Sequence data from the run, using sgRNAs designed to capture a region of interest (ROI) (chr22:42,122,115-41,161,320), includes four different structural events: (1) a deletion of CYP2D6 in one allele; (2) a tandem hybrid allele with CYP2D6 in one allele; (3) a duplication event in one allele; and (4) a deletion of CYP2D6 in one allele and a duplication of CYP2D6 in a second allele.

[0034] [Figure 16] FIG. 16 illustrates a non-limiting example of a computer system according to embodiments presented herein.

[0035] [Figure 17]FIG. 17 shows a non-limiting example of a nested enrichment approach for analyzing complex genomic regions of interest according to embodiments presented herein.

[0036] [Figure 18] Figure 18 shows non-limiting representative fold change data for ROIs when using nested enrichment methodology to analyze complex genomic regions of interest.As shown in this figure, performing nested enrichment using different pairs of outer gRNAs before DNA digestion and then CRISPR reaction with a second inner gRNA results in significant enrichment of ROIs for downstream applications compared to samples that only receive inner gRNA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Detailed Description Disclosed herein is a method for analyzing a genomic region of interest (ROI) (e.g., from genomic DNA). The region of interest can be, for example, a complex (e.g., highly complex) genomic region. A complex genomic region can include, for example, a highly polymorphic region, a region that includes a target gene and one or more pseudogenes with high sequence homology to the target gene, a region that includes one or more repetitive elements, one or more inversions, one or more insertions, one or more duplications, one or more tandem repeats, one or more retrotransposons, etc. The methods presented herein generally involve excising a region of interest from genomic DNA using a clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease and two or more guide RNAs (gRNAs).

[0038] In one aspect, the present disclosure provides a nested enrichment approach for enriching and analyzing complex genomic regions of interest. Nested enrichment approaches generally involve using a CRISPR-associated endonuclease in combination with a pair of outer gRNAs (e.g., a first outer gRNA and a second outer gRNA) and / or a pair of inner gRNAs (e.g., a first inner gRNA and a second inner gRNA). The method involves using a CRISPR-associated endonuclease and the pair of outer gRNAs to excise a fragment from genomic DNA containing the genomic region of interest to generate a first excised fragment that includes the genomic region of interest. The method further includes using a CRISPR-associated endonuclease and the pair of inner gRNAs to excise a smaller fragment from the first excised fragment to generate a second excised fragment that includes the genomic region of interest. In some cases, the method further involves using one or more exonucleases to digest background DNA.

[0039] The method presented herein further comprises analyzing (e.g., by sequencing, for example, by long-read sequencing, by genotyping, by performing structural analysis) the genomic region of interest (e.g., located on the second fragment). A method of analyzing the CYP2D6 locus (e.g., including the target gene CYP2D6 and pseudogenes CYP2D7 and CYP2D8) is further presented herein. Advantageously, in some embodiments, the method does not involve the use of DNA amplification (e.g., no amplification). The method can improve the accuracy of sequencing (e.g., reduce the error rate of sequencing) of complex (e.g., highly complex) genomic regions (e.g., compared to traditional methods) and / or can reduce the time for sequencing of complex (e.g., highly complex) genomic regions (e.g., compared to traditional methods) and / or can reduce the cost of sequencing of complex genomic (e.g., highly complex) regions (e.g., compared to traditional methods). Furthermore, the method presented herein may allow for the use of higher starting materials (e.g., higher amounts of genomic DNA) than standard CRISPR-based methods.Further presented herein are systems for carrying out the methods presented herein, as well as compositions and kits that include CRISPR-associated endonuclease and two or more gRNAs that excise genomic regions of interest (e.g., CYP2D6 locus (e.g., for excising CYP2D6 locus from genomic DNA)).

[0040] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is also noted that the claims may be drafted to exclude any necessary elements. Thus, this statement serves as a premise for the use of exclusive terminology, such as "solely," "only," or the use of a "negative" limitation in connection with the recitation of claim elements.

[0041] Certain ranges or numbers are presented herein with numerical values ​​preceded by the term "about". The term "about" is used herein to mean plus or minus 1%, 2%, 3%, 4%, or 5% of the number to which the term refers. As used herein, the terms "subject" and "individual" are used interchangeably and may be any animal, including a mammal (e.g., a human or non-human animal).

[0042] As used herein, the term "CYP2D6" may refer to the CYP2D6 gene or any of its structural variants or single gene copy variants. Structural variants of CYP2D6 may include gene fusions, hybrids with nearby highly homologous pseudogenes (e.g., CYP2D7 and CYP2D8), copy number variations (CNVs), gene duplications and multiplications, tandem repeats, and rearrangements. One example of CYP2D6 structural variants is the presence of CYP2D7-derived sequences in exon 9 of CYP2D6 (referred to as "exon 9 conversion"). Single gene copy variants may include single nucleotide polymorphisms (SNPs) or nucleotide insertions or deletions (indels). The allele of CYP2D6 can be a structural variant or single gene copy variant, including but not limited to any one of *1, *1xN, *2, *2xN, *2A, *2AxN, *35, *35xN, *9, *9xN, *10, *10xN, *17, *17xN, *29, *29xN, *36-*10, *36-*10xN, *36xN-*10, *36xN-*10xN, *41, *41xN, *3, *3xN, *4, *4xN, *4N, *5, *6, *6xN, *36 and *36xN.In some cases, each allele of CYP2D6 is a different structural variant or single gene copy variant.In some cases, each allele of CYP2D6 is identical.

[0043] The term "CYP2D6 locus" as used herein refers to a genomic region that includes the CYP2D6 gene and highly homologous pseudogenes CYP2D7 and CYP2D8. In humans, the CYP2D6 locus is found on chromosome 22. In some embodiments, the methods provided herein involve analyzing (e.g., sequencing, genotyping, performing structure analysis) a portion or the entire CYP2D6 locus (e.g., including the CYP2D6 gene and highly homologous pseudogenes CYP2D7 and CYP2D8). In some embodiments, the methods provided herein involve excising (e.g., by using a CRISPR-associated endonuclease and two or more gRNAs that target genomic sequences adjacent to the CYP2D6 locus) a portion or the entire CYP2D6 locus (e.g., including the CYP2D6 gene and highly homologous pseudogenes CYP2D7 and CYP2D8) from genomic DNA.

[0044] As used herein, the term "CRISPR / Cas nuclease system" refers to a complex that includes a guide RNA (gRNA) and a CRISPR-associated endonuclease (Cas protein). The term "CRISPR" can refer to clustered regularly interspaced short palindromic repeats and its associated systems. The CRISPR / Cas nuclease system can be a class 1 or class 2 CRISPR / Cas nuclease system. The CRISPR / Cas nuclease system can be a type I, type II, type III, type IV, type V, or type VI CRISPR / Cas nuclease system. The gRNA can interact with the Cas protein to direct the nuclease activity of the Cas protein to the target sequence. The target sequence can include a "protospacer" and a "protospacer adjacent motif" (PAM), both of which domains can be required for Cas-mediated activity (e.g., cleavage). The gRNA pairs with (or hybridizes to) a binding site on the opposite strand of the protospacer to direct Cas to the target sequence. A PAM site may refer to a short sequence recognized by the Cas protein and, in some cases, may be necessary for Cas protein activity.

[0045] As used herein, the term "Cas" or "Cas protein" refers to a protein of the CRISPR / Cas system or a protein derived from the CRISPR / Cas system that has endonuclease activity. In some cases, a CRISPR-associated endonuclease is a Cas protein as used herein. A Cas protein can be a naturally occurring Cas protein, a non-naturally occurring Cas protein, or a fragment thereof. In some cases, a Cas protein is a mutant of a naturally occurring Cas protein (e.g., has one or more amino acid substitutions, insertions, deletions, etc. compared to a naturally occurring Cas protein). In some cases, a Cas protein is a class I Cas protein, non-limiting examples of which include Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, and Csf1. In some cases, the Cas protein is a class II Cas protein, non-limiting examples of which include Cas9, Csn2, Cas4, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas13a (C2c2), Cas13b, Cas13c, and Cas13d. In some cases, the Cas protein is Cas9. In some cases, the Cas protein is Cas12a.

[0046] The terms "guide RNA" or "gRNA" are used interchangeably herein and generally refer to an RNA molecule (or collectively a group of RNA molecules) that can bind to a Cas protein and assist in targeting the Cas protein to a specific location within a target polynucleotide (e.g., DNA). A guide RNA may include a CRISPR RNA (crRNA) segment and, optionally, a trans-activating crRNA (tracrRNA) segment. The term "crRNA" as used herein may refer to an RNA molecule or a portion thereof that includes a polynucleotide-targeting guide sequence, a stem sequence, and, optionally, a 5'-overhang sequence. The crRNA can bind to a binding site. The term "tracrRNA" as used herein may refer to an RNA molecule or a portion thereof that includes a protein-binding segment (e.g., the protein-binding segment is capable of interacting with a CRISPR-associated protein, such as Cas9). The term "guide RNA" may refer to a single guide RNA (sgRNA), where the crRNA segment and the optional tracrRNA segment are located within the same RNA molecule. The term "guide RNA" may also collectively refer to a group of two or more RNA molecules, where the crRNA and tracrRNA are located in separate RNA molecules.

[0047] The term "long-read sequencing" (also referred to as "third generation sequencing"), as used herein, generally refers to any sequencing method capable of generating substantially longer sequencing reads (>10,000bp) than second generation sequencing. In some embodiments, the methods presented herein involve the use of long-read sequencing (e.g., for genotyping complex genomic regions of interest). Non-limiting examples of long-read sequencing systems include those developed by Pacific Biosciences, Oxford Nanopore Technology, Quantapore, Stratos, and Helicos. In some cases, the long-read sequencing method is single molecule real-time sequencing (SMRT) (e.g., those developed by Pacific Biosciences). In some cases, the long-read sequencing method is nanopore sequencing (e.g., MinION, GridION, and PromethION developed by Oxford Nanopore Technology). In some cases, long-read sequencing encompasses any long-read sequencing method or system currently under development or that will be developed in the future (e.g., a third generation sequencing method or system).

[0048] The term "nucleic acid amplification" as used herein generally refers to any method of generating multiple copies of a target nucleic acid (e.g., DNA) from a single nucleic acid molecule. The target nucleic acid may be DNA (e.g., DNA amplification) or RNA (e.g., RNA amplification). Nucleic acid amplification includes polymerase chain reaction (PCR) and any and all variations or modifications thereof, as well as alternative types of nucleic acid amplification methods, such as, but not limited to, loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), multiple displacement amplification (MDA), rolling circle amplification (RCA), ligase chain reaction (LCR), helicase-dependent amplification, and branched amplification (RAM). In various aspects of the present disclosure, the methods presented herein do not involve the use of nucleic acid (e.g., DNA) amplification (e.g., no amplification).

[0049] Methods of the Disclosure

[0050] The disclosure herein generally provides a nested enrichment approach for enriching and analyzing (e.g., sequencing, genotyping, structural analysis) a genomic region of interest (e.g., a complex genomic region of interest). In various aspects, the method includes contacting genomic DNA comprising the genomic region of interest (e.g., a complex genomic region of interest) with a clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease and a pair of outer guide RNAs (gRNAs), thereby generating a first excised fragment comprising the genomic region of interest. In various aspects, the method further includes contacting the first excised fragment with a CRISPR-associated endonuclease and a pair of inner gRNAs, thereby generating a second (e.g., smaller) excised fragment comprising the genomic region of interest. In various aspects, the method further includes analyzing (e.g., sequencing, genotyping, structural analysis) the genomic region of interest (e.g., present within the second excised fragment).

[0051] In various embodiments, the method involves contacting genomic DNA containing a genomic region of interest (e.g., a complex genomic region of interest) with a clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease and a pair of outer guide RNAs (gRNAs). The pair of outer gRNAs can include a first outer gRNA and a second outer gRNA.

[0052] The first outer gRNA and the second outer gRNA comprise a nucleotide sequence that is substantially complementary to a nucleotide sequence present in the genomic DNA. Generally, the first outer gRNA and the second outer gRNA are substantially complementary to different nucleotide sequences present in the genomic DNA. The first outer gRNA sequence and the second outer gRNA sequence are selected such that they are substantially complementary to nucleotide sequences adjacent to the genomic region of interest. For example, the first outer gRNA can be substantially complementary to a nucleotide sequence upstream of the genomic region of interest, and the second outer gRNA can be substantially complementary to a nucleotide sequence downstream of the genomic region of interest, or vice versa. Generally, contacting the genomic DNA with the CRISPR-associated endonuclease and the pair of outer gRNAs results in excision of a fragment of the genomic DNA (e.g., the first excised fragment) that contains the genomic region of interest (e.g., the complex genomic region of interest).

[0053] The first outer gRNA and the second outer gRNA can be substantially complementary to a nucleotide sequence (e.g., present in genomic DNA) that is up to about 30 kilobases long (e.g., upstream and / or downstream) from the genomic region of interest. For example, the first outer gRNA and the second outer gRNA can be substantially complementary to a nucleotide sequence (e.g., present in genomic DNA) that is at least about 5 kilobases, at least about 10 kilobases, at least about 15 kilobases, at least about 20 kilobases, at least about 25 kilobases, or more long (e.g., upstream and / or downstream) from the genomic region of interest.

[0054] Without wishing to be bound by theory, it is believed that after the first fragment is excised, the CRISPR-associated endonuclease and the pair of outer gRNAs remain associated with the 5'-end and 3'-end of the first excised fragment, blocking it. Advantageously, this feature can be used to remove background genomic DNA. In a preferred embodiment, the first excised fragment (and remaining genomic DNA) is contacted with one or more exonucleases. The one or more exonucleases can digest background DNA, while leaving the blocked fragment intact. The one or more exonucleases can be selected from the group consisting of exonuclease I, exonuclease II, exonuclease III, exonuclease IV, exonuclease V, exonuclease VI, exonuclease VII, exonuclease VIII, and any combination thereof.

[0055] In various embodiments, the method further comprises contacting the first excised fragment (e.g., containing the genomic region of interest) with a CRISPR-associated endonuclease and a pair of inner gRNAs.In some cases, the contacting step is performed after contacting the first excised fragment (and remaining genomic DNA) with one or more exonucleases as described herein.The pair of inner gRNAs can include a first inner gRNA and a second inner gRNA.

[0056] The first inner gRNA and the second inner gRNA comprise a nucleotide sequence that is substantially complementary to a nucleotide sequence present in the first excised fragment (e.g., generated by contacting genomic DNA with a CRISPR-associated endonuclease and a pair of outer gRNAs, as described herein). Generally, the first inner gRNA and the second inner gRNA are substantially complementary to different nucleotide sequences present in the first excised fragment (e.g., generated by contacting genomic DNA with a CRISPR-associated endonuclease and a pair of outer gRNAs, as described herein). The first inner gRNA sequence and the second inner gRNA sequence are selected such that they are substantially complementary to nucleotide sequences that flank the genomic region of interest. For example, the first inner gRNA can be substantially complementary to a nucleotide sequence upstream of the genomic region of interest and the second inner gRNA can be substantially complementary to a nucleotide sequence downstream of the genomic region of interest, or vice versa. Generally, a first excised fragment containing the genomic region of interest (e.g., generated by contacting genomic DNA with a CRISPR-associated endonuclease and a pair of outer gRNAs as described herein) is contacted with a CRISPR-associated endonuclease and a pair of inner gRNAs to excise a second fragment (e.g., a second excised fragment) containing the genomic region of interest.

[0057] The first inner gRNA and the second inner gRNA can be substantially complementary to a nucleotide sequence (e.g., present in the first excised fragment) that is about 0.06 kilobases to about 200 kilobases in length (e.g., upstream and / or downstream) from the genomic region of interest. In general, the pair of inner gRNAs is nested and thus substantially complementary to a nucleotide sequence that is closer in length to the genomic region of interest than the pair of outer gRNAs. In other words, the pair of inner gRNAs is used in conjunction with the CRISPR-associated endonuclease described herein to excise a smaller fragment (e.g., the second excised fragment) from the first excised fragment. The second excised fragment preferably includes the genomic region of interest (e.g., its entirety).

[0058] In various aspects, the method involves isolating genomic DNA comprising the genomic region of interest. In some embodiments, the method involves isolating high molecular weight genomic DNA. In some embodiments, the method involves enriching high molecular weight genomic DNA. In some embodiments, the high molecular weight genomic DNA is at least about 10 kilobases in length. For example, the high molecular weight genomic DNA is at least about 10 kilobases in length, at least about 15 kilobases in length, at least about 20 kilobases in length, at least about 30 kilobases in length, at least about 35 kilobases in length, at least about 40 kilobases in length, at least about 45 kilobases in length, at least about 50 kilobases in length, at least about 55 kilobases in length, at least about 60 kilobases in length, at least about 65 kilobases in length, at least about 70 kilobases in length, at least about 75 kilobases in length, at least about 80 kilobases in length, at least about 85 kilobases in length, at least about 90 kilobases in length, at least about 95 kilobases in length, or longer. In some embodiments, isolating high molecular weight genomic DNA ensures that the whole intact genomic region of interest is contained in the sample.In some embodiments, isolating and / or enriching high molecular weight genomic DNA is carried out before the first CRISPR reaction (e.g., before contacting genomic DNA with CRISPR-associated endonuclease and pair of outer gRNAs).In some embodiments, isolating and / or enriching high molecular weight genomic DNA is carried out after carrying out the first CRISPR reaction (e.g., after contacting genomic DNA with CRISPR-associated endonuclease and pair of outer gRNAs).

[0059] In various embodiments, the method involves any method for isolating high molecular weight genomic DNA.Non-limiting examples of the method for isolating high molecular weight genomic DNA include NucleoBond (registered trademark) Genomic DNA and RNA purification system (manufactured by Takara Bio) and Nanobind CBB Big DNA kit (manufactured by Circulomics).

[0060] In some embodiments, the step of isolating the genomic DNA comprising the genomic region of interest can be carried out before the step of contacting the genomic DNA with CRISPR-associated endonuclease and guide RNA.In other embodiments, the step of isolating the genomic DNA comprising the genomic region of interest can be carried out after the step of contacting the genomic DNA with CRISPR-associated endonuclease and guide RNA (e.g., after the genomic region of interest is excised from the genomic DNA).

[0061] In various embodiments, the starting amount of genomic DNA used in the method is higher than that generally used in CRISPR-based techniques.In some cases, the starting amount of genomic DNA used in any method presented herein is at least about 1 μg (e.g., at least about 5 μg, at least about 10 μg, at least about 20 μg, at least about 50 μg, at least about 100 μg, at least about 500 μg or more).

[0062] In various embodiments, the genomic region of interest is a complex genomic region or a highly complex genomic region. In some cases, the genomic region of interest is a highly polymorphic genomic region. In some cases, the genomic region of interest contains a large number of repetitive elements or regions. In some cases, the genomic region of interest contains one or more target genes and one or more additional genes that have high sequence identity to the target gene (e.g., have at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or higher sequence identity to the target gene). In some cases, the genomic region of interest contains one or more target genes and one or more pseudogenes with high sequence identity to the target genes (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or higher sequence identity to the target genes). In some cases, the genomic region of interest includes one or more repeat regions, one or more duplications, one or more insertions, one or more inversions, one or more tandem repeats, one or more retrotransposons, or any combination thereof. In some cases, the genomic region of interest is a genomic region that is generally difficult or difficult to accurately analyze by traditional methods (e.g., by short-read sequencing).

[0063] In some cases, the complex genomic region of interest is at least about 10 kilobases in length. For example, the genomic region of interest may be at least about 10 kilobases in length, at least about 15 kilobases in length, at least about 20 kilobases in length, at least about 25 kilobases in length, at least about 30 kilobases in length, at least about 35 kilobases in length, at least about 40 kilobases in length, at least about 45 kilobases in length, at least about 50 kilobases in length, at least about 55 kilobases in length, at least about 60 kilobases in length, at least about 65 kilobases in length, at least about 70 kilobases in length, at least about 75 kilobases in length, at least about 80 kilobases in length, at least about 85 kilobases in length, at least about 90 kilobases in length, at least about 100 kilobases in length, at least about 150 kilobases in length, at least about 160 kilobases in length, at least about 170 kilobases in length, at least about 180 kilobases in length, at least about 190 kilobases in length, at least about 200 kilobases in length, at least about 210 kilobases in length, at least about 220 kilobases in length, at least about 230 kilobases in length, at least about 240 kilobases in length, at least about 250 kilobases in length, at least about 260 kilobases in length, at least about 270 kilobases in length, at least about 280 kilobases in length, at least about 290 kilobases in length, at least about 300 kilobases in length, at least about 310 kilobases in length, at least about 320 kilobases in length, at least about 330 kilobases in length, at least about 340 kilobases The length may be about 95 kilobases, at least about 100 kilobases, at least about 110 kilobases, at least about 120 kilobases, at least about 130 kilobases, at least about 140 kilobases, at least about 150 kilobases, at least about 160 kilobases, at least about 170 kilobases, at least about 180 kilobases, at least about 190 kilobases, at least about 200 kilobases, at least about 210 kilobases, at least about 220 kilobases, at least about 230 kilobases, at least about 240 kilobases, or at least about 250 kilobases. In some embodiments, the genomic region of interest is longer than about 10 kilobases. In some embodiments, the genomic region of interest is shorter than about 250 kilobases.

[0064] CRISPR-associated endonuclease can be any CRISPR-associated endonuclease described herein.In some cases, CRISPR-associated endonuclease is class I or class II CRISPR-associated endonuclease.Non-limiting examples of Cas I CRISPR-associated endonuclease include Cas3, Cas5, Cas8a, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Csx11, Csx10 and Csf1.Non-limiting examples of class II CRISPR-associated endonuclease include Cas9, Cas12a, Csn2, Cas4, Cas12b, Cas12c, Cas13a, Cas13b, Cas13c and Cas13d. In some cases, the CRISPR-associated endonuclease is a Cas protein or polypeptide. In some embodiments, the CRISPR-associated endonuclease is a Cas12a protein or polypeptide.

[0065] In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein or polypeptide. In some cases, the Cas9 protein or polypeptide is derived from the bacterial species Streptococcus pyogenes. In some cases, the Cas9 protein or polypeptide has an amino acid sequence identical to the wild-type Cas9 amino acid sequence. In other cases, the Cas9 protein or polypeptide has an amino acid sequence that is modified compared to the wild-type Cas9 amino acid sequence. In some cases, the Cas9 protein or polypeptide has one or more mutations (e.g., compared to the wild-type Cas9 protein or polypeptide). In some cases, the one or more mutations are substitutions, deletions, or insertions. The Cas9 protein or polypeptide may have an amino acid sequence that has at least about 50% sequence identity compared to the wild-type Cas9 protein or polypeptide. For example, a Cas9 protein or polypeptide may have at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity relative to a wild-type Cas9 protein or polypeptide. In some cases, the Cas9 variant may comprise one or more point mutations relative to a wild-type S. pyogenes Cas9. For example, the Cas9 mutant may comprise a point mutation selected from the group consisting of R780A, K810A, K848A, K855A, H982A, K1003A, R1060A, D1135E, N497A, R661A, Q695A, Q926A, L169A, Y450A, M495A, M694A and M698A compared to wild-type S. pyogenes Cas9.

[0066] In various aspects, the method involves the use of gRNAs (e.g., a pair of outer gRNAs and / or a pair of inner gRNAs). The gRNAs can be CRISPR RNAs (crRNAs) or single guide RNAs (sgRNAs). In some embodiments, the gRNAs include a nucleotide sequence that is complementary or substantially complementary to a target nucleotide sequence, such that the gRNAs can bind to the target nucleotide sequence and direct the CRISPR complex to a desired cut site. In some embodiments, each of the gRNAs (e.g., inner gRNA, outer gRNA) binds to a different target nucleotide sequence. In some embodiments, at least one of the gRNAs is complementary or substantially complementary to a region upstream of the genomic region of interest, and at least one of the gRNAs is complementary or substantially complementary to a region downstream of the genomic region of interest. For example, at least one of the outer gRNAs is complementary or substantially complementary to a region upstream of the genomic region of interest, and at least one of the outer gRNAs is complementary or substantially complementary to a region downstream of the genomic region of interest. Similarly, at least one of the inner gRNAs is complementary or substantially complementary to the upstream region of the genomic region of interest, and at least one of the inner gRNAs is complementary or substantially complementary to the downstream region of the genomic region of interest.In some embodiments, gRNA pair (e.g., inner gRNA pair, outer gRNA pair) binds to the target sequence adjacent to the genomic region of interest.Generally, gRNA is designed to target the genomic sequence outside the genomic region of interest, and thus, by contact (e.g., with CRISPR-associated endonuclease and outer gRNA pair or inner gRNA pair), the whole genomic region of interest is excised.

[0067] In various aspects, the method further comprises analyzing the genomic region of interest. In some cases, the analyzing comprises genotyping the genomic region of interest. Genotyping can comprise the process of identifying the difference in the genetic composition of the genomic region of interest by using one or more assays to investigate the sequence of the genomic region of interest, and in some cases by comparing the sequence with another sequence (e.g., a reference sequence). Genotyping can be carried out by any known method, including but not limited to DNA sequencing, restriction fragment length polymorphism identification (RFLPI), random amplified polymorphism detection (RAPD), amplified fragment length polymorphism detection (AFLPD), polymerase chain reaction (PCR), allele-specific oligonucleotide (ASO) probe, and hybridization to DNA microarrays or beads. In some cases, the analyzing comprises carrying out structural analysis on the genomic region of interest.

[0068] In some cases, the analyzing step includes sequencing the genomic region of interest. In some cases, the sequencing is a long-read sequencing method (e.g., a third generation sequencing method). The long-read sequencing method can be any sequencing method capable of generating substantially longer sequencing reads than short-read sequencing methods (e.g., a second generation sequencing method). In some cases, the long-read sequencing method is a sequencing method capable of generating at least 10,000 kilobases of sequencing reads. In some cases, the long-read sequencing method is single molecule real-time sequencing (e.g., SMRT sequencing, Pacific Biosciences). In some cases, the long-read sequencing method is nanopore sequencing (e.g., MinION, GridION, and PromethION developed by Oxford Nanopore Technologies). In some aspects, prior to sequencing, the method further involves ligating adaptors (e.g., sequencing adaptors) to the ends of the genomic region of interest. The methods may in some cases involve any other processing methods suitable for sequencing applications, including tailing steps, dephosphorylation steps, and the like.

[0069] In various aspects, the methods presented herein are amplification-free (e.g., do not involve a nucleic acid amplification (e.g., DNA amplification) step). In some cases, the methods presented herein do not involve polymerase chain reaction (PCR). In some cases, the methods presented herein do not involve isothermal amplification. In some cases, the methods presented herein do not involve any one of loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), multiple displacement amplification (MDA), rolling circle amplification (RCA), ligase chain reaction (LCR), helicase-dependent amplification, and branched amplification (RAM). Nucleic acid amplification techniques often introduce errors into the sequencing template. Advantageously, the methods presented herein avoid the use of nucleic acid amplification methods that may introduce errors into the sequencing template.

[0070] In various embodiments, the method does not involve fragmentation, shearing, or digestion of genomic DNA.In some cases, the method does not involve digestion of genomic DNA, for example, with restriction enzyme.In other words, the method is carried out directly on genomic DNA that has not been sheared, digested, or fragmented.In other cases, the method involves digestion with exonuclease (for example, after contacting genomic DNA with CRISPR-associated endonuclease and a pair of outer gRNAs to remove background genomic DNA, as described herein).

[0071] In various embodiments, the complex genomic region comprises a target gene and one or more pseudogenes with high sequence identity to the target gene.In some cases, one or more pseudogenes can have at least about 75% sequence identity to the target gene (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%).In a particular embodiment, the locus comprises the target gene CYP2D6 and the pseudogenes CYP2D7 and CYP2D8.

[0072] In various embodiments, the complex genomic region comprises a target gene and one or more additional genes with high sequence identity to the target gene.In some cases, the one or more additional genes can have at least about 75% sequence identity to the target gene (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%).In a particular embodiment, the locus comprises genes CYP2C8, CYP2C9, CYP2C18 and CYP2C19.In some cases, the locus is generally difficult or difficult to accurately sequence by traditional methods (e.g., by short-read sequencing).

[0073] In various embodiments, the complex genomic region is a highly polymorphic locus. In various embodiments, the complex genomic region comprises one or more repeat regions, one or more duplications, one or more insertions, one or more inversions, one or more tandem repeats, one or more retrotransposons, or any combination thereof.

[0074] In some cases, the complex genomic region of interest is at least about 10 kilobases in length. For example, the genomic region of interest may be at least about 10 kilobases in length, at least about 15 kilobases in length, at least about 20 kilobases in length, at least about 25 kilobases in length, at least about 30 kilobases in length, at least about 35 kilobases in length, at least about 40 kilobases in length, at least about 45 kilobases in length, at least about 50 kilobases in length, at least about 55 kilobases in length, at least about 60 kilobases in length, at least about 65 kilobases in length, at least about 70 kilobases in length, at least about 75 kilobases in length, at least about 80 kilobases in length, at least about 85 kilobases in length, at least about 90 kilobases in length, at least about 100 kilobases in length, at least about 150 kilobases in length, at least about 160 kilobases in length, at least about 170 kilobases in length, at least about 180 kilobases in length, at least about 190 kilobases in length, at least about 200 kilobases in length, at least about 210 kilobases in length, at least about 220 kilobases in length, at least about 230 kilobases in length, at least about 240 kilobases in length, at least about 250 kilobases in length, at least about 260 kilobases in length, at least about 270 kilobases in length, at least about 280 kilobases in length, at least about 290 kilobases in length, at least about 300 kilobases in length, at least about 310 kilobases in length, at least about 320 kilobases in length, at least about 330 kilobases in length, at least about 340 kilobases The length may be about 95 kilobases, at least about 100 kilobases, at least about 110 kilobases, at least about 120 kilobases, at least about 130 kilobases, at least about 140 kilobases, at least about 150 kilobases, at least about 160 kilobases, at least about 170 kilobases, at least about 180 kilobases, at least about 190 kilobases, at least about 200 kilobases, at least about 210 kilobases, at least about 220 kilobases, at least about 230 kilobases, at least about 240 kilobases, or at least about 250 kilobases. In some embodiments, the genomic region of interest is longer than about 10 kilobases. In some embodiments, the genomic region of interest is shorter than about 250 kilobases.

[0075] In some cases, at least one of the gRNAs (e.g., at least one of the first outer gRNA, the second outer gRNA, the first inner gRNA, and the second inner gRNA) comprises a nucleotide sequence according to any of the nucleotide sequences presented in Table 1 below (e.g., SEQ ID NOs: 1-418). In some cases, at least one of the gRNAs (e.g., at least one of the first outer gRNA, the second outer gRNA, the first inner gRNA, and the second inner gRNA) comprises a nucleotide sequence having at least about 90% (e.g., at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) sequence identity to any of the nucleotide sequences presented in Table 1 below (e.g., SEQ ID NOs: 1-418). In some embodiments, for a pair of gRNAs, the first gRNA is selected to be complementary or substantially complementary to a nucleotide sequence upstream of CYP2D6 present on genomic DNA, and the second gRNA is selected to be complementary or substantially complementary to a nucleotide sequence downstream of CYP2D8 present on genomic DNA. Table 1 provides a non-limiting list of gRNAs that can be used in the present disclosure (e.g., to excise a fragment of genomic DNA containing the entire CYP2D6 locus), along with their location relative to the CYP2D6 locus (e.g., upstream of CYP2D6 or downstream of CYP2D8). In some cases, the first gRNA comprises a nucleotide sequence of any one of SEQ ID NOs:1, 2, 13-16, 27-67, 78-81, and 215-343, or a nucleotide sequence having at least 90% sequence identity (e.g., at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) to any one of SEQ ID NOs:1, 2, 13-16, 27-67, 78-81, and 215-343.In some cases, the second gRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 3-12, 17-26, 68-77, 82-214, 344-418, or a nucleotide sequence having at least 90% sequence identity (e.g., at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) to any one of SEQ ID NOs: 3-12, 17-26, 68-77, 82-214, and 344-418. In some cases, at least one of the gRNAs is a crRNA. In some cases, at least one of the gRNAs is an sgRNA. Table 1. Guide RNA sequences [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11]

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

[0076] In various embodiments, the method further comprises identifying one or more genetic variations of CYP2D6. In some cases, the genetic variation is a pharmacogenetically relevant variation in CYP2D6 (e.g., star allele haplotype). In some cases, the genetic variation is a structural variation of CYP2D6. In some cases, based on the genetic variation, the subject is identified as having a loss or loss of CYP2D6 function. In some cases, the subject is identified as having an increase or gain of CYP2D6 function.

[0077] In various aspects, the method further comprises recommending a treatment for the subject based on the identifying step. In various aspects, the method further comprises treating the subject based on the identifying step. In various aspects, the method involves recommending an alternative treatment based on the identifying step. In various aspects, the method involves recommending a dosage of the drug based on the identifying step. In various aspects, the method involves modifying (or recommending a modification of) the dosage of a drug (e.g., activated or metabolized by CYP2D6) administered to the subject. In some cases, the drug (or therapeutic drug) is a drug activated or metabolized by CYP2D6.

[0078] Compositions and Kits

[0079] In one aspect, provided herein are compositions and kits comprising: (a) a clustered regularly interspaced short palindromic repeats (CRISPR) associated endonuclease; (b) an outer gRNA pair comprising (i) a first outer gRNA comprising a nucleotide sequence substantially complementary to a first nucleotide sequence present upstream of a genomic region of interest in genomic DNA, and (ii) a second outer gRNA comprising a nucleotide sequence substantially complementary to a second nucleotide sequence present downstream of said genomic region of interest in genomic DNA; (c) an inner gRNA pair comprising (iii) a first inner gRNA comprising a nucleotide sequence substantially complementary to a third nucleotide sequence present upstream of said genomic region of interest in genomic DNA, and (iv) a second inner gRNA comprising a nucleotide sequence substantially complementary to a fourth nucleotide sequence present downstream of said genomic region of interest in genomic DNA, wherein the third nucleotide sequence and the fourth nucleotide sequence are present on the genomic DNA at a base length closer to the genomic region of interest than the first nucleotide sequence and the second nucleotide sequence.

[0080] In some cases, the composition and / or kit further comprises an exonuclease. The exonuclease can be selected from the group consisting of exonuclease I, exonuclease II, exonuclease III, exonuclease IV, exonuclease V, exonuclease VI, exonuclease VII, and exonuclease VIII.

[0081] CRISPR-associated endonuclease can be any CRISPR-associated endonuclease described herein.In some cases, CRISPR-associated endonuclease is class I or class II CRISPR-associated endonuclease.Non-limiting examples of Cas I CRISPR-associated endonuclease include Cas3, Cas5, Cas8a, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Csx11, Csx10 and Csf1.Non-limiting examples of class II CRISPR-associated endonuclease include Cas9, Cas12a, Csn2, Cas4, Cas12b, Cas12c, Cas13a, Cas13b, Cas13c and Cas13d. In some cases, the CRISPR-associated endonuclease is a Cas protein or polypeptide. In some embodiments, the CRISPR-associated endonuclease is a Cas12a protein or polypeptide.

[0082] In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein or polypeptide. In some cases, the Cas9 protein or polypeptide is derived from the bacterial species Streptococcus pyogenes. In some cases, the Cas9 protein or polypeptide has an amino acid sequence identical to the wild-type Cas9 amino acid sequence. In other cases, the Cas9 protein or polypeptide has an amino acid sequence that is modified compared to the wild-type Cas9 amino acid sequence. In some cases, the Cas9 protein or polypeptide has one or more mutations (e.g., compared to the wild-type Cas9 protein or polypeptide). In some cases, the one or more mutations are substitutions, deletions, or insertions. The Cas9 protein or polypeptide may have an amino acid sequence that has at least about 50% sequence identity compared to the wild-type Cas9 protein or polypeptide. For example, a Cas9 protein or polypeptide may have at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity relative to a wild-type Cas9 protein or polypeptide. In some cases, the Cas9 variant may comprise one or more point mutations relative to a wild-type S. pyogenes Cas9. For example, the Cas9 mutant may comprise a point mutation selected from the group consisting of R780A, K810A, K848A, K855A, H982A, K1003A, R1060A, D1135E, N497A, R661A, Q695A, Q926A, L169A, Y450A, M495A, M694A and M698A compared to wild-type S. pyogenes Cas9.

[0083] In some cases, the genomic region of interest is a locus including CYP2D6, CYP2D7, and CYP2D8. In some cases, at least one of the gRNAs (e.g., at least one of the first inner gRNA, the second inner gRNA, the first outer gRNA, and the second outer gRNA) comprises a nucleotide sequence according to any of the nucleotide sequences presented in Table 1 (e.g., SEQ ID NOs: 1-418). In some cases, at least one of the gRNAs (e.g., at least one of the first inner gRNA, the second inner gRNA, the first outer gRNA, and the second outer gRNA) comprises a nucleotide sequence having at least about 90% (e.g., at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) sequence identity to any of the nucleotide sequences presented in Table 1 (e.g., SEQ ID NOs: 1-418). In some cases, at least one of the gRNAs is a crRNA. In some cases, at least one of the gRNAs is an sgRNA. In some cases, the first outer guide RNA, the first inner guide RNA, or both, comprise the nucleotide sequence of any one of SEQ ID NOs: 3-12, 17-26, 68-77, 82-214, and 344-418. In some cases, the second outer guide RNA, the second inner guide RNA, or both, comprise the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 13-16, 27-67, 78-81, and 215-343.

[0084] In some embodiments, the kit further comprises instructions for using the kit in any of the methods presented herein.In some cases, the kit further comprises instructions for using the kit in nested CRISPR reaction (e.g., as described herein).In some cases, the kit further comprises instructions for using the kit in a method for excising a genomic region of interest from genomic DNA (e.g., as described herein).In some cases, the kit further comprises instructions for using the kit in a method for excising a CYP2D6 locus from genomic DNA (e.g., as described herein).

[0085] Subjects and biological samples

[0086] A subject can provide a biological sample for genetic analysis. A biological sample can be any material produced by a subject. In general, a biological sample is any tissue obtained from a subject or any material produced by a subject. A biological sample can be a bodily fluid, such as blood (e.g., whole blood, plasma, serum), urine, saliva, bone marrow, spinal fluid, sputum, ascites, lymphatic fluid, pleural fluid, amniotic fluid, semen, vaginal fluid, sweat, stool, glandular secretions, intraocular fluid, breast milk, etc. A biological sample can be a cell and / or a solid tissue (e.g., buccal tissue (e.g., from a buccal swab), feces, skin, hair, organ tissue, etc.). In some cases, a biological sample is a solid tumor or a biopsy of a solid tumor. In some cases, a biological sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample. A biological sample can be any biological sample that contains genomic DNA.

[0087] A biological sample can be drawn from a subject. The subject can be a mammal, reptile, amphibian, bird, or fish. The mammal can be a human, ape, orangutan, monkey, chimpanzee, cow, pig, horse, rodent, bird, reptile, dog, cat, or other animal. The reptile can be a lizard, snake, alligator, turtle, crocodile, and tortoise. The amphibian can be a toad, frog, newt, and salamander. Examples of birds include, but are not limited to, ducks, geese, penguins, ostriches, and owls. Examples of fish include, but are not limited to, catfish, eels, sharks, and swordfish. The subject is preferably a human. The subject can have a disease or condition. The subject can be prescribed a therapeutic agent. The therapeutic agent can be a therapeutic agent that is activated and / or metabolized by CYP2D6.

[0088] System of the present disclosure

[0089] Further provided herein is a system for carrying out the methods provided herein. In one aspect, a system is provided that includes: (a) at least one memory location configured to receive a data input comprising data generated by any method described herein; and (b) a computer processor operably coupled to the at least one memory location, the computer processor being programmed to generate an output based on the data.

[0090] In various aspects, the output is a report. In various aspects, the output is a genotype of the complex genomic region of interest. In various aspects, the output is a gene sequence of the complex genomic region of interest. In various aspects, the output is a structural analysis of the complex genomic region of interest. In various aspects, the analyzing step comprises genotyping the complex genomic region of interest. In various aspects, the analyzing step comprises performing a structural analysis of the complex genomic region of interest. In various aspects, the analyzing step comprises sequencing the complex genomic region of interest.

[0091] In various embodiments, the output identifies a genetic variation in CYP2D6. In various embodiments, the output identifies a reduction, loss, or increase in function of CYP2D6. In various embodiments, the report recommends a treatment for the subject based on the genetic variation. In various embodiments, the report recommends a dosage of a therapeutic agent for the subject based on the genetic variation. In various embodiments, the report recommends modifying a dosage of a therapeutic agent based on the genetic variation. In some cases, the therapeutic agent is a therapeutic agent that is activated or metabolized by CYP2D6.

[0092] The present disclosure further provides a computer-based system for carrying out the methods described herein. In some aspects, the system can be used to analyze data generated by the methods presented herein. The system can include one or more client components. The one or more client components can include a user interface. The system can include one or more server components. The server components can include one or more memory locations. The one or more memory locations can be configured to receive data input. The data input can include sequencing data. The sequencing data can be generated from a nucleic acid sample (e.g., genomic DNA) from a subject. Non-limiting examples of sequencing data suitable for use with the system of the present disclosure are described. The system can further include one or more computer processors. The one or more computer processors can be operably coupled to the one or more memory locations. The one or more computer processors can be programmed to generate an output for display on a screen. The output can include one or more reports.

[0093] The systems described herein may include one or more client components. The one or more client components may include one or more software components, one or more hardware components, or a combination thereof. The one or more client components can access one or more services through one or more server components. The one or more services can be accessed by one or more client components through a network. The network may be the Internet, an Internet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. The network, in some cases, is a telecommunications and / or data network. The network may include one or more computer servers, thereby enabling distributed computing such as cloud computing. The network, in some cases, may implement a peer-to-peer network with the aid of a computer system, thereby enabling devices coupled to the computer system to function as clients or servers.

[0094] The system may include one or more memory locations (e.g., random access memory, read only memory, flash memory), electronic storage (e.g., hard disk), communication interfaces (e.g., network adapters) for communicating with one or more other systems, and peripherals such as cache, other memory, data storage and / or electronic display adapters. The memory, storage, interfaces and peripherals communicate with the CPU through a communication bus, such as a motherboard. The storage may be a data storage device (or data repository) for storing data. In one embodiment, the received sequencing data may be stored in one or more memory locations.

[0095] The system may include one or more computer processors, which may be operatively coupled to one or more memory locations, for example to access stored data, and which may implement machine-executable code to perform the methods described herein.

[0096] Machine-executable or machine-readable code may be provided in the form of software. During use, the code may be executed by a processor. In some cases, the code may be retrieved from a storage device and stored in memory for ready access by the processor. In some situations, electronic storage may be omitted, and machine-executable instructions may be stored in memory.

[0097] The code may be precompiled and configured for use on a machine having a processor adapted to execute the code, may be compiled at run time, or may be interpretively executed at run time. The code may be provided in a programming language that may be selected to enable the code to be executed in a precompiled, compiled-at-once, or interpretive manner.

[0098] Aspects of the systems and methods presented herein, such as computer systems, can be embodied in programming. Various aspects of the technology can be considered as "products" or "articles of manufacture" generally in the form of machine (or processor) executable code and / or associated data embodied or embodied in one type of machine-readable medium. The machine executable code can be stored in electronic storage devices such as memory (e.g., read-only memory, random access memory, flash memory) or hard disks. "Storage" type media can include any or all of the tangible memory of the computer, processor, etc., or associated modules thereof, such as various semiconductor memories, tape drives, disk drives, etc., which can provide non-transitory storage at any time for software programming. All or parts of the software can be communicated from time to time over the Internet or various other telecommunications networks. Such communication can, for example, allow the software to be loaded from one computer or processor to another, for example, from a management server or host computer to the computer platform of an application server. Thus, other types of media that may carry software elements include light waves, radio waves, and electromagnetic waves, such as those used across physical interfaces between local devices, through wired and optical communication networks, and through various air links. The physical elements that carry such waves, such as wired or wireless links, optical links, etc., may also be considered software-bearing media. As used herein, except where limited to non-transitory, terms such as tangible "storage" media, computer or machine "readable media," etc., refer to any medium that participates in providing instructions to a processor for execution.

[0099] Thus, machine-readable media such as computer executable code may take many forms, including but not limited to tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, any of the storage devices of any computer(s) that can be used to implement, for example, the databases shown in the figures, and the like. Volatile storage media include dynamic memories such as the main memory of such computer platforms. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus in a computer system. Carrier wave transmission media may take the form of electric or electromagnetic signals, or sound or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer readable media include, for example, a floppy disk, a flexible disk, a hard disk, a magnetic tape, any other magnetic medium, a CD-ROM, a DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with a pattern of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave carrying data or instructions, a cable or link which transports such a carrier wave, or any other medium from which programming code and / or data may be read by a computer. 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.

[0100] The systems disclosed herein may include or communicate with one or more electronic displays. The electronic display may be part of a computer system, or may be coupled to the computer system directly or through a network. The computer system may include a user interface (UI) for providing the various features and functionality disclosed herein. Examples of UIs include, without limitation, graphical user interfaces (GUIs) and web-based user interfaces. The UI may provide interaction tools by which a user may utilize the methods and systems described herein. By way of example, the UI envisioned in the present invention may be a web-based tool by which a health care practitioner may order genetic tests, customize a list of genetic variants to be tested, and receive and view reports.

[0101] The methods disclosed herein may include biomedical databases, genomic databases, biomedical reports, disease reports, case-control analyses, and rare variant discovery analyses based on data and / or information from one or more databases, one or more assays, one or more data or results, one or more outputs based on or derived from one or more assays, one or more outputs based on or derived from one or more data or results, or combinations thereof.

[0102] As described herein, one or more computer processors can implement machine executable code to perform the disclosed method. The machine executable code can include any number of open source or closed source software. The machine executable code can be implemented to analyze data input. The data input can be sequencing data generated by one or more sequencing reactions. The computer processor can be operably coupled to at least one memory location. The computer processor can access data (e.g., sequencing data) from the at least one memory location. In some cases, the computer processor can implement machine executable code to map the sequencing data to a reference sequence. In some cases, the computer processor can implement machine executable code to determine the presence or absence of a genetic variant from the sequencing data. In some cases, the computer processor can implement machine executable code to generate an output (e.g., a report) for display on a screen.

[0103] The machine executable code may include one or more algorithms. The one or more algorithms may be used to implement the methods of the present disclosure.

[0104] The system of the present disclosure may include one or more computer systems. Figure 16 illustrates a computer system (also referred to herein as "system") 1601 programmed or otherwise configured to implement the methods of the present disclosure, such as, for example, receiving data and generating output based on said data. The system 1601 includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") 1605, which may be a single-core processor or a multi-core processor, or multiple processors for parallel processing. The system 1601 also includes memory 1610 (e.g., random access memory, read-only memory, flash memory), electronic storage 1615 (e.g., hard disk), communication interface 1620 (e.g., network adapter) for communicating with one or more other systems, and peripherals 1625, such as cache, other memory, data storage, and / or electronic display adapters. The memory 1610, storage 1615, interface 1620, and peripherals 1625 communicate with the CPU 1605 through a communication bus (solid lines) such as a motherboard. The storage device 1615 may be a data storage device (or data repository) for storing data. The system 1601 is operatively coupled to a computer network ("network") 1630 with the aid of a communication interface 1620. The network 1630 may be the Internet, an Internet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. The network 1630 is, in some cases, a telecommunications and / or data network. The network 1630 may include one or more computer servers, thereby enabling distributed computing, such as cloud computing.The network 1630, in some cases, with the aid of the system 1601, may implement a peer-to-peer network, thereby enabling devices coupled to the system 1601 to function as clients or servers.

[0105] System 1601 is in communication with a processing system 1640. Processing system 1640 can be configured to implement methods disclosed herein, such as, for example, mapping sequencing data to a reference sequence or assigning classifications to genetic variants. Processing system 1640 can be in communication with system 1601 through a network 1630 or by a direct (e.g., wired, wireless) connection. Processing system 1640 can be configured for analysis, such as nucleic acid sequence analysis.

[0106] The methods and systems described herein may be implemented by machine (or computer processor) executable code (or software) stored in an electronic storage location of the system 1601, such as memory 1610 or electronic storage 1615. During use, the code may be executed by the processor 1605. In some embodiments, the code may be retrieved from storage 1615 and stored in memory 1610 for ready access by the processor 1605. In some circumstances, the electronic storage 1615 may be omitted, with machine executable instructions stored in memory 1610.

[0107] The code may be precompiled and configured for use on a machine having a processor adapted to execute the code, may be compiled at run time, or may be interpretively executed at run time. The code may be provided in a programming language that may be selected to enable the code to be executed in a precompiled, compiled-at-once, or interpretive manner.

[0108] Aspects of the systems and methods presented herein can be embodied in programming. Various aspects of the technology can be considered as "products" or "articles of manufacture" generally in the form of machine (or processor) executable code and / or associated data embodied or embodied in one type of machine-readable medium. The machine executable code can be stored in electronic storage such as memory (e.g., read-only memory, random access memory, flash memory) or hard disk. A "storage" type medium can include any or all of the tangible memory of a computer, processor, etc., or associated modules thereof, such as various semiconductor memories, tape drives, disk drives, etc., which can provide non-transitory storage at any time for software programming. All or part of the software can be communicated from time to time over the Internet or various other telecommunications networks. Such communication can, for example, allow the software to be loaded from one computer or processor to another, for example, from a management server or host computer to the computer platform of an application server. Thus, other types of media that may carry software elements include light waves, radio waves, and electromagnetic waves, such as those used across physical interfaces between local devices, through wired and optical communication networks, and through various air links. The physical elements that carry such waves, such as wired or wireless links, optical links, etc., may also be considered software-bearing media. As used herein, except where limited to non-transitory, terms such as tangible "storage" media, computer or machine "readable media," etc., refer to any medium that participates in providing instructions to a processor for execution.

[0109] Thus, a machine-readable medium such as a computer-executable code may take many forms, including but not limited to tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, any of the storage devices of any computer(s) that can be used to implement, for example, a database, etc. Volatile storage media include dynamic memories such as the 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 in a computer system. Carrier wave transmission media may take the form of electric or electromagnetic signals, or sound or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer readable media include, for example, a floppy disk, a flexible disk, a hard disk, a magnetic tape, any other magnetic medium, a CD-ROM, a DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with a pattern of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave carrying data or instructions, a cable or link which transports such a carrier wave, or any other medium from which programming code and / or data may be read by a computer. 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.

[0110] The computer system 1601 may include or be in communication with an electronic display that includes a user interface (UI). Examples of UIs include, without limitation, graphical user interfaces (GUI) and web-based user interfaces.

[0111] In some embodiments, the system 1601 includes a display for providing visual information to a user. In some embodiments, the display is a cathode ray tube (CRT). In some embodiments, the display is a liquid crystal display (LCD). In further embodiments, the display is a thin film transistor liquid crystal display (TFT-LCD). In some embodiments, the display is an organic light emitting diode (OLED) display. In various further embodiments, the OLED display is a passive matrix OLED (PMOLED) or an active matrix OLED (AMOLED) display. In some embodiments, the display is a plasma display. In other embodiments, the display is a video projector. In yet another embodiment, the display is a combination of devices such as those disclosed herein. The display is capable of providing one or more biomedical reports to an end user once generated by the methods described herein.

[0112] In some embodiments, the system 1601 includes an input device to receive information from a user. In some embodiments, the input device is a keyboard. In some embodiments, the input device is a pointing device, including, by way of non-limiting examples, a mouse, a trackball, a trackpad, a joystick, a game controller, or a stylus. In some embodiments, the input device is a touch screen or a multi-touch screen. In other embodiments, the input device is a microphone for capturing voice or other sound input. In other embodiments, the input device is a video camera for capturing motion or visual input. In yet another embodiment, the input device is a combination of devices, such as those disclosed herein.

[0113] The system 1601 may include or be operatively coupled to one or more databases. The databases may include genomic databases, proteomic databases, pharmacogenomic databases, biomedical databases, and scientific databases. The databases may be publicly available databases. Alternatively or in addition, the databases may include proprietary databases. The databases may be commercially available databases. The databases include, but are not limited to, MendelDB, PharmGKB, Varimed, Regulome, curated BreakSeq junctions, Online Mendelian Inheritance in Man (OMIM), Human Genome Mutation Database (HGMD), NCBI dbSNP, NCBI RefSeq, GENCODE, GO (gene ontology), and Kyoto Encyclopedia of Genes and Genomes (KEGG).

[0114] The data may be generated and / or transmitted from a geographic location, including the same country as the user of the data. The data may be generated and / or transmitted from a geographic location in, for example, one country, and the user of the data may be in a different country. In some cases, the data accessed by the system of the present disclosure may be transmitted to the user from one of multiple geographic locations. The data may be transmitted back and forth between multiple geographic locations, for example, by a network, a secure network, an unsecured network, the Internet, or an intranet. EXAMPLES

[0115] The following examples are presented for the purpose of illustrating various embodiments of the present disclosure, and are not intended to limit the present disclosure in any way. The examples, together with the methods described herein, are currently representative preferred embodiments, are illustrative, and are not intended to limit the scope of the embodiments of the present disclosure. Variations in the examples and other uses encompassed within the spirit of the present disclosure as defined by the scope of the claims will occur to those skilled in the art.

[0116] Example 1

[0117] CYP2D6 and Clinical Trials

[0118] CYP2D6 Gene Structure: CYP2D6 is a small gene (4382 bp) with nine exons. However, genetic analysis of this highly polymorphic locus is difficult due to the presence of highly similar non-functional CYP2D7 and CYP2D8 pseudogenes within the locus, as shown in Figure 1. The similarity between CYP2D6 and CYP2D7 and the presence of large repeat regions have led to gene deletions and duplications as well as complex gene hybrids containing either 3'CYP2D7 and 5'CYP2D6 or 3'CYP2D6 and 5'CYP2D7. Currently, multiple test assays are required to detect the presence of these structural variants.

[0119] Current Testing Platforms: One of the common methods for analyzing CYP2D6 is by sequence analysis of long-range allele-specific PCR products. Briefly, allele-specific primers are used to amplify the targeted region. Single nucleotide variants (SNVs) found in the PCR products represent the haplotypes of that allele. Allele-specific amplicons can also result from duplicated gene copies as well as CYP2D6-2D7 and CYP2D7-2D6 hybrid genes. More recently, long-read sequencing techniques such as single molecule real-time (SMRT) sequencing or nanopore sequencing have also been used to more precisely characterize CYP2D6 haplotypes; however, library generation for long-read sequencing of CYP2D6 remains limited. The XL-PCR reaction currently used to generate CYP2D6 templates for sequencing is limited in the size of the products that can be generated, primer-specific, and does not capture complex hybrids or many known CNVs unless the mutation has been previously characterized and is known to be present in the sample of interest.

[0120] In summary, CYP2D6 is a highly polymorphic gene that is directly involved in the metabolism of approximately 25% of all prescription drugs. Genetic variations in this gene, including copy number changes, can directly affect the drug metabolism status of patients. Accurate genotyping, including copy number, is crucial, and current methodologies are unable to fully assay the complexity of this genetic region.

[0121] A method is proposed herein to develop a diagnostic quality methodology for CYP2D6 analysis by utilizing CRISPR / Cas9 technology and site-specific adapter ligation in combination with long-read sequencing.In this approach, a single, sample-agnostic CRISPR cutting step is utilized to isolate the entire CYP2D6 locus for long-read sequencing.This methodology allows both single nucleotide polymorphism (SNP) and CNV to be accurately detected, and to assign the most accurate possible phased CYP2D6 genotype and metabolizer status.

[0122] Both in vitro and in vivo, CRISPR technology can be used to target and excise a genomic region of interest (ROI). Briefly, when CRISPR-C associated protein 9 (Cas9) forms a complex with a synthetically generated target-specific guide RNA (sgRNA), a double-stranded cut is created in a sequence that has complementarity to the target-specific sequence of the guide RNA. By designing sgRNAs to target sequences at both ends of the ROI, CRISPR-Cas9 can be used to excise DNA that can be up to several megabases in length.

[0123] Long-Read Sequencing: The development of short-read next-generation sequencing (NGS) has revolutionized human genetics, but its limitations are well recognized. Long-read sequencing of isolated HMW DNA fragments has recently attracted attention because it allows obtaining phasing information, identifying small structural variations, and better assembling highly complex regions of the genome, including tandem repeats. The use of CRISPR technology for targeted isolation of DNA fragments provides an innovative and superior method to target relevant regions of the genome for long-read sequencing.

[0124] GeT-RM cohort: As part of a major effort to systematically characterize the CYP2D6 gene structure, CYP2D6 genotyping data were provided to establish a state-of-the-art set of well-characterized reference materials for assay development, validation, quality control, and proficiency testing. This effort was conducted in collaboration with the Genetic Testing Reference Materials Coordination Program (GeT-RM) at the Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, the Coriell Institute for Medical Research, and other PGx community members. As part of this study, Pharmacoscan™-based CYP2D6 genotyping was provided for several samples containing complex structural arrangements and / or rare CYP2D6 genotypes. This data was used in conjunction with XL-PCR-based NGS analysis to determine the most accurate genotype possible for these samples with current analytical methodologies. All cell line and consensus genotyping information and annotation data will provide a basis for validation of the proposed new sequencing and analytical approaches.

[0125] Research design and methods

[0126] Objective 1 (Method Development): (a) Optimization of a specific CRISPR / Cas9 methodology to generate high molecular weight DNA segments containing the CYP2D6-D7 genomic locus for subsequent size analysis (e.g., gel) in genomic human DNA (e.g., blood samples). (b) Generation of XL-libraries for isolation / enrichment and sequencing of targeted regions. (c) Establishment of NGS methods (e.g., PacBio, MinION) for long template sequencing of genomic variants of the CYP2D6-D7 genomic locus. A schematic of the proposed workflow is shown in Figure 2.

[0127] Isolation of HMW DNA: The typical length of the ROI (CYP2D6 and CYP2D7) is 28-35 kb. To ensure that the entire ROI is intact for downstream analysis, a protocol was developed to isolate high molecular weight gDNA (up to 70 kb) using the NucleoBond® Genomic DNA and RNA purification system. The modified protocol allows for the extraction of gDNA with molecular weights greater than 50 kb, compared to the 10 kb-50 kb range observed using other methodologies (Figure 3).

[0128] Design and validation of highly specific sgRNAs: Due to the complex and highly polymorphic nature of the CYP2D6 locus, traditional PCR and array-based techniques require multiple assays to perform both CNV and SNP analysis. CRISPR Cas9 approaches that target only the CYP2D6 gene cannot capture alleles containing structural mutations, such as D6 / D7 hybrid alleles or CYP2D6 duplication events. To overcome this limitation, we identified unique sequences flanking the region encompassing both CYP2D6 and CYP2D7. By designing sgRNAs targeting these unique regions, one CRISPR / Cas9 cleavage reaction was performed to isolate the entire CYP2D6 / CYP2D7 region (Figure 4A).

[0129] To confirm the specificity and efficacy of sgRNA, XL-PCR products containing the targeted sgRNA binding sites were generated from gDNA. The XL-PCR products were incubated with Cas9 but without sgRNA (Figure 4B, sample A) or with Cas9 and different sgRNAs (Figure 4B, samples B and C). All PCR products incubated with Cas9 and sgRNA were cleaved to generate DNA fragments of the expected size, but different sgRNAs showed different degrees of cleavage efficiency.

[0130] Cutting the CYP2D6-CYP2D7 locus in genomic DNA: sgRNA must bind with high efficiency and specificity to gDNA that may contain off-target recognition sites. To examine the cutting efficiency and specificity of CRISPR, genomic DNA was incubated with Cas9 but without sgRNA (negative control) or with Cas9 and a pool of two sgRNAs that cut 5' of CYP2D6 and 3' of CYP2D7. PCR reactions were performed with primers flanking each of the predicted cleavage sites. If the sgRNA binds to the correct binding site and cleavage occurs, a decrease in PCR product is expected. Indeed, this is observed (Figure 5A, Figure 5B). PCR was also performed on the CYP2D6 locus using primers internal to the sgRNA binding site to determine whether Cas9-mediated off-target cleavage occurs within the CYP2D6 gene. No evidence of off-target cleavage within CYP2D6 was observed (Figure 5A, Figure 5B).

[0131] In summary, XL-PCR and genomic DNA interrogation demonstrated that the Cas9-sgRNA complex cut both sides of the targeted CYP2D6-CYP2D7 locus with high efficiency and without significant off-target activity within the locus. Cleavage created the predicted 28 kb fragment, which, after enrichment, can be utilized for downstream long-read NGS.

[0132] Example 2 Further optimization of the CRISPR / Cas9 methodology

[0133] Other sgRNAs and Cas enzymes were developed and tested. Standard software is used to identify and design sgRNAs to be tested as described above. The goal is to obtain sgRNAs that cut with high efficiency and specificity in the ROI. Shorter DNA fragments are preferred, but still contain the entire ROI. Shorter fragments may have the benefit of reduced sequencing and processing costs. We also attempt to cut the same region using CRISPR Cas12a enzyme. Cas12a endonuclease functions similarly to Cas9, but has a different PAM sequence requirement (TTTV), which creates a 5'-sticky overhang after cutting. In contrast, Cas9 creates blunt ends, which is important for later steps.

[0134] Example 3 Enrichment of the CYP2D6-CYP2D7 locus in genomic DNA

[0135] As a proof of concept, 5 μg of gDNA was cut with Cas9-sgRNA targeting the 5' of CYP2D6 and 3' of CYP2D7 cut sites described above. The cut DNA was run on a BluePippen (Sage Science) instrument using a 0.75% agarose gel cassette that allows for size selection in the range of 1-50 kb. PCR was used to confirm that the eluted samples contained the desired CYP2D6-CYP2D7 locus. While this gel-based approach allows for the isolation of HMW samples, it does have several drawbacks, including time (approximately 10-12 hours per Blue Pippen run), limited sample numbers (4-5 samples per run), significant loss of material / poor recovery, and high cost per sample (approximately $50.00).

[0136] To overcome these limitations, several approaches to enrich the target are tested. This allows identifying the advantages and disadvantages of the various methods and ultimately identifying the most appropriate approach for further clinical trial development. This is a typical approach for clinical diagnostic test development. The following long-read sequencing discussion refers to Oxford Nanopore (ONT) sequencing, but any protocol can be adapted with minor modifications to fit PacBio sequencing requirements.

[0137] Method 1: Amplification-free enrichment of targets

[0138] DNA Preparation: This amplification-free library preparation method involves dephosphorylation and 3'-end capping of DNA samples, followed by CRISPR treatment and site-specific ONT adaptor ligation. In the first step, gDNA is treated with shrimp alkaline phosphatase, which removes the phosphate group from the 5'-end of the DNA fragment, and terminal transferase, which adds a single thymidine dideoxynucleotide to the 3'-end. This step ensures that the gDNA ends are incapable of ligation. The DNA is then treated with CRISPR Cas9:gRNA complex, resulting in blunt-ended CYP2D6 / CYP2D7 fragments of approximately 28-35 kb (see previous paragraph for details). This is followed by an "A-tailing" step, in which a DNA polymerase is used to add an adenosine nucleotide to the free 3'-end of the DNA (e.g., the end not capped with ddTTP). Finally, ONT adaptors with thymidine overhangs are added to the DNA. The DNA end generated by CRISPR-Cas9 cleavage is the only end with a complementary 3' overhang and 5' phosphate group, and therefore only this DNA end will ligate to the adapter.

[0139] Sequencing: The resulting libraries are sequenced directly on an ONT instrument. If the amount of DNA library generated by this method proves too challenging for ONT sequencing, this can be overcome by multiplexing the samples prior to sequencing and / or by increasing the amount of input gDNA. Additionally, background can be reduced by treating the samples with exonuclease (ONT adapters are resistant to exonuclease III and lambda exonuclease), resulting in degradation of all background DNA.

[0140] Method 2: Enrichment using in vitro transcription

[0141] Rationale: When previous methods have failed to generate sufficient DNA or when excessive background DNA is present, an alternative method, targeted amplification by in vitro transcription (IVT), is evaluated. IVT has several advantages over PCR: (1) transcription is less likely to introduce error propagation; (2) transcription can produce RNA molecules as long as 20-30 kb, which is longer than the size of the longest-range PCR product.

[0142] DNA preparation: After cleavage by CRISPR, the DNA is treated with exonuclease to generate sticky ends, and a double-stranded DNA fragment containing overhangs complementary to the T7 promoter and the sticky ends of the CYP26-CYP2D7 locus is ligated to the target fragment. DNA polymerase and DNA ligase are used to fill gaps and seal any nicks. Transcripts as long as about 20 kb can be produced by phage T7 RNA polymerase. Because promoters ligate to both ends of the locus of about 28 kb, the longest transcripts produced by T7 RNA polymerase from promoters at the ends of the locus can be long enough to cover the entire region. However, the majority of T7 products are generally less than 4 kb in length. The recently discovered Syn5 cyanophage RNA polymerase can produce transcripts as long as 30 kb. The Syn5 promoter is tested together with the T7 promoter.

[0143] In vitro transcription: IVT is performed using T7 RNA polymerase and Syn5 RNA polymerase. The former enzyme is commercially available, while the latter enzyme was expressed and purified in our laboratory. There are several commercially available T7 RNA polymerase IVT kits optimized to produce long RNA transcripts. Previous studies have shown that T7 promoter sequences randomly inserted into the human genome produce a significant fraction of RNA transcripts larger than 5 kb during IVT. Total RNA yield, percentage of large transcripts (>15 kb) and error rate are important factors in determining which polymerase and IVT method is the better choice. Since a wide range of RNA transcript lengths can be produced, SPRI beads can be used to select the largest transcripts. RNA is sequenced directly on the ONT instrument.

[0144] Method 3: Introducing promoters into multiple sites for in vitro transcription

[0145] Rationale: When the above techniques are insufficient, the T7 or Syn5 promoter is inserted at multiple sites across the targeted region. A potential problem with this approach is that fragmentation of the locus increases the difficulty of unambiguously assigning variants to CYP2D7 or CYP2D6 (because genes and pseudogenes share approximately 94% sequence identity) and deriving phasing information. To overcome this limitation, multiple cohesive insertion sites are used to generate overlapping fragments.

[0146] Promoter introduction: CRISPR cleavage occurs at sites adjacent to the ROI and at regularly spaced (~10 kb) distant sites within the locus. Cleavage is performed in two separate reactions, each with a different set of target sites, so that the resulting overlapping fragments can be used to splice the reads together after sequencing. Exonuclease treatment, ligation of promoter-containing adapters, IVT, and cDNA synthesis are described above. The promoter-containing adapters contain a short fixed sequence immediately downstream of the promoter. When cDNA synthesis is performed, a primer with complementarity to this fixed sequence is used for reverse transcription (RT). If the RNA produced by IVT spans the length between the two insertion sites, a RT primer specific to this sequence will select cDNA molecules spanning the same region.

[0147] Potential alternatives: If necessary, several cycles of long-range PCR using a fixed sequence at the beginning of each IVT product can be used to selectively amplify cDNA molecules spanning the insertion site.

[0148] Potential alternatives: RNA sequencing by ONT requires large amounts of RNA. If necessary, select for long transcripts by performing cDNA synthesis with a primer annealing to a site distant from the transcription start (15–20 kb). If a significant proportion of sequencing reads do not map to the target locus, try to prevent ligation of adapters to non-target sites. Dephosphorylation of gDNA before CRISPR treatment and capping the ends of gDNA with so-called "dumbbell" adapters are two possible options.

[0149] Example 4 Establishment of an NGS method for long template sequencing of mutants

[0150] Methods: Currently, there are two main commercially available platforms that can be applied to develop potential diagnostic tests. PacBio is the first and foremost technology for long-read sequencing, but the associated costs are significant. More recently, nanopore sequencing technology has emerged as a potentially viable cost-effective platform. Oxford Nanopore (ONT) as a platform continues to mature with respect to throughput, cost and accuracy. Therefore, given these advantages, the focus is on ONT. Nevertheless, the proposed methodology and methods are primarily platform agnostic and can be adapted to fit any of the two current or upcoming long-read platforms. Sequencing runs can be performed on the Oxford Nanopore MinION.

[0151] Objective 2 (Validation): (a) Perform sequence analysis using current software and platforms for long-read sequence alignment to perform variant calling, CNV analysis and phasing. (b) Compare CYP2D6-D7 long-read sequence analysis results with sequence / copy number variation and characterize consensus genotyping and annotation results together with results from the Get-RM project to estimate performance characteristics and guidance for further diagnostic test development. Test and compare the feasibility of each method in terms of time- and cost-effectiveness, minimization of required steps and quality of results. The overarching objective is to select the most suitable method for isolation, enrichment and sequencing of the entire CYP2D6 gene.

[0152] Selection of samples for validation: Once the sample preparation method is developed, an expanded set of additional samples with known genotypes and haplotypes will be analyzed. To evaluate the platform against the expanded data set, samples with complex structures such as duplications, hybrids, selected deletions, and complex rearrangements will be included. Samples will be selected from the GeT-RM project (see "The GeT-RM cohort" above). These cell lines and data provide a unique resource as they allow novel long-read sequence data to be evaluated against the current gold standard. For this proposal, a subset of these cell lines was obtained from LCL cell lines. Additional samples will be obtained to characterize other relevant variants and haplotypes from cell line repositories and existing collaborations. To further validate the methodology with additional samples, additional cell lines from the NIST Coriell cohort that have been extensively characterized, including by whole genome sequencing, will be utilized. Additionally, additional sample types representative of typical diagnostic specimens will be obtained, including whole blood and saliva. A total of 48 cell lines representing duplications, deletions, hybrids and tandem arrangements are selected for sequencing for this purpose. Analyses are performed in duplicate for a total of 96 sequenced samples.

[0153] Variant calling, CNV calling, and phasing: We use software packages developed specifically for long-read ONT data. Clair is the latest update to Clairvoyante, a multitask five-layer convolutional neural network model for predicting variant type, zygosity, alternative alleles, and insertion / deletion length. An additional package that has been recently developed is Megalodon. Megalodon's functionality is focused on linking informative neural network base calling to reference sequences. The performance characteristics of the Nanopore technology were recently evaluated by Bowden et al. for whole genome sequencing using standard reference samples. The consensus accuracy at 82× coverage was 99.9%, but the data also show some current limitations of the platform. Since only small targeted regions are proposed to be sequenced, and given the ability to sequence regions very deeply, we expect that the current analysis platform will generate sufficiently accurate data for targeted sequences. We will also monitor future software developments and take advantage of new methods as they become available.

[0154] Comparison with consensus data: Data is compared with GeT-RM consensus results (based on results from all platforms and variants scrutiny by a team of experts). Concordance is determined for haplotype calling SNPs and CNVs, and the ability of hybrid haplotypes to identify sequence features is evaluated, and the concordance is measured for determining metabolic status. Additional variants are then compared with genotyping data from the GeT-RM project. Data is analyzed together with phasing information (e.g., determined haplotypes) to determine whether the phased genotyping data is consistent with the results, since this provides non-attributed phasing information. Finally, any additional variants identified by sequencing alone are identified. Exploratory sequence comparisons for sequence similarity between CYP2D6 and its pseudogenes are also performed.

[0155] Expected problems: One of the problems concerns the overall accuracy of the sequencing platform. The first approach is to sequence very deeply. This should allow for the determination of non-systematic sequencing errors, but it is more difficult to determine the inherent errors due to the technical limitations of the platform. Comparison with consensus data of CYP2D6 reference samples makes it possible to estimate this effect. Furthermore, further benchmarking of the ONT platform and improved sequence analysis methods is expected to increase sequence annotation for long-read data.

[0156] Future Directions: In pharmacogenetics, CYP2D6 stands out as one of the most widely tested genes, but is technically challenging to analyze using current testing techniques. The ultimate goal is to develop a unified clinical trial method that can replace the current platforms, which are imperfect and prone to errors. This application serves as a proof of concept that CRISPR-based sequence targeting, innovative fragment enrichment and long-read sequencing are viable approaches.

[0157] Example 5

[0158] Targeting specific genomic loci for analysis

[0159] Compared to traditional methods such as PCR or oligonucleotide hybridization, this technique uses the CRISPR / CAS9 system and locus-specific guide RNAs to make targeted cuts only in the region of interest (ROI). The novel method of enriched region selection and sgRNA design allows for the capture of the entire locus, including highly similar pseudogenes and repetitive regions. An example of such a region is shown in Figure 1.

[0160] Current Issues

[0161] Common DNA extraction methodologies and sequencing approaches for highly polymorphic genes such as CYP2D6, which contain repetitive regions (e.g., REP6) and share high sequence similarity with nearby pseudogenes, have many drawbacks. These issues include errors introduced by PCR, limited size that can be captured by PCR, off-target array hybridization, the need for multiple assays (e.g., CNV analysis using sequencing + qPCR), off-target alignment, lack of variant phasing, and high financial and time burdens. In Figure 6, IGV alignments of six example traditionally prepared libraries sequenced by NGS are highlighted. These libraries (A-F) were generated from CYP2D6 long-range PCR (XL-PCR) amplicons. Amplicons were subjected to fragmentation (100-300 bp), adapter ligation, and PCR amplification prior to NGS analysis. There are several limitations to this approach. First, as shown for CYP2D6, the copy number status of CYP2D6 and whether hybrid alleles are present must be known prior to XL-PCR in order to amplify the CYP2D6 gene in each sample. Specific primers must be used for normal, duplicated, deleted, and hybrid alleles, respectively. This requires an additional copy number assay to be performed prior to NGS. Furthermore, XL-PCR amplification times are typically 0.5–1 h per kb of target amplicon length.

[0162] Analysis of short-read sequence data is also hampered by reduced phasing capacity and is prone to off-target alignments with highly similar pseudogenes or homologous regions (e.g., CYP2D7 pseudogene, which is 94% similar to CYP2D6, as shown in Figure 1). Furthermore, different haplotypes of the same gene may have different levels of similarity to pseudogenes, making it impossible to align variants correctly.

[0163] PCR-free libraries offer significant advantages over traditional PCR-based approaches: they remove the potential for the introduction of PCR-derived sequence errors and overcome the current limitation of maximum PCR product size, they eliminate the XL-PCR reaction time, which represents a significant time saving, and the approach allows for heterozygous variant phasing and copy number variation (CNV) detection.

[0164] sgRNA design

[0165] As mentioned above, due to the complex and highly polymorphic nature of the CYP2D6 locus, traditional PCR and array-based techniques require multiple assays to be performed for both CNV and SNP analysis. Due to DNA shearing during extraction and sample handling, one intuitively selects the smallest possible CRISPR / Cas9 target region to capture the gene of interest to maximize the amount of intact target region for enrichment. However, CRISPR / Cas9 approaches that target only the CYP2D6 gene fail to capture alleles containing structural mutations, such as D6 / D7 hybrid alleles or CYP2D6 duplication events, which constitute at least 20% of the detected alleles. Examples of the highly complex requirements for reasonable guide RNA design are shown in Figures 7A-7C.

[0166] The first design limitation is that RNA for targeting the Cas9 complex to the ROI cannot be designed near the CYP2D6 gene itself. There are two main reasons for this. The first reason is that there are limited sites of unique sequences adjacent to CYP2D6 that are not identical to CYP2D7. Such unique sequence sites contain repetitive regions that are not fully functional and cannot capture mutations in important promoter regions. The second reason is that in the presence of CYP2D6 CNVs or D6 / D7 or D7 / D6 hybrid alleles, additional cuts will occur, preventing accurate CNV analysis and sequence alignment (Figure 7A). Similar limitations of the approach to cut near CYP2D7 and CYP2D8 are shown in Figures 7B and 7C, respectively.

[0167] To overcome these limitations, we identified unique sequences that flank the regions encompassing both CYP2D6, CYP2D7, and CYP2D8, yet still generate cut fragments of reasonable size for long-range sequence analysis. By designing sgRNAs to target these unique regions, one CRISPR / Cas9 cleavage reaction is performed to isolate the entire CYP2D6 / CYP2D7 / CYP2D8 region (Figure 8). Furthermore, depending on the downstream application, the design must target the correct strand (+ or -) depending on whether the sgRNA is to target the 5' or 3' end of the ROI. Non-limiting examples of sgRNA sequences to be tested are shown in Table 2 below. CYP2D6 is encoded on the -strand, but the position of the guide RNA (upstream or downstream) is referenced to the +strand. Sequences with a lower chromosomal position are considered to be further upstream, and sequences with a higher chromosomal position are considered to be downstream. Table 2. Guide RNA sequences [Table 2-1] [Table 2-2] [Table 2-3]

[0168] sgRNA performance analysis and validation

[0169] To confirm the specificity and efficacy of sgRNA, XL-PCR products containing the targeted sgRNA binding sites were generated from gDNA. The XL-PCR products were incubated with Cas9 + no sgRNA (or off-target sgRNA) or Cas9 + sgRNA of interest. Figure 9A shows a representative agarose gel showing the cutting efficiency of two different sgRNAs (T_1 and T_2) at multiple reaction time points. All PCR products incubated with Cas9 and sgRNA were cut to generate DNA fragments of the expected size, but different sgRNAs showed different degrees of cutting efficiency.

[0170] After determining the cleavage efficiency of the XL-PCR amplicons, the cleavage efficiency on genomic DNA was analyzed. This was done by performing Cas-mediated cuts with specific sgRNAs and then performing quantitative PCR reactions on the cut DNA. Primers were designed on either side of the predicted sgRNA target cut sites. PCR reactions were performed on 100 ng of total genomic DNA from either the Cas9 reaction or the uncut control. If the DNA was cut at the correct site, a reduction in PCR product would be observed compared to the amount of PCR product generated in the uncut control sample (e.g., Cas9 reaction with an sgRNA against an off-target region). As shown in Figure 9B and Figure 9C, this approach was used to determine whether the sgRNA was able to target the desired ROI in the genomic DNA and determine the efficiency of its cut. XL-PCR of the entire CYP2D6 gene showed no difference between the cut and uncut controls. This indicates that the reduction in the amount of PCR product observed at the cut sites across reactions is due to targeted Cas9-mediated cuts of these specific regions, rather than random cuts of DNA.

[0171] High molecular weight (HMW) DNA isolation

[0172] Isolation of long segments (≧50 kb) of high molecular weight genomic (HMW) DNA allows for the generation of sequencing libraries without PCR amplification. As shown in FIG. 10, HMW DNA was extracted in-house from lymphoblastoid cells (18959 and 19213) using the Nanobind CCB Dig DNA kit (Circulomics, Madison Wi). Extracted DNA was run on a 2% agarose gel and compared in size to lambda HINDIII ladder (upper band, 23.1 kb), lambda DNA (48.5 kb), and previously extracted genomic DNA obtained from the Corriel Institute (extracted by alternative methodologies). The size of the in-house extracted DNA was significantly larger than DNA extracted by other methodologies (e.g., Coriell gDNA 18996), with the majority of runs exceeding the 48.5 kb lambda DNA. Further enrichment of high molecular weight DNA was performed using the Short Read Eliminator Kit (Circulomics, Madison Wis.).

[0173] CRISPR / Cas9 enrichment and library preparation

[0174] CRISPR / Cas9 enrichment with the above sgRNAs was performed using a modified version of the Nanopore Cas-mediated protocol (VNR_9084_v109_revK_04Dec2018). Modifications to the volume and concentration of sgRNA used in the process were made to achieve optimal results (specifically 33.3 μl of sgRNA (3 μM) per sgRNA). Amplicons were used to ligate adapters via a ligation protocol (SQK-LSK109) and run the prepared libraries for sequencing on the MinION sequencing platform (Oxford Nanopore, UK) and data analysis was performed.

[0175] Proof of concept

[0176] Sequencing using sgRNAs enriching the entire CYP2D6-CYP2D7-CYP2D8 region (chr22:42,122,115-42,161,317) confirms three important things: (1) the sgRNA design successfully captures the entire target region, (2) this strategy allows for significant enrichment of the entire ROI relative to off-target reads, and (3) this method provides the ability to successfully perform long-read sequencing of the entire ROI (approximately 40 kb).

[0177] As shown in Figure 11A, across the genome, significant sequence enrichment was observed only for chromosome 22 (chr22), which contains the targeted ROI. All other genomic regions showed minimal coverage. Further analysis of chr22 found that only the region containing the ROI was enriched and had >10x coverage (Figure 11B). In total, 121 of the 176 reads mapped to chr22 were full-length reads aligned with the ROI (68.75%). The average accuracy and identity per read for all chromosome 22 reads are shown in Figure 11B.

[0178] Execution Alignment and Time

[0179] The median aligned read length is about 39.35 kb (Figure 12A), indicating that the target design size was successfully sequenced and aligned. Notably, all aligned reads were captured within the first 2.5 hours of sequencing on the minION (Figure 12B). This indicates that the sequencing time using the method described herein can be significantly reduced compared to standard long-read sequencing run times. This is of great value in terms of both turnaround time and instrument throughput results.

[0180] IGV analysis

[0181] Further IGV analysis of the sequence data alignment showed that the sequence reads aligned to the correct genomic location (chr22:42,122,115-42,161,317) with uniform depth and coverage across the entire ROI. Figure 13 shows the IGV alignment of 121 38.5 kb reads aligned to the target CYP2D6 region. To further probe the specificity of this approach, sgRNA enrichment in the target region of the reverse DNA strand (+ or -) was performed and the sequence data alignment was compared to sgRNA enrichment in the original strand design. As shown in Figure 14, 100% sequence enrichment occurred in the ROI of either the CYP2D6-CYP2D7-CYP2D8 region (chr22:42,122,115-42,161,317 - shown in the alignment at the top of the figure) or the adjacent region (shown in the alignment at the bottom of the figure) depending on the sgRNA strand target. No overlap with adjacent off-target regions was observed depending on the design. This demonstrates two crucial points of this approach: (1) no significant off-target cut occurs within our designed ROI, and (2) the enrichment approach does not lead to significant shearing of the ROI.

[0182] Figure 15 shows a Sashimi plot showing sgRNA specificity for multiple complex structural arrangements. The plot shows aligned regions for four sequencing runs. Sequence data from the run, using sgRNAs designed to capture a region of interest (ROI) (chr22:42,122,115-41,161,320), includes four different structural events: (1) a deletion of CYP2D6 in one allele; (2) a tandem hybrid allele with CYP2D6 in one allele; (3) a duplication event in one allele; and (4) a deletion of CYP2D6 in one allele and a duplication of CYP2D6 in a second allele. This data demonstrates successful enrichment of structural variants in ROIs for all orientations of recombination involving CYP2D6 CNVs or D6 / D7 or D7 / D6 hybrid alleles, including those with upstream and downstream CYP2D6-like or CYP2D7-like regions. No off-target cuts occurred between the upstream region of CYP2D6 and the downstream region of CYP2D8, regardless of the structural variants present, overcoming the limitations of the design described in Figure 7 and validating the approach described in Figure 8.

[0183] Example 6 A nested CRISPR-Cas9 method for enriching genomic regions of interest

[0184] In this embodiment, nested CRISPR-Cas9 method is used to enrich (e.g., complex) genomic regions of interest.This method has many advantages over current methods, including: (1) increasing the specificity of enrichment to regions of interest; and (2) increasing the capacity of input DNA material to increase the overall enrichment of ROI.Figure 17 provides an example of the schematic diagram for carrying out nested enrichment as described herein.

[0185] In this example, the CRISPR-Cas9 reaction is carried out using the amount of genomic DNA desired for downstream use. The outer set of guide RNAs is designed to be up to 30 kb downstream and upstream of the region of interest (e.g., the CYP2D6 locus) that is targeted. The Cas9-guide RNA complex cuts the genomic region of interest from the genomic DNA, blocking the ends of the excised DNA fragments containing the region of interest. Exonuclease digestion is then carried out to digest unprotected DNA (e.g., DNA that does not contain the region of interest). Because the ends of the DNA fragments containing the genomic region of interest are protected from exonuclease digestion (e.g., by steric hindrance caused by the bound Cas9-guide RNA complex), the excised DNA fragments containing the region of interest are left intact. This step allows additional enrichment of the region of interest, thereby increasing specificity and the ability to use larger amounts of genomic DNA (e.g., >10 μg) than are typically used during Cas-based enrichment protocols.

[0186] After performing exonuclease digestion, the enriched large undigested fragments are used in a CRISPR-Cas9 reaction that uses an inner set of guide RNAs that target the desired region of interest of a size suitable for long-read sequencing. This step adds additional specificity to the first enrichment protocol and provides more ends of the region of interest for downstream library generation.

[0187] The efficiency of the nested CRISPR-Cas9 approach is shown in FIG. 18 for two representative sets of sgRNAs. As shown in FIG. 18, an initial enrichment was performed using two representative sets of outer gRNAs located either 10 kb upstream (set 1) or 20 kb upstream (set 2) of the inner gRNA cut site. No outer gRNA enrichment was performed on the uncut sample. The same sets of inner gRNAs were then used for set 1, set 2, and the uncut sample, and libraries were prepared as described above. As shown in FIG. 18, the fold enrichment observed relative to uncut was approximately 1.7-fold for set 2 and approximately 3.4-fold for set 1.

[0188] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein can be used to implement the embodiments of the present disclosure. It is intended that the following claims define the scope of the disclosure, and that methods and structures falling within the scope of these claims and their equivalents are covered thereby.

Claims

1. 1. A method for analyzing a genomic region of interest, comprising: a) contacting genomic DNA comprising the genomic region of interest with a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated endonuclease and a pair of outer guide RNAs (gRNAs), thereby generating a first excised fragment comprising the genomic region of interest; b) contacting the first excised fragment with a CRISPR-associated endonuclease and a pair of inner gRNAs, thereby generating a second excised fragment that includes the genomic region of interest; c) analyzing the genomic region of interest contained within the second excised fragment; A method comprising:

2. The method of claim 1, wherein the CRISPR-associated endonuclease and the outer gRNA pair of a) associate with and block the 5' and 3' ends of the first excised fragment.

3. 3. The method of claim 2, further comprising the step of contacting the product of a) with one or more exonucleases prior to b), such that background genomic DNA is digested and the first excised fragment is not digested.

4. 4. The method of claim 3, wherein the one or more exonucleases are selected from the group consisting of Exonuclease I, Exonuclease II, Exonuclease III, Exonuclease IV, Exonuclease V, Exonuclease VI, Exonuclease VII, Exonuclease VIII, and any combination thereof. (i) the pair of outer gRNAs comprises a first outer gRNA and a second outer gRNA; (ii) the first outer gRNA comprises a nucleotide sequence substantially complementary to a first nucleotide sequence present in the genomic DNA, and the second outer gRNA comprises a nucleotide sequence substantially complementary to a second nucleotide sequence present in the genomic DNA; (iii) the first nucleotide sequence and the second nucleotide sequence are different; and (iv) the first nucleotide sequence and the second nucleotide sequence flank the genomic region of interest; The method of claim 1.

6. 6. The method of claim 5, wherein the first nucleotide sequence, the second nucleotide sequence, or both, are present within the genomic DNA at a length up to about 100 kilobases from the genomic region of interest. (i) the pair of inner gRNAs comprises a first inner gRNA and a second inner gRNA; (ii) the first inner gRNA comprises a nucleotide sequence substantially complementary to a third nucleotide sequence present in the genomic DNA, and the second inner gRNA comprises a nucleotide sequence substantially complementary to a fourth nucleotide sequence present in the genomic DNA; (iii) the third nucleotide sequence and the fourth nucleotide sequence are different; (iv) the third nucleotide sequence and the fourth nucleotide sequence flank the genomic region of interest; (v) the third nucleotide sequence and the fourth nucleotide sequence are present in the genomic DNA at a base length closer to the genomic region of interest than the first nucleotide sequence and the second nucleotide sequence; and (vi) the base length of the second excised fragment is shorter than that of the first excised fragment; The method according to claim 5.

8. The analyzing step includes: (i) sequencing the genomic region of interest contained within the second excised fragment; (ii) genotyping the genomic region of interest contained within the second excised fragment; (iii) performing a structural analysis on the genomic region of interest contained within the second excised fragment; or (iv) any combination of (i) to (iii) The method of claim 1 , comprising:

9. The method of claim 1, wherein the genomic DNA is provided in an amount of about 10 μg or more. (i) prior to b), isolating the first excised fragment; (ii) prior to c), isolating the second excised fragment; or (iii) both (i) and (ii) The method of claim 1 further comprising:

11. The method of claim 1 , which does not involve DNA amplification.

12. 2. The method of claim 1, further comprising, prior to c), attaching one or more adaptors to the 5' end, the 3' end, or both, of the second excised fragment.

13. 2. The method of claim 1, wherein the CRISPR-associated endonuclease is Cas9 or a mutant thereof.

14. 2. The method of claim 1, wherein the genomic DNA is not fragmented, digested or sheared prior to a).

15. 2. The method of claim 1, wherein the genomic region of interest is a complex genomic region.

16. The complex genomic region (i) a gene of interest and one or more of its pseudogenes; (ii) one or more repeat regions; (iii) one or more overlaps; (iv) one or more insertions; (v) one or more inversions, (vi) one or more tandem repeats, (vii) one or more retrotransposons; (viii) a highly polymorphic locus, or (ix) any combination of (i) to (viii) 16. The method of claim 15, comprising:

17. 2. The method of claim 1, wherein the sequencing comprises long-read sequencing.

18. 18. The method of claim 17, wherein the long-read sequencing comprises single molecule real-time sequencing or nanopore sequencing.

19. 10. The method of claim 1, wherein the method does not involve either polymerase chain reaction (PCR) or isothermal amplification.

20. The method of claim 1 , wherein the genomic DNA is provided or obtained in a biological sample.

21. 21. The method of claim 20, wherein the biological sample comprises a body fluid, a solid tissue sample, or a diagnostic sample.

22. A kit for analyzing a genomic region of interest, comprising: a) Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-related endonucleases; b) i) a first outer gRNA comprising a nucleotide sequence substantially complementary to a first nucleotide sequence present upstream of the genomic region of interest in genomic DNA; ii) a second outer gRNA comprising a nucleotide sequence substantially complementary to a second nucleotide sequence present downstream of said genomic region of interest in genomic DNA; The outer gRNA pair containing c) iii) a first inner gRNA comprising a nucleotide sequence substantially complementary to a third nucleotide sequence present upstream of the genomic region of interest in genomic DNA; iv) a second, inner gRNA comprising a nucleotide sequence substantially complementary to a fourth nucleotide sequence present downstream of the genomic region of interest in the genomic DNA; The inner gRNA pair containing wherein the third nucleotide sequence and the fourth nucleotide sequence are present at a base length closer to the genomic region of interest on the genomic DNA than the first nucleotide sequence and the second nucleotide sequence. kit.

23. 1. A system for analyzing a genomic region of interest, comprising: (a) (i) contacting genomic DNA comprising the genomic region of interest with a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated endonuclease and a pair of outer guide RNAs (gRNAs), thereby generating a first excised fragment comprising the genomic region of interest; (ii) contacting the first excised fragment with a CRISPR-associated endonuclease and a pair of inner gRNAs, thereby generating a second excised fragment that includes the genomic region of interest; (iii) analyzing the genomic region of interest contained within the second excised fragment; at least one memory location configured to receive a data input including data generated by a method including: (b) a computer processor operably coupled to the at least one memory location, the computer processor being programmed to generate an output based on the data. A system including:

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