Polynucleotide Amplification Using the CRISPR-CAS System

Abstract

This application relates to polynucleotide amplification using CRISPR-CAS systems. A method for amplifying a target nucleic acid, the method comprising providing a system having a crRNA or a derivative thereof and a Cas protein or a variant thereof. The crRNA or the derivative thereof comprises a target-specific nucleotide region that is substantially complementary to a region of the target nucleic acid, and contacting the target nucleic acid with the system to form a complex.

Description

[0001] This application is a divisional application of an application with an application date of November 10, 2015, an application number of 201580072952.7, and a title of "Polynucleotide Amplification Using CRISPR-Cas Systems".

[0002] The present disclosure generally relates to methods for amplifying polynucleotides, and more particularly to methods for amplifying polynucleotides using CRISPR-Cas systems and their applications. Background

[0003] Nucleic acid amplification is a key step in many nucleic acid-based methods such as nucleic acid sequencing. Currently, most nucleic acid amplification methods in use, such as those used for cluster generation in next-generation sequencing, require both temperature cycling and fluid exchange. On the other hand, isothermal amplification can be time and energy efficient by eliminating the temperature ramp and equilibration time. Several isothermal amplification methods have been developed, such as isothermal amplification based on recombinase polymerase amplification (RPA). These isothermal amplification systems generally lack the desired speed and efficiency that are ideally suited for some applications. In addition, some systems require additional enzymes and reagents, including ATP. Accordingly, there remains a need in the art for convenient, rapid, and efficient isothermal nucleic acid amplification methods. The present disclosure addresses this need by providing methods for amplifying nucleic acids using CRISPR-Cas systems. Related advantages are also provided.

[0004] Clustered regularly interspaced short palindromic repeats (CRISPR) are involved in an interference pathway that protects cells from phages and conjugative plasmids in many bacteria and archaea (Marraffini and Sontheimer, 2010, Nat Rev Genet. 11(3):181-190). CRISPR consists of an array of short repeat sequences spaced by unique variable DNA sequences called spacers of similar size that typically originate from phage or plasmid DNA (Barrangou et al., 2007, Science 315:1709-12; Bolotin et al., 2005, Microbiology 151:2551-61; Mojica et al., 2005, J Mol Evol 60:174-82). Thus, CRISPR sequences provide an adaptive genetic record of past infections and can be transcribed into CRISPR RNAs (crRNAs) - small RNAs that target invasive nucleic acids (Marraffini and Sontheimer, 2010, Nat Rev Genet 11(3):181-190). CRISPR is typically associated with CRISPR-associated (Cas) genes that encode proteins related to CRISPR. Cas proteins can provide a mechanism for destroying invading foreign nucleic acids targeted by crRNAs. CRISPR together with Cas (CRISPR-associated) genes constitutes an adaptive immune system that provides acquired tolerance to invasive foreign nucleic acids in bacteria and archaea (Barrangou et al., 2007, Science 315:1709-12).

[0005] Overview

[0006] The present disclosure provides methods for amplifying polynucleotides, and more particularly relates to methods for amplifying target DNA sequences using a CRISPR-Cas system and their applications.

[0007] In one aspect, the present disclosure provides a method for amplifying a target double-stranded nucleic acid, the method comprising: (a) providing a system having: a clustered regularly interspaced short palindromic repeat (CRISPR) RNA (crRNA) or a derivative thereof, and a CRISPR-associated (Cas) protein or a variant thereof, wherein the crRNA or the derivative thereof comprises a target-specific nucleotide region complementary to a region of the first strand of the target double-stranded nucleic acid; (b) contacting the target double-stranded nucleic acid with the system to form a complex; (c) hybridizing a primer to the second strand of the target double-stranded nucleic acid, the primer comprising a sequence complementary to a region of the second strand of the target double-stranded nucleic acid, and (d) using a polymerase to extend a nucleic acid complementary to the second strand of the target double-stranded nucleic acid from the primer.

[0008] In some embodiments, the method provided herein further comprises repeating step (c) to step (d) one or more times, e.g., until a desired degree of amplification is achieved. In some embodiments, the method provided herein further comprises repeating step (a) to step (d) until a desired degree of amplification is achieved.

[0009] In some embodiments, the target nucleic acid provided herein is double-stranded DNA (dsDNA). In some embodiments, the target nucleic acid is double-stranded RNA (dsRNA).

[0010] In some embodiments, the system is a type I CRISPR-Cas system or a derivative thereof. In some embodiments, the system is a type II CRISPR-Cas system or a derivative thereof. In some embodiments, the system is a type III CRISPR-Cas system or a derivative thereof.

[0011] In some embodiments, the system further comprises a trans-activating crRNA (tracrRNA) or a derivative thereof. In some embodiments, the crRNA or the derivative thereof is a polynucleotide comprising a crRNA polynucleotide fused to a tracrRNA polynucleotide.

[0012] In some embodiments, the first strand of the target double-stranded nucleic acid comprises a sequence complementary to a 5'-NGG protospacer adjacent motif (PAM).

[0013] In some embodiments, the first strand of the target double-stranded nucleic acid comprises a universal sequence, and wherein the crRNA or the derivative thereof comprises a sequence complementary to a region of the universal sequence. In some embodiments, the primer comprises a sequence of a region of the universal sequence.

[0014] In some embodiments, the universal sequence has the sequence of SEQ ID No. 3. In some embodiments, the crRNA comprises the sequence of SEQ ID No. 7. In some embodiments, the universal sequence has the sequence of SEQ ID No. 5.

[0015] In some embodiments, the universal sequence has the sequence of SEQ ID No. 4. In some embodiments, the crRNA comprises the sequence of SEQ ID No. 8. In some embodiments, the universal sequence has the sequence of SEQ ID No. 6.

[0016] In some embodiments, the Cas protein or its variant is a Cas9 protein or its variant. In some embodiments, the Cas9 protein comprises two inactivated nuclease domains. In some embodiments, the two inactivated nuclease domains comprise a first mutation in the domain that cleaves the strand complementary to the crRNA and a second mutation in the domain that cleaves the strand non-complementary to the crRNA. In some embodiments, the first mutation is D10A and the second mutation is H840A. In some embodiments, the Cas protein or its variant is a Cascade protein or its variant. In some embodiments, the Cas protein or its variant is a Cas3 protein or its variant.

[0017] In some embodiments, the polymerase is a strand displacement polymerase. In some embodiments, the polymerase is selected from the group consisting of: Bst, Bsu, and Phi29.

[0018] In some embodiments, the methods provided herein further comprise: applying at least one transposase and a transposon end composition to a sample comprising a target nucleic acid under conditions in which the target nucleic acid and at least one transposon end composition comprising a transfer strand undergo a transposition reaction to produce a mixture, wherein the target nucleic acid is fragmented to produce a plurality of target nucleic acid fragments, and incorporating a universal primer sequence into each of the plurality of target nucleic acid fragments, wherein the crRNA or its derivative comprises a target-specific nucleotide region complementary to a region of the universal primer.

[0019] In some embodiments, the universal primer is incorporated into the plurality of target nucleic acid fragments by a PCR reaction. In some embodiments, the universal primer has a sequence complementary to the 5'-NGG protospacer adjacent motif (PAM).

[0020] In some embodiments, the universal sequence has the sequence of SEQ ID No. 3. In some embodiments, the crRNA comprises the sequence of SEQ ID No. 7. In some embodiments, the universal sequence has the sequence of SEQ ID No. 5.

[0021] In some embodiments, the universal primer has the sequence of SEQ ID No. 4. In some embodiments, the crRNA comprises the sequence of SEQ ID No. 8. In some embodiments, the universal sequence has the sequence of SEQ ID No. 6.

[0022] In some embodiments, two universal primers are incorporated into both ends of each of a plurality of target nucleic acid fragments. In some embodiments, the two universal primers have the sequences of SEQ ID No.3 and SEQ ID No.4. In some embodiments, the two universal primers have the sequences of SEQ ID No.5 and SEQ ID No.6.

[0023] In some embodiments, the target double-stranded nucleic acid is linearly amplified. In some embodiments, the target double-stranded nucleic acid is exponentially amplified.

[0024] In some embodiments, provided herein is a method for amplifying a target double-stranded nucleic acid, the method comprising: (a) providing a first system having: a first clustered regularly interspaced short palindromic repeat (CRISPR) RNA (crRNA) or a derivative thereof, and a first CRISPR-associated (Cas) protein or a variant thereof, wherein the first crRNA or its derivative comprises a target-specific nucleotide region complementary to a region of the first strand of the target double-stranded nucleic acid; (b) providing a second system having:

[0025] a second clustered regularly interspaced short palindromic repeat (CRISPR) RNA (crRNA) or a derivative thereof, and a second CRISPR-associated (Cas) protein or a variant thereof, wherein the second crRNA or its derivative comprises a target-specific nucleotide region complementary to a region of the second strand of the target double-stranded nucleic acid; (c) contacting the target double-stranded nucleic acid with the first system and the second system;

[0026] (d) hybridizing a first primer to the second strand of the target double-stranded nucleic acid, the first primer comprising a sequence complementary to a region of the second strand of the target double-stranded nucleic acid, and hybridizing a second primer to the first strand of the target double-stranded nucleic acid, the second primer comprising a sequence complementary to a region of the first strand of the target double-stranded nucleic acid, and (e) extending the 3'-ends of the first primer and the second primer with one or more polymerases to produce first and second double-stranded target nucleic acids. In some embodiments, the method provided herein further comprises repeating step (a) and step (e) one or more times, e.g., until a desired degree of amplification is achieved.

[0027] In some embodiments, the target nucleic acid is double-stranded DNA (dsDNA). In some embodiments, the target nucleic acid is double-stranded RNA (dsRNA).

[0028] In some embodiments, the first system or the second system is a type I CRISPR-Cas system or a derivative thereof. In some embodiments, the first system or the second system is a type II CRISPR-Cas system or a derivative thereof. In some embodiments, the first system or the second system is a type III CRISPR-Cas system or a derivative thereof.

[0029] In some embodiments, the first system or the second system further comprises a trans-activating crRNA (tracrRNA) or a derivative thereof. In some embodiments, the crRNA or a derivative thereof of the first system or the second system is a polynucleotide comprising a crRNA polynucleotide fused to a tracrRNA polynucleotide. In some embodiments, the first strand and the second strand of the target double-stranded nucleic acid comprise sequences complementary to a 5'-NGG protospacer adjacent motif (PAM).

[0030] In some embodiments, the first strand of the target double-stranded nucleic acid comprises a first universal sequence, and wherein the crRNA or a derivative thereof of the first system comprises a sequence complementary to a region of the first universal sequence, and the second strand of the target double-stranded nucleic acid comprises a second universal sequence, and wherein the crRNA or a derivative thereof of the second system comprises a sequence complementary to a region of the second universal sequence.

[0031] In some embodiments, the first primer comprises a sequence of a region of the first universal sequence, and the second primer comprises a sequence of a region of the second universal sequence. In some embodiments, the first universal sequence (which comprises the first primer) has the sequence of SEQ ID No.3, the crRNA or a derivative thereof of the first system comprises the sequence of SEQ ID No.7, and the first primer comprises the sequence of SEQ ID No.5, and the second universal sequence (which comprises the second primer) has the sequence of SEQ ID No.4, the crRNA or a derivative thereof of the second system comprises the sequence of SEQ ID No.8, and the second primer comprises the sequence of SEQ ID No.6.

[0032] In some embodiments, the Cas protein or a variant thereof of the first system or the second system is a Cas9 protein or a variant thereof. In some embodiments, the Cas9 protein comprises two inactivated nuclease domains. In some embodiments, the two inactivated nuclease domains comprise a first mutation in the domain that cleaves the strand complementary to the crRNA and a second mutation in the domain that cleaves the strand non-complementary to the crRNA. In some embodiments, the first mutation is D10A, and the second mutation is H840A. In some embodiments, the Cas protein or a variant thereof of the first system or the second system is a cascade protein or a variant thereof. In some embodiments, the Cas protein or a variant thereof of the first system or the second system is a Cas3 protein or a variant thereof.

[0033] In some embodiments, the polymerase is a strand displacement polymerase. In some embodiments, the polymerase is selected from the group consisting of: Bst, Bsu, and Phi29.

[0034] In some embodiments, the target nucleic acid is genomic DNA. In some embodiments, the target nucleic acid comprises chromosomal DNA or a fragment thereof. In some embodiments, the target nucleic acid comprises a genome or a portion of a genome.

[0035] In some embodiments, the methods provided herein further comprise sequencing the target nucleic acid or a fragment of the target nucleic acid. In some embodiments, the sequencing comprises using one or more of sequencing by synthesis, bridge PCR, chain termination sequencing, hybridization sequencing, nanopore sequencing, and ligation sequencing. Sequence Listing <110> Illumina, Inc. <120> Polynucleotide Amplification Using CRISPR-CAS Systems <130> 12957-170-228 <140> To be assigned <141> 2015-11-10 <150> 62 / 078,355 <151> 2014-11-11 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Standard P5 sequence <400> 1 aatgatacgg cgaccaccga gatctacac 29 <210> 2 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Standard P7 sequence <400> 2 caagcagaag acggcatacg agat 24 <210> 3 <211> 32 <212> DNA <213> Artificial sequence <220> <223> PAM-modified P5 sequence <220> <221> misc_feature <222> (3)..(3) <223> n is a, c, g or t <400> 3 ccnaatgata cggcgaccac cgagatctac ac 32 <210> 4 <211> 27 <212> DNA <213> Artificial sequence <220> <223> PAM-modified P7 sequence <220> <221> misc_feature <222> (3)..(3) <223> n is a, c, g or t <400> 4 ccncaagcag aagacggcat acgagat 27 <210> 5 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Truncated PAM-modified P5 sequence <220> <221> misc_feature <222> (3)..(3) <223> n is a, c, g or t <400> 5 ccnaatgata cggcgaccac c 21 <210> 6 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Truncated PAM-modified P7 sequence <220> <221> misc_feature <222> (3)..(3) <223> n is a, c, g or t <400> 6 ccncaagcag aagacggcat a 21 <210> 7 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Guide RNA targeting P5 <400> 7 ucgguggucg ccguaucauu 20 <210> 8 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Guide RNA targeting P7 <400> 8 cguaugccgu cuucugcuug 20 Brief description of the drawings

[0036] Figure 1A Primers for amplifying DNA fragments designed according to the present method are shown. The primers contain all or part of the Illumina universal sequencing primer adaptors P5 and P7. The Nextera (Illumina, Inc.) library preparation method can be used to add the primers. Figure 1BIllustrated is one round of Cas9-mediated linear amplification of crRNA using a targeted modified P5 primer. 1B(I) depicts the DNA to be amplified having appropriate primer sequences P5 and P7. The guide RNA targeting P5 bound to Cas9 is also shown as P5'. 1B(II) shows the R-loop created by Cas9 after the guide RNA hybridizes to the first strand. 1B(III) illustrates the immobilized P5 primer hybridizing to the displaced second strand, followed by polymerase extension. 1B(IV) shows the resulting extended primer P5. As shown in step 1B(II), the resulting amplicon can be re-targeted by Cas9+crRNA. Figure 1C Illustrated is one round of Cas9-mediated linear amplification of crRNA using a targeted modified P7 primer. 1C(I) depicts the DNA to be amplified having appropriate primer sequences P5 and P7. The guide RNA targeting P7 bound to Cas9 is also shown as P7'. 1C(II) shows the R-loop created by Cas9 after the guide RNA hybridizes to the first strand. 1C(III) illustrates the immobilized P7 primer hybridizing to the displaced second strand, followed by polymerase extension. 1C(IV) shows the resulting extended primer P7. As shown in step 1C(II), the resulting amplicon can be re-targeted by Cas9+crRNA. As the two sides of the amplicon undergo amplification ( Figure 1B and 1C ), exponential amplification can be achieved. DETAILED DESCRIPTION

[0037] The present disclosure provides methods for rapidly and efficiently amplifying target nucleic acids using a CRISPR-Cas system.

[0038] Nucleic acid amplification is a step in many nucleic acid-based methods such as next-generation sequencing. Currently, polymerase chain reaction (PCR) is the most widely used method for DNA amplification for, e.g., detecting and identifying infectious diseases, genetic disorders, and other research purposes. The PCR reaction typically uses two oligonucleotide primers that hybridize to the 3' ends of a double-stranded target nucleic acid sequence and a DNA polymerase that can extend the annealed primers by adding deoxyribonucleoside triphosphates (dNTPs) to produce a double-stranded nucleic acid product. Gill and Ghaemi, Nucleosides, Nucleotides, and Nucleic Acids, 2008, 27:224-243. However, the PCR reaction requires thermal cycling to separate the two DNA strands. Similarly, many currently used nucleic acid amplification methods, such as those used for cluster generation in next-generation sequencing, require both temperature cycling and fluid exchange.

[0039] Several isothermal amplification methods have been developed to eliminate temperature ramps and equilibration times, such as recombinase polymerase amplification (RPA)-based isothermal amplification, transcription-mediated amplification, nucleic acid sequence-based amplification, signal-mediated RNA amplification, strand displacement amplification, rolling circle amplification, loop-mediated DNA amplification, isothermal multiple displacement amplification, helicase-dependent amplification, single primer isothermal amplification, and circular helicase-dependent amplification, as described in Gill and Ghaemi, Nucleosides, Nucleotides, and Nucleic Acids, 2008, 27:224-243.

[0040] For example, in transcription-mediated amplification (TMA), an RNA polymerase is used to prepare RNA from a promoter built into the primer region, and then a reverse transcriptase synthesizes cDNA from the primer. Then, a third enzyme, e.g., ribonuclease H, can be used to degrade the target RNA from the cDNA without a heat denaturation step. This amplification technique is very similar to self-sustained sequence replication (3SR) and nucleic acid sequence-based amplification (NASBA), but uses different enzymes. Ibid. As another example, helicase-dependent amplification (HDA) utilizes a thermostable helicase (Tte-UvrD) without heating to unwind dsDNA to create single strands, which can then be used to hybridize and extend primers by a polymerase. It has been shown that the reaction time for products with an amplified length of 70-120 base pairs exceeds 1 hour. Ibid. As yet another example, loop-mediated amplification (LAMP) employs a thermostable polymerase with strand displacement ability and a set of four or more specially designed primers. Each primer is designed to have a hairpin end that snaps into a hairpin after being displaced to facilitate self-priming and further polymerase extension. In the LAMP reaction, although the reaction is carried out under isothermal conditions, the double-stranded target requires an initial heat denaturation step. Additionally, the amplification produces products of various lengths in a ladder pattern. Ibid. As yet another example, strand displacement amplification (SDA) combines the ability of a restriction endonuclease to nick the unmodified strand of its target DNA with the ability of an exonuclease-deficient DNA polymerase to extend the 3' end at the nick and displace the downstream DNA strand. Ibid. Other exemplary isothermal amplification methods include, but are not limited to, those described in Craw and Balachandran, Lab Chip, 2012, 12:2469-2486.

[0041] However, these currently developed isothermal amplification systems generally lack the desired speed and efficiency that are ideally suited for some applications. In addition, some systems require additional enzymes and reagents, including ATP. Thus, there remains a need in the art for convenient, rapid, and effective isothermal nucleic acid amplification methods. The present disclosure addresses this need by providing methods for amplifying nucleic acids using CRISPR-Cas systems. For example, one advantage provided by the methods herein is that Cas proteins recognize target nucleic acids without consuming ATP or energy input, and thus the methods herein provide cost- and time-effective isothermal amplification methods.

[0042] Definitions

[0043] As used herein, the terms “includes,” “including,” “includes,” “including,” “contains,” “containing,” “have,” “having,” and any variations thereof are intended to cover non-exclusive inclusion such that a recited method, method, method-defined product, or composition that includes, includes, or contains an element or list of elements does not include only those elements but may include other elements not expressly listed or other elements inherent to such recited method, method, method-defined product, or composition.

[0044] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural referents. Thus, for example, reference to “a protein” includes mixtures of two or more proteins, and the like.

[0045] As used herein, the term “about” or “approximately” means within 5% of a given value or range.

[0046] As used herein, the term “nucleic acid” means single-stranded polymers and double-stranded polymers of nucleotide monomers, including 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by internucleotide phosphodiester bonds or internucleotide analog linkages, and associated counterions such as H + , NH 4+ , trialkylammonium, tetraalkylammonium, Mg 2+ , Na +etc. The nucleic acid can be a polynucleotide or an oligonucleotide. The nucleic acid can be composed entirely of deoxyribonucleotides, composed entirely of ribonucleotides, or can be a chimeric mixture thereof. The nucleotide monomer units can include any nucleotide described herein, including but not limited to naturally occurring nucleotides and nucleotide analogs. The size of the nucleic acid generally ranges from a few monomer units such as 5 - 40 to several thousand monomer nucleotide units. Nucleic acids include but are not limited to genomic DNA, cDNA, hnRNA, mRNA, rRNA, tRNA, fragmented nucleic acids, nucleic acids obtained from subcellular organelles such as mitochondria or chloroplasts, and nucleic acids obtained from microorganisms or DNA or RNA viruses that may be present on or in a biological sample.

[0047] As used herein, the term "target nucleic acid" is intended to mean a nucleic acid that is the object of analysis or action. Analysis or action includes subjecting the nucleic acid to procedures such as copying, amplification, sequencing, and / or other procedures for nucleic acid interrogation. The target nucleic acid can include nucleotide sequences other than the target sequence to be analyzed. For example, the target nucleic acid can contain one or more adapters, including adapters that flank the target nucleic acid sequence to be analyzed and function as primer binding sites. The target nucleic acid hybridized to a capture oligonucleotide or a capture primer can contain nucleotides that extend beyond the 5' or 3' end of the capture oligonucleotide in such a way that not all of the target nucleic acid is suitable for extension.

[0048] As used herein, the term "target specificity" when referring to guide RNAs, crRNAs, or their derivatives or other nucleotides is intended to mean a polynucleotide that contains a nucleotide sequence specific for a target polynucleotide sequence, i.e., a nucleotide sequence capable of selectively annealing to an identified region of the target polynucleotide (e.g., target DNA). The target-specific nucleotides can be of a single kind of oligonucleotide, or the target-specific nucleotides can include two or more kinds of oligonucleotides having different sequences. Thus, the target-specific nucleotides can be two or more sequences, including 3, 4, 5, 6, 7, 8, 9, or 10 or more different sequences. In one embodiment, the crRNA or its derivative contains a target-specific nucleotide region complementary to a region of the target DNA sequence. In one embodiment, the crRNA or its derivative can contain other nucleotide sequences in addition to the target-specific nucleotide region. In one embodiment, the other nucleotide sequences can be from the tracrRNA sequence.

[0049] As used herein, when referring to the use of polynucleotides, the term "complementary" is intended to mean a polynucleotide comprising a nucleotide sequence capable of selectively annealing, under certain conditions, to an identified region of a target polynucleotide. As used herein, the terms "substantially complementary" and grammatical equivalents are intended to mean a polynucleotide comprising a nucleotide sequence capable of specifically annealing, under certain conditions, to an identified region of a target polynucleotide. Annealing refers to the nucleobase pairing interaction of one nucleic acid with another nucleic acid, which results in the formation of a duplex, triplex, or other higher order structure. The primary interactions are generally nucleobase specific through Watson-Crick and Hoogsteen type hydrogen bonding, e.g., A:T, A:U, and G:C. In certain embodiments, base stacking and hydrophobic interactions can also contribute to duplex stability. The conditions for annealing of a polynucleotide to a complementary or substantially complementary region of a target nucleic acid are well known in the art, e.g., the conditions described in: Nucleic Acid Hybridization, A Practical Approach, Hames and Higgins, eds., IRL Press, Washington, D.C. (1985) and Wetmur and Davidson, Mol. Biol. 31:349 (1968). The annealing conditions will depend on the particular application and can be routinely determined by one of ordinary skill in the art without undue experimentation.

[0050] As used herein, the term "hybridization" refers to the process by which two single-stranded polynucleotides non-covalently bind to form a stable double-stranded polynucleotide. The resulting double-stranded polynucleotide is a "hybrid" or "duplex". Hybridization conditions will generally include a salt concentration of less than about 1 M, more typically less than about 500 mM, and can be less than about 200 mM. Hybridization buffers include buffer salt solutions such as 5% SSPE or other such buffers known in the art. The hybridization temperature can be as low as 5°C, but is typically greater than 22°C, more typically greater than about 30°C, and usually exceeds 37°C. Hybridization is typically carried out under stringent conditions, i.e., conditions under which the probe will hybridize to its target sequence, but will not hybridize to other non-complementary sequences. Stringent conditions are sequence-dependent and will be different in different circumstances and can be routinely determined by one of ordinary skill in the art.

[0051] In the context of "polynucleotide", as used herein, the terms "variant" and "derivative" refer to such polynucleotides that contain a nucleotide sequence or polynucleotide fragment of a polynucleotide that has been altered by the introduction of nucleotide substitutions, deletions or additions. A variant or derivative of a polynucleotide can be a fusion polynucleotide that comprises a portion of the nucleotide sequence of the polynucleotide. The terms "variant" or "derivative" as used herein also refer to, for example, a polynucleotide or a fragment thereof that has been chemically modified by covalent attachment of any type of molecule to the polynucleotide. For example, but not limited to, a polynucleotide or a fragment thereof can be chemically modified, such as by acetylation, phosphorylation, methylation, and the like. The variant or derivative is modified in a manner such that the type or location of the attached molecule is different from that of the naturally occurring or starting nucleotide or polynucleotide. Variants or derivatives also include the deletion of one or more chemical groups that are naturally present on the nucleotide or polynucleotide. Variants or derivatives of a polynucleotide or polynucleotide fragment can be chemically modified by chemical alteration using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, and the like. In addition, variants or derivatives of a polynucleotide or polynucleotide fragment can contain one or more dNTPs or nucleotide analogs. Polynucleotide variants or derivatives can have functions similar or identical to those of the polynucleotides or polynucleotide fragments described herein. Compared to the polynucleotides or polynucleotide fragments described herein, polynucleotide variants or derivatives can have additional or different functions.

[0052] As used herein, the term "dNTP" refers to deoxynucleoside triphosphate. NTP refers to ribonucleoside triphosphate, such as those used for the synthesis of crRNA or tracrRNA. Purine bases (Pu) include adenine (A), guanine (G), and their derivatives and analogs. Pyrimidine bases (Py) include cytosine (C), thymine (T), uracil (U), and their derivatives and analogs. By way of illustration and not limitation, examples of such derivatives or analogs are those modified with a reporter group, biotinylated, amine-modified, radiolabeled, alkylated, etc., and also include phosphorothioates, phosphites, derivatives with modified ring atoms, etc. Reporter groups can be fluorescent groups such as fluorescein, chemiluminescent groups such as luminol, terbium chelates such as N-(hydroxyethyl)ethylenediaminetriacetic acid capable of detection by delayed fluorescence, etc.

[0053] As used herein, the term "nucleotide analog" refers to a synthetic analog having a modified nucleobase moiety, a modified pentose moiety, and / or a modified phosphate moiety, and in the case of polynucleotides, a modified internucleoside linkage, as is generally described elsewhere (e.g., Scheit, Nucleotide Analogs, John Wiley, New York, 1980; Englisch, Angew. Chem. Int. Ed. Engl. 30:613-29, 1991; Agarwal, Protocols for Polynucleotides and Analogs, Humana Press, 1994; and S. Verma and F. Eckstein, Ann. Rev. Biochem. 67:99-134, 1998). Exemplary phosphoester analogs include, but are not limited to, phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoranilidates, phosphoramidates, boronophosphates, including associated counterions such as H + , NH4 + , Na +(If such counterions are present). Exemplary modified nucleobase moieties include, but are not limited to, 5-methylcytosine (5mC); C-5-propynyl analogs, including but not limited to C-5 propynyl-C and C-5 propynyl-U; 2,6-diaminopurine, also known as 2-aminoadenine or 2-amino-dA); inosine, pseudouridine, 2-thiopyrimidine, isocytosine (isoC), 5-methylisoC, and isoguanine (isoG; see, e.g., U.S. Patent No. 5,432,272). Exemplary modified pentose moieties include, but are not limited to, locked nucleic acid analogs including but not limited to Bz-A-LNA, 5-Me-Bz-C-LNA, dmf-G-LNA, and T-LNA (see, e.g., The Glen Report, 16(2):5, 2003; Koshkin et al., Tetrahedron 54:3607-30, 1998) and 2'- or 3'-modifications, where the 2'- or 3'-position is hydrogen, hydroxy, alkoxy (e.g., methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy, and phenoxy), azido, amino, alkylamino, fluoro, chloro, or bromo. Modified internucleotide linkages include phosphorothioate analogs, analogs having achiral and uncharged subunit linkages (e.g., Sterchak, E.P. et al., Organic Chern., 52:4202, 1987), and uncharged morpholine-based polymers having achiral subunit linkages (see, e.g., U.S. Patent No. 5,034,506). Some internucleotide linkage analogs include morpholidate, acetal, and polyamide-linked heterocycles.

[0054] As used herein, the term "polymerase chain reaction" or "PCR" refers to a procedure in which a small amount of nucleic acid, e.g., RNA and / or DNA, is amplified, as described, e.g., in U.S. Patent No. 4,683,195 to Mullis. Generally, sequence information from the ends or outside of the region of interest needs to be available such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to the opposite strands of the template to be amplified. The 5'-terminal nucleotides of the two primers can coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage, or plasmid sequences transcribed from total cellular RNA, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263 (1987); Erlich, ed., PCR Technology, (Stockton Press, NY, 1989).

[0055] As used herein, the terms "ligation," "ligating," and their grammatical equivalents are intended to mean the formation of a covalent bond or linkage between the ends of two or more nucleic acids (e.g., oligonucleotides and / or polynucleotides), typically in a template-driven reaction. The nature of the bond or linkage can vary widely, and ligation can be carried out enzymatically or chemically. As used herein, ligation is typically carried out enzymatically to form a phosphodiester linkage between the 5'-carbon terminal nucleotide of one oligonucleotide and the 3'-carbon of another nucleotide. Template-driven ligation reactions are described in the following references: U.S. Patent Nos. 4,883,750; 5,476,930; 5,593,826; and 5,871,921, which are incorporated herein by reference in their entirety. The term "ligation" also includes the non-enzymatic formation of phosphodiester bonds, as well as the formation of non-phosphodiester covalent bonds between the ends of oligonucleotides, such as phosphorothioate bonds, disulfide bonds, and the like.

[0056] As used herein, the term "adapter" is a single-stranded or double-stranded nucleic acid molecule that can be ligated to the ends of other nucleic acids. In one embodiment, an adapter is a short chemically synthesized double-stranded nucleic acid molecule that can be used to ligate the ends of two other nucleic acid molecules. In one embodiment, an adapter is a double-stranded nucleic acid (e.g., oligonucleotide) that contains single-stranded nucleotide overhangs at the 5' and / or 3' ends. In some embodiments, the single-stranded overhangs are 1, 2, or more nucleotides. In some embodiments, an adapter contains additional nucleic acid sequences for cloning or analyzing "inserts." In some embodiments, an adapter includes a label or affinity tag for analyzing or purifying an "insert." The term "insert" refers to a nucleic acid sequence of interest. In some embodiments, an insert is a double-stranded DNA that contains single-stranded nucleotide overhangs at the 5' end and / or 3' end. In some embodiments, the single-stranded overhangs are 1, 2, or more nucleotides.

[0057] As used herein, the term "CRISPR-Cas system" refers to an enzymatic system that includes a guide RNA sequence comprising a nucleotide sequence that is complementary or substantially complementary to a region of a target polynucleotide and a protein having nuclease activity. CRISPR-Cas systems include type I CRISPR-Cas systems, type II CRISPR-Cas systems, type III CRISPR-Cas systems, and their derivatives. CRISPR-Cas systems include engineered and / or programmed nuclease systems derived from naturally occurring CRISPR-Cas systems. A CRISPR-Cas system can comprise an engineered and / or mutated Cas protein. A CRISPR-Cas system can comprise an engineered and / or programmed guide RNA.

[0058] As used herein, the term "guide RNA" refers to an RNA that contains a sequence that is complementary or substantially complementary to a region of a target DNA sequence. The guide RNA can contain nucleotide sequences other than the region that is complementary or substantially complementary to the region of the target DNA sequence. The guide RNA can be a crRNA or a derivative thereof, e.g., a crRNA:tracrRNA chimera.

[0059] As used herein, the term "nuclease" refers to an enzyme capable of cleaving the phosphodiester bond between the nucleotide subunits of a nucleic acid; the term "endonuclease" refers to an enzyme capable of cleaving the phosphodiester bond within a polynucleotide chain; and the term "nickase" refers to an endonuclease that cleaves only one strand of a DNA duplex. The term "Cas9 nickase" refers to a nickase obtained from a Cas9 protein, typically by inactivating one of the nuclease domains of the Cas9 protein.

[0060] In the context of a polypeptide, as used herein, the terms "variant" and "derivative" refer to a polypeptide that contains the amino acid sequence of a polypeptide or polypeptide fragment that has been altered by the introduction of amino acid residue substitutions, deletions, or additions. A variant or derivative of a polypeptide can be a fusion protein that contains a portion of the amino acid sequence of the polypeptide. As used herein, the terms "variant" or "derivative" also refer to a polypeptide or polypeptide fragment that has been chemically modified, e.g., by covalent attachment of any type of molecule to the polypeptide. For example, but not limited to, a polypeptide or polypeptide fragment can be chemically modified, e.g., by glycosylation, acetylation, polyethylene glycolylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, conjugation to a cellular ligand or other protein, etc. A variant or derivative is modified in a way that is different from the native or starting peptide or polypeptide in the type or location of the attached molecule. A variant or derivative also includes the deletion of one or more chemical groups that are naturally present on the peptide or polypeptide. A variant or derivative of a polypeptide or polypeptide fragment can be chemically modified by chemical modification using techniques known to those of skill in the art, which include, but are not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis with tunicamycin, etc. In addition, a variant or derivative of a polypeptide or polypeptide fragment can contain one or more non-classical amino acids. A polypeptide variant or derivative can have a function that is similar or identical to the polypeptide or polypeptide fragment described herein. A polypeptide variant or derivative can have additional or different functions compared to the polypeptide or polypeptide fragment described herein.

[0061] As used herein, the term "detecting" a nucleic acid molecule or a fragment thereof refers to determining the presence of the nucleic acid molecule, typically when the nucleic acid molecule or a fragment thereof is completely or partially separated from other components of a sample or composition, and can also include determining the charge-to-mass ratio, mass, quantity, absorbance, fluorescence, or other properties of the nucleic acid molecule or a fragment thereof.

[0062] As used herein, the term "primer" refers to a natural or synthetic oligonucleotide primer that can serve as an initiation point for nucleic acid synthesis when placed under conditions where primer extension (not limited to the number of extended bases) is initiated. The primer can be a single-stranded oligodeoxyribonucleotide. The length of the primer can range from about 10 to 50 nucleotides, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides. The primer does not need to reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with the template so that primer extension occurs. If desired, the primer can be labeled by, for example, spectroscopic, photochemical, biochemical, immunochemical, or chemical means by incorporating a detectable label. Exemplary labels include, but are not limited to, biotin, amines, radiolabels (e.g., 32P), fluorescent dyes, electron-dense reagents, enzymes (commonly used in ELISAs), or biotin.

[0063] As used herein, the term "linear amplification" refers to an amplification process that amplifies a target nucleic acid using multiple cycles of primer extension reactions. With linear amplification, the abundance of the transcript increases proportionally with the number of cycles and is linearly amplified. Examples of linear amplification procedures are LCR, the aRNA method of Phillips and Eberwine above, and the linear amplification methods described herein. Different from exponential amplification, the amount of the amplification product does not increase exponentially. For example, in an ideal 4-hour linear amplification reaction with a copy ratio of 2000 copies / minute, 2000 copies of template DNA will produce 960,000,000 copies.

[0064] As used herein, the term "exponential amplification" refers to an amplification procedure in which the product (i.e., amplicon) doubles with each reaction cycle. "Exponential amplification" is a non-linear amplification that results in an exponential increase in the number of copies of the nucleic acid present. For example, exponential amplification can occur when primer extension starts from both ends of the amplicon in one amplification cycle. For example, PCR is an exponential amplification procedure. For example, in an ideal PCR reaction with 30 cycles, 2 copies of template DNA will produce 2^30 or 1,073,741,824 copies.

[0065] As used herein, the term "polymerase" refers to a protein capable of catalyzing the specific incorporation of nucleotides to extend the 3' hydroxyl terminus of a primer molecule (such as, for example, a template oligonucleotide) against a nucleic acid target sequence. The polymerase can be, for example, thermophilic such that it is active at elevated reaction temperatures. For example, it can also have strand displacement ability.

[0066] Method for amplifying polynucleotides

[0067] In one aspect, the present disclosure provides a method for CRISPR-Cas system-mediated amplification. The method provided herein is based in part on the binding of a guide RNA to a region of a target double-stranded nucleic acid disrupting the interaction between the two strands of the target nucleic acid and thereby creating a loop structure (also referred to as an "R-loop"), exposing the strand that is not complementary to the guide RNA. This exposed strand can undergo hybridization with a primer and be extended, for example, by a suitable polymerase during nucleic acid amplification. As Figures 1A-1C explained, this loop structure created by a CRISPR-Cas system (such as a system comprising Cas9 or a cascade protein) can be accessed by other enzymes. Thus, the loop structure can further be used as a template to initiate primer hybridization and interact with a polymerase for amplification.

[0068] In some embodiments, the present disclosure provides a method for amplifying a target double-stranded nucleic acid, the method comprising providing a system having: a clustered regularly interspaced short palindromic repeat (CRISPR) RNA (crRNA) or a derivative thereof, and a CRISPR-associated (Cas) protein or a variant thereof, wherein the crRNA or its derivative comprises a target-specific nucleotide region complementary to a region of the first strand of the target double-stranded nucleic acid; contacting the target double-stranded nucleic acid with the system to form a complex; hybridizing a primer to the second strand of the target double-stranded nucleic acid, the primer comprising a sequence complementary to a region of the second strand of the target double-stranded nucleic acid, and using a polymerase to extend a nucleic acid complementary to the second strand of the target double-stranded nucleic acid from the primer.

[0069] The methods provided herein can be used in a variety of amplification methods, including but not limited to, linear nucleic acid amplification and exponential nucleic acid amplification.

[0070] In some embodiments, the target nucleic acid is linearly amplified according to the methods provided herein. For example, in some embodiments, the methods provided herein further comprise repeating the following one or more times, such as until a desired amplification amount is achieved: hybridizing a primer to the second strand of the target double-stranded nucleic acid and using a polymerase to extend a nucleic acid complementary to the second strand of the target double-stranded nucleic acid from the primer.

[0071] In other embodiments, the target nucleic acid is exponentially amplified. In an exemplary exponential nucleic acid amplification, the product (i.e., the amplicon) doubles with each reaction cycle. "Exponential amplification" is a non-linear amplification that results in an exponential increase in the number of copies of the nucleic acid present. Typically, in exponential amplification, primer extension (or copying) occurs from both ends of the amplicon. To ensure that the newly created strands have primer binding sites at both ends, in exponential amplification, the 3' end of the newly synthesized nucleic acid contains the reverse complement of the primer, and the ends of the template are typically copied in each amplification cycle.

[0072] The number of cycles of the reaction according to the method depends on the application and the desired amount of amplification product. In some embodiments, the number of cycles is from 5 to 100. In some embodiments, the number of cycles is from 10 to 90. In some embodiments, the number of cycles is from 20 to 80. In some embodiments, the number of cycles is from 30 to 70. In some embodiments, the number of cycles is from 40 to 60. Exemplary numbers of cycles include 10, 15, 20, 25, 30, 35, 40, 45, and 50. The cycles do not need to be synchronized between amplicons because they are in a PCR reaction where the initiation of each cycle is controlled by changing the temperature. Thus, as used herein, a cycle refers to the average number of amplification rounds that an amplicon undergoes.

[0073] In some embodiments, the initialization step involves providing multiple CRISPR-Cas systems to the target nucleic acid to open the double-stranded nucleic acid structure at two or more target sequences and form two or more R-loop structures. The initialization step does not require heating the reaction as needed in a PCR reaction because the initialization step is enzyme-driven. The next step according to the method involves providing primers that target the R-loop region and annealing the primers to form a relatively stable nucleic acid-nucleic acid interaction, e.g., a DNA-DNA hybrid. Generally, a stable nucleic acid-nucleic acid hybrid is formed when the primer sequence has substantial complementarity to the template sequence. Then one or more polymerases bind to the primer-template hybrid and initiate nucleic acid synthesis in an extension / elongation step. In some embodiments, the temperature of this extension / elongation step depends on the polymerase used. At this step, the polymerase synthesizes a new nucleic acid strand complementary to the template strand by adding dNTPs that are complementary to the template. In some embodiments, when using a DNA polymerase, the reaction condenses the 5'-phosphate group of the dNTP with the 3'-hydroxyl group at the end of the nascent (extended) DNA strand. The extension time depends on both the polymerase used and the length of the nucleic acid fragment to be amplified. One or more of these steps can be repeated one or more times, e.g., until the desired amount of amplification is achieved.

[0074] To allow for exponential growth of the amplification products, it is advantageous for the newly created nucleic acid products in each cycle to contain primer binding sites at both ends. In some embodiments, the 3' end of the newly synthesized molecule is the reverse complement of the primer. In some embodiments, two primers each targeting one end of the target nucleic acid can be used for exponential amplification. In some embodiments, it is beneficial to design the primers in such a way that they can be targeted by the CRISPR-Cas systems provided herein - i.e., the primers can be targeted by the guide RNAs of the CRISPR-Cas systems, e.g., crRNAs, such that the CRISPR-Cas systems can be repeatedly used to bind to the target nucleic acid to initiate a new round of amplification.

[0075] Thus, in some embodiments, two or more CRISPR-Cas systems are used to initiate primer binding at both ends of the target nucleic acid. In some embodiments, provided herein is a method for amplifying a target double-stranded nucleic acid, the method comprising: (a) providing a first system having a first clustered regularly interspaced short palindromic repeat (CRISPR) RNA (crRNA) or a derivative thereof, and a first CRISPR-associated (Cas) protein or a variant thereof, wherein the first crRNA or a derivative thereof comprises a target-specific nucleotide region complementary to a region of the first strand of the target double-stranded nucleic acid; (b) providing a second system having a second clustered regularly interspaced short palindromic repeat (CRISPR) RNA (crRNA) or a derivative thereof, and a second CRISPR-associated (Cas) protein or a variant thereof, wherein the second crRNA or a derivative thereof comprises a target-specific nucleotide region complementary to a region of the second strand of the target double-stranded nucleic acid; (c) contacting the target double-stranded nucleic acid with the first system and the second system; (d) hybridizing a first primer to the second strand of the target double-stranded nucleic acid, the first primer comprising a sequence complementary to a region of the second strand of the target double-stranded nucleic acid, and hybridizing a second primer to the first strand of the target double-stranded nucleic acid, the second primer comprising a sequence complementary to a region of the first strand of the target double-stranded nucleic acid, and (e) extending the 3' ends of the first primer and the second primer with one or more polymerases to produce first and second double-stranded target nucleic acids. In some embodiments, the primer hybridization is repeated one or more times by the polymerase extension step, e.g., until the desired degree of amplification is achieved.

[0076] In other embodiments, the methods provided herein are for multiplex amplification. As used herein, the term "multiplex amplification" refers to the amplification of more than one nucleic acid of interest, e.g., the amplification of multiple sequences from the same sample. The term "multiplex amplification" also refers to the amplification of one or more sequences present in multiple samples, either simultaneously or in a stepwise amplification manner. Thus, in some embodiments, two or more target nucleic acid sequences are amplified in an amplification reaction, and the amplification reaction includes appropriate templates and enzymes to amplify at least two target nucleic acid sequences. One application of the multiplex amplification provided herein is the detection of two or more target sequences in a sample, since multiplex amplification is capable of amplifying two or more target sequences. When only one of the target sequences is actually present in the test sample, the result of multiplex amplification may be the amplification of only the one sequence that is present. Multiplex amplification can utilize the same primer pair to amplify one or more intervening amplicon sequences. Alternatively, multiplex amplification can utilize one or more primer pairs.

[0077] In some embodiments, the double-stranded nucleic acid provided herein is double-stranded DNA. In other embodiments, the double-stranded nucleic acid provided herein is double-stranded RNA. In some embodiments, the target nucleic acid is genomic DNA. In other embodiments, the target nucleic acid comprises chromosomal DNA or a fragment thereof. In still other embodiments, the target nucleic acid includes a genome or a portion of a genome.

[0078] In some embodiments, the systems provided herein are derived from CRISPR-Cas systems. CRISPR-Cas systems can generally be divided into three main types (types I-III), which are further subdivided into ten subtypes based on core element content and sequence (Makarova et al., 2011, Nat Rev Microbiol 9:467-77). Two key elements of these CRISPR-Cas systems are the Cas protein and the CRISPR RNA (crRNA). The crRNA consists of short repeat sequences interspersed with spacer sequences derived from invading DNA. The Cas protein has multiple activities, such as nuclease activity. Thus, the CRISPR-Cas system provides a mechanism for targeting a specific sequence and certain enzymatic activities on that sequence.

[0079] Typical type I CRISPR-Cas systems contain the Cas3 protein with separate helicase and DNase activities. For example, in type I-E systems, the crRNA is incorporated into a multi-subunit effector complex called Cascade (CRISPR-associated complex for antiviral defense) (Brouns et al., 2008, Science 321:960-4), which binds the target DNA and triggers degradation by the Cas3 protein (Sinkunas et al., 2011, EMBO J 30:1335-1342; Beloglazova et al., 2011, EMBO J 30:616-627).

[0080] Type II CRISPR-Cas systems include the signature Cas9 protein, a protein (about 160 kDa) capable of generating crRNA and cleaving target DNA. The Cas9 protein typically contains two nuclease domains, a RuvC-like nuclease domain near the amino terminus and an HNH (or McrA-like) nuclease domain near the middle of the protein. Each nuclease domain of the Cas9 protein is specialized for cleaving one strand of the double helix (Jinek et al., 2012, Science 337(6096):816-821).

[0081] Type III CRISPR-Cas systems contain polymerase and RAMP modules. Type III systems can be further divided into type III-A and type III-B subtypes. It has been shown that type III-A CRISPR-Cas systems target plasmids, and the polymerase-like protein of type III-A systems is involved in the cleavage of target DNA (Marraffini and Sontheimer, 2008, Science 322:1843–1845). It has also been shown that type III-B CRISPR-Cas systems target RNA (Hale et al., 2009, Cell 139:945–956).

[0082] Thus, in some embodiments, the system is a type I CRISPR-Cas system or a derivative thereof. In other embodiments, the system is a type II CRISPR-Cas system or a derivative thereof. In still other embodiments, the system is a type III CRISPR-Cas system or a derivative thereof.

[0083] Key elements of the CRISPR-Cas system include guide RNAs, such as crRNAs, and Cas proteins. The crRNA or its derivatives contain a target-specific nucleotide region that is complementary or substantially complementary to a region of the target nucleic acid. In some embodiments, the crRNA or its derivatives contain a user-selectable RNA sequence that allows for enzyme-specific targeting of complementary double-stranded DNA. In some embodiments, the user-selectable RNA sequence contains 20-50 nucleotides that are complementary or substantially complementary to a region of the target DNA sequence. In some embodiments, the user-selectable RNA sequence contains less than 20 nucleotides that are complementary or substantially complementary to a region of the target DNA sequence. Exemplary user-selectable RNA sequences contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotides that are complementary or substantially complementary to a region of the target DNA sequence. In some embodiments, the target-specific nucleotide region of the crRNA has 100% base pair match to a region of the target nucleic acid. In some embodiments, the target-specific nucleotide region of the crRNA has 90%-100%, 80%-100%, or 70%-100% base pair match to a region of the target nucleic acid. In some embodiments, there is one base pair mismatch between the target-specific nucleotide region of the crRNA and the region of the target nucleic acid. In some embodiments, there are two base pair mismatches between the target-specific nucleotide region of the crRNA and the region of the target nucleic acid. In some embodiments, there are three base pair mismatches between the target-specific nucleotide region of the crRNA and the region of the target nucleic acid. In some embodiments, there are four base pair mismatches between the target-specific nucleotide region of the crRNA and the region of the target nucleic acid. In some embodiments, there are five base pair mismatches between the target-specific nucleotide region of the crRNA and the region of the target nucleic acid.

[0084] In some embodiments, the systems provided herein further include a trans-activating crRNA (tracrRNA) or its derivatives.

[0085] The CRISPR-Cas systems provided herein include engineered and / or programmed nuclease systems derived from naturally occurring CRISPR-Cas systems. The CRISPR-Cas systems can include Cas proteins that are engineered and / or mutated. The CRISPR-Cas systems can also include engineered and / or programmed guide RNAs. In some embodiments, the crRNA or its derivative provided herein is a polynucleotide having a crRNA polynucleotide fused to a tracrRNA polynucleotide. Chimeric single guide RNAs (sgRNAs) are described in Jinek et al., 2012, Science 337, 816-821, which is incorporated herein by reference in its entirety. In one embodiment, the Cas protein or its variant provided herein can be directed by a chimeric sgRNA to any genomic locus followed by a 5'-NGG protospacer adjacent motif (PAM). For example, in some embodiments, crRNA and tracrRNA are synthesized by in vitro transcription using a synthetic double-stranded DNA template containing a T7 promoter. The tracrRNA has a fixed sequence, while the target sequence dictates a portion of the crRNA sequence. Equimolar concentrations of crRNA and tracrRNA are mixed and heated at 55 °C for 30 seconds. Cas9 is added at the same molar concentration at 37 °C and incubated with the RNA mixture for 10 minutes. Then, a 10- to 20-fold molar excess of the Cas9 complex is added to the target DNA. The binding reaction can occur within 15 minutes.

[0086] In some embodiments, the Cas protein or its variant is a Cas9 protein or its variant. Isolated Cas9-crRNA complexes from the Streptococcus thermophilus CRISPR-Cas system and complexes assembled in vitro from the individual components demonstrated its binding to both synthetic oligodeoxynucleotides and plasmid DNA carrying nucleotide sequences complementary to the crRNA. Cas9 has been shown to have two nuclease domains - the RvvC- and HNH- active site / nuclease domains, and these two nuclease domains are responsible for cleaving opposite DNA strands. In some embodiments, the Cas9 protein is derived from the Cas9 protein of the Streptococcus thermophilus CRISPR-Cas system. In some embodiments, the Cas9 protein is a multidomain protein having approximately 1,409 amino acid residues.

[0087] In some embodiments, the Cas9 protein or a variant thereof is a nuclease-null variant of the Cas9 protein, in which both the RuvC- and HNH-active sites / nuclease domains are mutated. The nuclease-null variant of the Cas9 protein binds to double-stranded DNA but does not cleave the DNA, and thus it can also be used for target-specific DNA enrichment. In some embodiments, the Cas9 protein has two inactivated nuclease domains, the two inactivated nuclease domains having a first mutation in the domain that cleaves the strand complementary to the crRNA and a second mutation in the domain that cleaves the strand non-complementary to the crRNA. In some embodiments, the Cas9 protein has a first mutation D10A and a second mutation H840A.

[0088] In some embodiments, the Cas protein or a variant thereof is the Cascade protein or a variant thereof. The Cascade complex in Escherichia coli (E. coli) recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. The E. coli Cascade complex is a 405-kDa complex that contains five functionally essential CRISPR-associated (Cas) proteins (CasA1B2C6D1E1, also known as the Cascade protein) and a 61-nucleotide crRNA. The crRNA directs the Cascade complex to the dsDNA target sequence by forming base pairs with the complementary DNA strand while displacing the non-complementary strand to form an R-loop. The Cascade recognizes the target DNA without consuming ATP, indicating that continuous invasive DNA surveillance occurs without energy input. Matthijs et al., Nature Structural & Molecular Biology, 2011, 18, 529–536.

[0089] In some embodiments, the Cas protein or a variant thereof is the Cas3 protein or a variant thereof. E. coli Cas3 can catalyze the ATP-independent annealing of RNA to DNA, forming an R-loop and a hybrid of RNA base-pairing to double-stranded DNA. The Cas3 protein can use a gRNA that is longer than the gRNA of Cas9. Howard et al., Biochem J., 2011, 439(1):85-95. Such a longer RNA can allow other elements to more easily access the target DNA, for example, access to a primer to be extended by a polymerase. Another advantage provided by the Cas3 protein is that, unlike Cas9, the Cas3 protein does not require a PAM sequence, and thus provides more flexibility in targeting a desired sequence. Formation of an R-loop by Cas3 may require magnesium as a cofactor. Howard et al., Biochem J., 2011, 439(1):85-95. Thus, in some embodiments, the systems provided herein also include magnesium.

[0090] It should be understood that any CRISPR-Cas system capable of breaking double-stranded nucleic acids and creating a loop structure can be used in the present method. For example, the Cas proteins provided herein can include, but are not limited to, the Cas proteins described in Haft et al., PLoS Comput Biol., 2005, 1(6):e60, and Zhang et al., Nucl. Acids Res., 2013, 10.1093 / nar / gkt1262. Some of these CRISPR-Cas systems require the presence of specific sequences that recognize and bind to the target sequence for these CRISPR-Cas systems. For example, Cas9 requires the presence of a 5'-NGG protospacer adjacent motif (PAM). Thus, in some embodiments, a PAM sequence or a sequence complementary to the PAM sequence is engineered into the target nucleic acid to initiate the binding of the CRISPR-Cas system to the target nucleic acid.

[0091] In some embodiments, the primers provided herein are single-stranded oligodeoxyribonucleotides. In some embodiments, the primer length ranges from about 10 to about 50 nucleotides. Exemplary primer lengths provided herein are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.

[0092] In some embodiments, the primers provided herein have 100% base pair match to a region of the target nucleic acid. In some embodiments, the primers provided herein have 90%-100%, 80%-100%, or 70%-100% base pair match to a region of the target nucleic acid. In some embodiments, there is one base pair mismatch between the primer and the region of the target nucleic acid. In some embodiments, there are two base pair mismatches between the primer and the region of the target nucleic acid. In some embodiments, there are three base pair mismatches between the primer and the region of the target nucleic acid. In some embodiments, there are four base pair mismatches between the primer and the region of the target nucleic acid. In some embodiments, there are five base pair mismatches between the primer and the region of the target nucleic acid.

[0093] In some embodiments, the primers provided herein are labeled, e.g., for enrichment or detection of amplification products. In some embodiments, the primers provided herein are labeled for detection by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. In some embodiments, the primers are labeled with biotin, amine, radiolabel (e.g., 32P), fluorescent dye, electron-dense reagent, enzyme (commonly used in ELISAs), or biotin.

[0094] In some embodiments, the polymerase provided herein catalyzes the incorporation of nucleotides to extend the 3'-hydroxyl terminus of a primer molecule relative to a nucleic acid target sequence. In some embodiments, the reaction by the polymerase provided herein is carried out under isothermal conditions. In some embodiments, the polymerase is a strand-displacement polymerase. Exemplary polymerases include, but are not limited to, Bst DNA polymerase, 9°N m DNA polymerase, Phi29 DNA polymerase, DNA polymerase I (E. coli), DNA polymerase I (Large fragment), (Klenow) fragment, Klenow fragment (3'-5' exo-), T4 DNA polymerase, T7 DNA polymerase, Deep VentR TM (exo-) DNA polymerase, Deep VentR TM DNA polymerase, DyNAzyme TM EXT DNA, DyNAzyme TM II Hot Start DNA polymerase, Phusion TM High-Fidelity DNA polymerase, Therminator TM DNA polymerase, Therminator TM II DNA polymerase, VentR DNA polymerase, VentR (exo-) DNA polymerase, RepliPHI TM Phi29 DNA polymerase, rBst DNA polymerase, rBst DNA polymerase (Large fragment), Fragment (IsoTherm TM DNA polymerase), MasterAmp TM AmpliTherm TMDNA polymerases, Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Tgo DNA polymerase, SP6 DNA polymerase, Tbr DNA polymerase, DNA polymerase β, and ThermoPhi DNA polymerase. In some embodiments, the polymerase is selected from the group consisting of: Bst, Bsu, and Phi29. When the polymerase extends the hybridized strand, it may be beneficial to include a single-stranded binding protein (SSB). The SSB can stabilize the displaced (non-template) strand. Accordingly, in some embodiments, the methods provided herein may also include an SSB protein.

[0095] As discussed, one advantage provided by the linear or exponential amplification provided herein is that amplification can be carried out at a constant temperature or under isothermal conditions. As used herein, the terms "constant temperature," "isothermal conditions," or "isothermally" refer to a set of reaction conditions under which the reaction temperature is maintained substantially constant during the course of the amplification reaction. However, the temperature need not be maintained precisely at one temperature. If the equipment used to maintain the elevated temperature allows the temperature of the reaction mixture to vary by a few degrees, this is harmless to the amplification reaction and it can still be considered an isothermal reaction. In some embodiments, the amplification reaction is carried out at a constant temperature between 20°C and 70°C. In some embodiments, the amplification reaction is carried out at a constant temperature between 25°C and 60°C. In some embodiments, the amplification reaction is carried out at a constant temperature between 40°C and 55°C. In some embodiments, the amplification reaction is carried out at a constant temperature between 30°C and 40°C. Exemplary amplification reaction temperatures include 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, and 60°C. In one embodiment, the amplification reaction temperature is about 37°C.

[0096] In some embodiments, the time for which the amplification reaction runs can vary from, for example, 1 minute to several hours until the desired amount of amplification is achieved. In some embodiments, the time for the amplification reaction is 5 minutes to 1 hour. Exemplary amplification reaction times include 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, and 60 minutes.

[0097] In some embodiments, the methods herein can be used in certain applications where it is desired to detect and / or quantify the amplified nucleic acid product. The amplified target sequence can be detected by any method known to those of ordinary skill in the art.

[0098] In some embodiments, dyes that specifically stain double-stranded DNA can be used to detect or quantify amplification products. In some embodiments, intercalating dyes exhibit enhanced fluorescence upon binding to DNA or RNA. In some embodiments, the dye can be, for example, a DNA or RNA intercalating fluorophore. Exemplary DNA or RNA intercalating fluorophores include, but are not limited to, acridine orange, ethidium bromide, Hoechst dyes, PicoGreen, propidium iodide, SYBR I (an asymmetric cyanine dye), SYBR II, TOTO (thiazole orange dimer), and YOYO (oxazole yellow dimer).

[0099] In some embodiments, amplification products of a specific size can be detected by gel electrophoresis. In some embodiments, the nucleotides used in the amplification reaction can be labeled, for example, with biotin. Biotin-labeled amplification sequences can be captured using avidin that is conjugated to a signal-generating enzyme, such as peroxidase.

[0100] In some embodiments, labeled nucleotides can be directly incorporated into the target sequence or into primers that contain a complementary sequence to the target of interest. Such labels can be radioactive and / or fluorescent in nature and can be resolved in any of the ways discussed herein.

[0101] Methods for detecting and / or continuously monitoring the amplification of nucleic acid products are also well known to those skilled in the art. For example, in some embodiments, the production or presence of a target nucleic acid and nucleic acid sequences can be detected and monitored by a Molecular Beacon. A Molecular Beacon is a hairpin-shaped oligonucleotide containing a fluorophore at one end and a quenching dye at the opposite end. The loop of the hairpin contains a probe sequence complementary to the target sequence, and the stem is formed by the annealing of complementary arm sequences located on either side of the probe sequence. When the Molecular Beacon encounters the target molecule, hybridization occurs; the loop structure is converted to a stable and more rigid conformation, causing the separation of the fluorophore and quencher molecules, generating fluorescence. Tyagi et al., Nature Biotechnology, 1996, 303 - 308. Thus, the generation of fluorescence indicates the synthesis of the expected amplification product. As another example, in some embodiments, the production or presence of a target nucleic acid and nucleic acid sequences can be detected and monitored by fluorescence resonance energy transfer (FRET). For example, in DNA-DNA interactions, FRET is a useful tool for quantifying molecular dynamics. To monitor the production of a specific product, the probe can be labeled with a donor molecule at one end and an acceptor molecule at the other end. Probe-target hybridization results in a change in the distance or orientation between the donor and acceptor, and a change in FRET is observed. Lakowicz, “Principles of Fluorescence Spectroscopy,” Plenum Publishing Corporation, 2nd Edition (Jul. 1, 1999). Other exemplary methods for detecting and / or continuously monitoring the amplification of nucleic acid products include, but are not limited to, mass spectrometry, capillary gel electrophoresis, sequencing, and various surface capture methods known to those skilled in the art. In some embodiments, running the amplification reaction with asymmetric amounts of primers (i.e., one primer is present at a higher concentration than the other) will allow for amplification biased towards one strand. The excess single-stranded DNA can facilitate detection by means such as Molecular Beacons that detect ssDNA.

[0102] The amplification methods provided herein can be used for a variety of applications. For example, in some embodiments, the methods provided herein can be used to isolate DNA fragments from genomic DNA by selectively amplifying specific DNA regions.

[0103] For another example, in some embodiments, the amplification methods provided herein can be used to diagnose a variety of diseases based on the production or presence of target nucleic acids associated with specific diseases. In some embodiments, the methods provided herein can be used for the early diagnosis of malignant diseases such as leukemia and lymphoma. In other embodiments, the methods provided herein can be directly used on genomic DNA samples to amplify and detect translocation-specific malignant cells.

[0104] In other embodiments, the methods provided herein can be used to diagnose infectious diseases, including those caused by bacteria or viruses. For example, the methods can be used to detect an infectious agent and distinguish non-pathogenic strains from pathogenic strains by specific nucleic acid sequences. In some embodiments, the amplification methods provided herein can amplify and identify non-culturable or slow-growing microorganisms from tissue culture assays and animal models, such as mycobacteria, anaerobic bacteria, or viruses. In other embodiments, the amplification methods provided herein can amplify and identify viral DNA in a sample from an individual.

[0105] In still other embodiments, the amplification methods can be used to generate nucleic acid material to enhance other procedures. For example, in some embodiments, the amplification methods can be used to generate hybridization probes for DNA hybridization or RNA hybridization and DNA cloning that require a relatively large amount of DNA representing a specific DNA region. As another example, the methods provided herein can be used for nucleic acid sequencing.

[0106] In certain embodiments, the CRISPR-Cas mediated nucleic acid amplification methods provided herein can be used to amplify, for example, nucleic acid fragment libraries generated using library preparation methods and / or kits purchased from Illumina, Inc. (San Diego, CA).

[0107] In some embodiments, the methods provided herein can be used to amplify nucleic acid fragment libraries generated from genomic DNA. In certain embodiments, nucleic acid fragment libraries are generated using tagmentation. In certain embodiments, nucleic acid fragment libraries are generated from genomic DNA using Illumina's Nextera library preparation method and kits (purchased from Illumina, Inc, San Diego, CA).

[0108] Thus, in some embodiments, the methods provided herein further comprise: applying at least one transposase and a transposon end composition to a sample comprising a target nucleic acid under conditions in which the target nucleic acid and at least one transposon end composition comprising a transfer strand undergo a transposition reaction to produce a mixture, wherein the target nucleic acid is fragmented to produce a plurality of target nucleic acid fragments, and thereby incorporating a universal primer sequence into each of the plurality of target nucleic acid fragments, wherein the crRNA or its derivative comprises a target-specific nucleotide region complementary to a region of the universal primer.

[0109] In some embodiments, the target nucleic acid undergoes transposase-mediated tagging, which results in fragmentation of the target nucleic acid and ligation of adapters to the 5' ends of both strands of the double-stranded DNA fragments. Optionally, tagging or transposition as described in U.S. Publication No. 2010 / 0120098, which is incorporated herein by reference in its entirety, can fragment the target nucleic acid and add adapters to the 5' and 3' ends. Briefly, a "transposition reaction" is a reaction in which one or more transposons are inserted into the target nucleic acid at random sites. The essential components in a transposition reaction are a transposase and a DNA oligonucleotide that presents the nucleotide sequence of the transposon (including the transferred transposon sequence and its complement (i.e., the untransferred transposon end sequence)) and other components required to form a functional transpososome complex or transposition complex. The DNA oligonucleotide can also contain additional sequences (e.g., adapter or primer sequences) as needed or desired. Exemplary transpososome complexes suitable for use in the methods provided herein include, but are not limited to, those formed by hyperactive Tn5 transposase and Tn5-type transposon ends, or those formed by MuA transposase and Mu transposon ends containing R1 and R2 end sequences (see, e.g., Goryshin and Reznikoff, J. Biol. Chem. 273:7367, 1998; and Mizuuchi, Cell 35:785, 1983; Savilahti et al., EMBO J. 14:4893, 1995; which are incorporated herein by reference in their entirety). However, any transposition system capable of inserting transposon ends into the tagged target nucleic acid with sufficient efficiency for its intended purpose can be used in the methods provided.Other examples of known transposon systems that can be used in the provided methods include, but are not limited to, Staphylococcus aureus Tn552, Tyl, transposon Tn7, Tn / O and IS10, Mariner transposase, Tel, P element, Tn3, bacterial insertion sequences, retroviruses, and yeast retrotransposons (see, e.g., Colegio et al., 2001, J. Bacteriol. 183:2384-8; Kirby et al., 2002, Mol. Microbiol. 43:173-86; Devine and Boeke, 1994, Nucleic Acids Res., 22:3765-72; International Patent Application No. WO 95 / 23875; Craig, 1996, Science 271:1512; Craig, 1996, reviewed in: Curr Top Microbiol Immunol. 204:27-48; Kleckner et al., 1996, Curr Top Microbiol Immunol. 204:49-82; Lampe et al., 1996, EMBO J. 15:5470-9; Plasterk, 1996, Curr Top Microbiol Immunol 204:125-43; Gloor, 2004, Methods Mol. Biol. 260:97-114; Ichikawa and Ohtsubo, 1990, J Biol Chem. 265:18829-32; Ohtsubo and Sekine, 1996, Curr. Top. Microbiol. Immunol. 204:1-26; Brown et al., 1989, Proc Natl Acad Sci USA 86:2525-9; Boeke and Corces, 1989, Annu Rev Microbiol. 43:403-34; which are incorporated herein by reference in their entirety). In some embodiments, the methods of the present disclosure further include removing the transposase and adding it to the ends of a suitable DNA fragment by PCR.

[0110] As used herein, the terms "tagmentation", "tagment", or "tagmenting" refer to the conversion of nucleic acids (e.g., DNA) into adapter-modified templates in solution, ready for cluster formation and sequencing by transposase-mediated fragmentation and tagging. The method typically involves modifying nucleic acids by a transposome complex that includes a transposase complexed with an adapter that contains transposon end sequences. Tagmentation results in simultaneous fragmentation of the nucleic acid and ligation of the adapter to the 5' ends of both strands of the duplex fragment. After a purification step to remove the transposase, additional sequences are added to the ends of the ligated fragments by PCR.

[0111] As used herein, the term "transposome complex" refers to a transposase non-covalently bound to double-stranded nucleic acid. For example, the complex can be a transposase pre-incubated with double-stranded transposon DNA under conditions that support non-covalent complex formation. The double-stranded transposon DNA can include, but is not limited to, Tn5 DNA, portions of Tn5 DNA, transposon end compositions, transposon end compositions, or mixtures of other double-stranded DNA that can interact with a transposase such as a hyperactive Tn5 transposase.

[0112] "Transposase" means an enzyme that can form a functional complex with a composition that contains transposon ends (e.g., a transposon, a transposon end, a transposon end composition), and that can, for example, catalyze the insertion or transposition of a composition containing transposon ends into a double-stranded target nucleic acid incubated with it in an in vitro transposition reaction. Transposases as shown herein can also include integrases from retrotransposons and retroviruses. Transposases, transposomes, and transposome complexes are generally known to those of skill in the art, as exemplified by the disclosure of US 2010 / 0120098, the content of which is incorporated herein by reference in its entirety. Although many of the embodiments described herein relate to Tn5 transposase and / or hyperactive Tn5 transposase, it will be understood that any transposon system that can insert transposon ends into 5'-tags and fragment target nucleic acids with sufficient efficiency for its intended purpose can be used in the present invention. In certain embodiments, a preferred transposon system can insert transposon ends into 5'-tags and fragment target nucleic acids in a random or nearly random manner.

[0113] As used herein, the term "transposition reaction" refers to a reaction in which one or more transposons are inserted into a target nucleic acid at, for example, random or near-random sites. The basic components in a transposition reaction are a transposase and a DNA oligonucleotide that presents the nucleotide sequence of the transposon (including the transferred transposon sequence and its complement (i.e., the non-transferred transposon end sequence)) and other components required to form a functional transposome complex or transpososome complex. Optionally or desirably, the DNA oligonucleotide may also contain additional sequences (e.g., adapter or primer sequences). In some embodiments, the methods provided herein are exemplified by using a transposome complex formed by a hyperactive Tn5 transposase and Tn5-type transposon ends (Goryshin and Reznikoff, 1998, J. Biol. Chem., 273:7367) or by a MuA transposase and Mu transposon ends containing R1 and R2 end sequences (Mizuuchi, 1983, Cell, 35:785; Savilahti et al., 1995, EMBO J., 14:4893). However, any transposition system capable of inserting transposon ends into the 5' tags and fragments of target DNA in a random or near-random manner with sufficient efficiency for its intended purpose can be used in the present invention.Examples of transposon systems known in the art that can be used in the present method include, but are not limited to, Staphylococcus aureus Tn552 (Colegio et al., 2001, J Bacteriol., 183:2384-8; Kirby et al., 2002, Mol Microbiol, 43:173-86), TyI (Devine and Boeke, 1994, Nucleic Acids Res., 22:3765-72 and International Patent Application No. WO 95 / 23875), transposon Tn7 (Craig, 1996, Science. 271:1512; Craig, 1996, reviewed in: Curr Top Microbiol Immunol, 204:27-48), TnIO and ISlO (Kleckner et al., 1996, Curr Top Microbiol Immunol, 204:49-82), Mariner transposase (Lampe et al., 1996, EMBO J., 15:5470-9), Tci (Plasterk, 1996, Curr Top Microbiol Immunol, 204:125-43), P Element (Gloor, 2004, Methods Mol Biol, 260:97-114), TnJ (Ichikawa and Ohtsubo, 1990, J Biol Chem. 265:18829-32), bacterial insertion sequences (Ohtsubo and Sekine, 1996, Curr.Top.Microbiol.Immunol.204:1-26), retroviruses (Brown et al., 1989, Proc Natl Acad Sci USA, 86:2525-9) and retrotransposons of yeast (Boeke and Corces, 1989, Annu Rev Microbiol. 43:403-34). The method for inserting transposon ends into a target sequence can be carried out in vitro using any suitable transposon system for which a suitable in vitro transposon system is available or the suitable transposon system can be developed based on knowledge in the art. Generally, a suitable in vitro transposon system for the methods provided herein requires at least: a transposase with sufficient purity, sufficient concentration and sufficient in vitro transposase activity; transposon ends that form a functional complex with the transposase, the functional complex having transposases capable of catalyzing the transposition reaction respectively.Suitable transposase transposon end sequences that can be used in the present invention include, but are not limited to, wild-type transposon end sequences, derived transposon end sequences, or mutated transposon end sequences that form a complex with a transposase, where the transposase is selected from a wild-type transposase, a derived form of a transposase, or a mutated form of a transposase.

[0114] The term "transposon end" (TE) refers to a double-stranded nucleic acid that exhibits only the nucleotide sequence ("transposon end sequence") necessary to form a complex with a transposase or integrase that is functional in an in vitro transposition reaction, e.g., double-stranded DNA. In some embodiments, the transposon end is capable of forming a functional complex with a transposase in a transposition reaction. As non-limiting examples, the transposon end can include a 19-bp outer end ("OE") transposon end, an inner end ("IE") transposon end, or a "mosaic end" ("ME") transposon end recognized by wild-type or mutated Tn5 transposase, or R1 and R2 transposon ends as described in the disclosure of US2010 / 0120098, the content of which is incorporated herein by reference in its entirety. The transposon end can include any nucleic acid or nucleic acid analogue suitable for forming a functional complex with a transposase or integrase in an in vitro transposition reaction. For example, the transposon end can include DNA, RNA, modified bases, unnatural bases, modified backbones, and can contain nicks in one or both strands. Although the term "DNA" is sometimes used in connection with the composition of the transposon end in this disclosure, it should be understood that any suitable nucleic acid or nucleic acid analogue can be utilized in the transposon end.

[0115] By an in vitro transposition reaction, the target nucleic acid fragment is tagged at the 5' end. In some embodiments, the methods provided herein further include the step of incorporating a 3' end tag into the 5'-tagged nucleic acid fragment to prepare a library of double-tagged nucleic acid fragments. Addition of the 3' end tag can be performed by a variety of methods, e.g., by using a DNA polymerase, a terminal transferase, and / or a ligase as described in WO 2010 / 048605, the content of which is incorporated herein by reference in its entirety.

[0116] In some embodiments, the double-tagged nucleic acid fragments are produced by using a polymerase having strand displacement or 5' nuclease activity, such as a DNA polymerase. In some embodiments, the methods provided herein include: incubating a population of annealed 5'-tagged nucleic acid fragments with a DNA polymerase having strand displacement or 5' nuclease activity under conditions of no thermal cycling and where the annealed 5'-tagged nucleic acid fragments are not denatured, where the DNA polymerase extends the 3' end of each strand of the annealed 5'-tagged nucleic acid fragment using the complementary strand as a template and displaces or digests the non-transferred strand, thereby producing a library of double-tagged double-stranded DNA fragments.

[0117] In other embodiments, the 5'-tagged nucleic acid fragment is incubated with a DNA polymerase consisting of a terminal transferase and at least one substrate of the terminal transferase for a sufficient time under conditions in which the terminal transferase ligates a second tag to the 3' end of the 5'-tagged nucleic acid fragment, thereby generating a library of double-tagged nucleic acid fragments. In some embodiments, the 3' end of the non-transferred transposon end that constitutes the transposon end composition is blocked (e.g., by using a non-transferred transposon end having a dideoxynucleotide or 3'-O-methyl nucleotide as the 3' end nucleotide).

[0118] In still other embodiments, the double-tagged nucleic acid fragment is generated by using a template-dependent ligase and a ligation-tagging oligonucleotide. In some embodiments, the 5'-tagged nucleic acid fragment is incubated with a template-dependent DNA ligase and a ligation-tagging oligodeoxynucleotide having a 3' portion and a 5' portion for a sufficient time under conditions in which the second tag is ligated to the annealed 5'-tagged DNA fragment, thereby generating a library of DNA fragments comprising the annealed double-tagged DNA fragments, wherein the 3' portion presents the second tag, the second tag presenting any sequence that is desired to be ligated to the 3' end of the 5'-tagged DNA fragment, and the 5' portion has a 5' monophosphate group and presents a random sequence.

[0119] In some embodiments, the generated nucleic acid fragments contain universal sequences at both ends of the nucleic acid fragment. For example, in a library, the universal sequences at both ends of the nucleic acid fragment can be targeted by a CRISPR-Cas system according to the methods provided herein, and thus, these fragments can be amplified, for example, in a cluster form reaction.

[0120] Such universal sequences can be introduced into both ends of the nucleic acid fragments in the library by PCR or a Nextera transposon reaction. In some embodiments, after generating a library of tagged nucleic acid fragments, the tagged nucleic acid fragments can be amplified, for example, using limited-cycle polymerase chain reaction (PCR) to introduce other terminal sequences or adapters, such as indices, universal primers, and other sequences required for cluster formation and sequencing. In certain embodiments, limited-cycle PCR amplification is performed to add index 1 (P7) and index 2 (P5) (purchased from Illumina, Inc, San Diego, CA) to both ends of the nucleic acid fragments.

[0121] In some embodiments, such amplification is performed on a library of 5'-tagged nucleic acid fragments. In some embodiments, such amplification is performed on a library of dual-tagged nucleic acid fragments. Exemplary amplification methods include: polymerase chain reaction (PCR), strand displacement amplification reaction, rolling circle amplification reaction, ligase chain reaction, transcription-mediated amplification reaction, and loop-mediated amplification reaction.

[0122] In some embodiments, the methods provided herein include amplifying a library of dual-tagged nucleic acid fragments using PCR. In some embodiments, the methods provided herein include amplifying a library of dual-tagged nucleic acid fragments using the Cas9-mediated amplification method provided herein. In some embodiments, the methods provided herein use single-primer PCR to amplify a library of dual-tagged DNA fragments. In some embodiments, the step of amplifying dual-tagged DNA fragments includes using a DNA polymerase and at least one primer complementary to the second tag. In some embodiments, the step of amplifying a library of dual-tagged DNA fragments includes amplifying a library of tagged DNA fragments by PCR using only one oligodeoxynucleotide that exhibits a sequence of at least a portion of the transfer strand as a PCR primer and using the dual-tagged DNA fragment as a template. In some embodiments, the primer comprises a 5' portion that comprises additional sequences, such as adapter sequences.

[0123] In some embodiments, two different PCR primers are used, each of which exhibits a sequence that constitutes at least a portion of the transferred transposon end of the transposon end composition. In some embodiments, each PCR primer comprises a 3' portion and a 5' portion, wherein the 3' portion exhibits the respective transferred transposon end sequence, and the 5' portion exhibits the sequence of the respective tag domain or linker for a particular purpose (e.g., a sequencing tag domain / linker or an amplification tag domain / linker for next-generation sequencing or amplification, and optionally an addressing tag domain / linker). For example, when a single transposon end composition is used in an in vitro transposition reaction to generate a library of double-tagged DNA fragments using a DNA polymerase with strand displacement or 5' nuclease activity, the double-tagged DNA fragments can be amplified by PCR using two different PCR primers. Each PCR primer comprises a 3' portion and a 5' portion, wherein the 3' portion exhibits the respective transferred transposon end sequence, and the 5' portion exhibits the sequence of the respective tag domain / linker for a particular purpose (e.g., a sequencing tag domain / linker or an amplification tag domain / linker for next-generation sequencing or amplification, and optionally an addressing tag domain / linker). In some embodiments, the 5' portion of each PCR primer is different from the 5' portion of the other primer, and thus the sequences of the two ends of the PCR product are different. For example, one end contains an index and / or a universal primer sequence, and the other end contains a different index and / or a universal primer sequence.

[0124] In some embodiments, the two ends of the double-tagged nucleic acid fragment are derived from two different transferred strand sequences. For example, in some embodiments, two different transposomes can be used in an in vitro transposition reaction, and each of the two transposomes contains the same transposase but different transposon end compositions. In some embodiments, two different transposomes are used, and each of the two different transposomes contains the same transposase while the transposon end composition contains different transferred strands. In some embodiments, two different transposomes are used, and each of the two transposomes contains a different transposase and a different transposon end composition, and each of the different transposon end compositions forms a functional complex with its respective transposase. In some embodiments, wherein two different transposon end compositions are used in an in vitro transposition reaction, and a library of double-tagged single-stranded nucleic acid fragments is generated using a DNA polymerase with strand displacement or 5' nuclease activity, the first tag exhibits the sequence of the transferred strand of one transposon end composition and the second tag exhibits the sequence of the non-transferred strand of the other transposon end composition.

[0125] In the above-mentioned embodiments and other embodiments in which two different transfer strands are linked to the 5' end of each opposite strand of a double-stranded nucleic acid, the methods provided herein may further include the step of PCR amplifying the double-tagged nucleic acid fragment using two different PCR primers. One of the PCR primers exhibits a sequence that constitutes at least a portion of one transfer strand of a transposon end composition, while the other of the PCR primers exhibits a sequence that constitutes at least a portion of the other transfer strand of another transposon end composition.

[0126] In some embodiments in which two primers are used, each PCR primer comprises a 3' portion and a 5' portion, where the 3' portion exhibits the respective transferred transposon end sequence and the 5' portion exhibits the sequence of the respective tag domain / linker for a particular purpose (e.g., a sequencing tag domain or an amplification tag domain for next-generation sequencing or amplification, and optionally an addressing tag domain). In some embodiments, the 5' portion of each PCR primer is different from the 5' portion of the other primer, and thus different sequences are introduced into the two ends of the PCR product. In some embodiments, the 5' portion of the first PCR primer or the 5' portion of the second PCR primer, or the 5' portions of both the first PCR primer and the second PCR primer, respectively, comprise a first sequencing tag / linker or a second sequencing tag / linker for generating a template for next-generation sequencing for a particular sequencing platform (e.g., a sequencing tag for the Illumina Nextera sequencing platform). In some embodiments, the 5' portion of the first PCR primer or the 5' portion of the second PCR primer further comprises an addressing tag domain / linker or another tag domain / linker for a particular purpose.

[0127] As is known to those skilled in the art, many enzymes and kits are available for performing amplification reactions by PCR. For example, in some embodiments, the FAILSAFE TM PCR System or the MASTERAMP TM Extra-Long PCR System from EPICENTRE Biotechnologies, Madison, WI is used for PCR amplification as described by the manufacturer. However, the present disclosure is not limited to using those products or conditions for the amplification reaction, and any suitable thermostable DNA polymerase and reaction mixture that allow sequence amplification between a primer annealing to the target sequence and a primer annealing to the transposon may be used.

[0128] The methods provided herein are not limited to using PCR to amplify a library of nucleic acid fragments with added tags. Any suitable amplification method that amplifies the same sequence and produces an amplification product with a suitable composition and amount for the intended purpose (e.g., rolling circle amplification, riboprimer amplification (e.g., U.S. Patent No. 7,413,857), ICAN, UCAN, ribospia, end-tagging (U.S. Patent Application No. 20050153333), Eberwine-type aRNA amplification, or strand displacement amplification) can be used in embodiments of the present invention. For example, some strand displacement methods that can be used are described in: PCT Patent Application No. WO 02 / 16639 by Takara Shuzo Company, Kyoto, Japan; WO 00 / 56877; and AU 00 / 29742; U.S. Patent Nos. 5,523,204; 5,536,649; 5,624,825; 5,631,147; 5,648,211; 5,733,752; 5,744,311; 5,756,702; and 5,916,779 by Becton Dickinson and Company; U.S. Patent Nos. 6,238,868; 6,309,833; and 6,326,173 by Nanogen / Becton Dickinson Partnership; U.S. Patent Nos. 5,849,547; 5,874,260; and 6,218,151 by Bio Merieux; U.S. Patent Nos. 5,786,183; 6,087,133; and 6,214,587 by Gen-Probe, Inc.; U.S. Patent No. 6,063,604 by Wick et al.; U.S. Patent No. 6,251,639 by Kurn; U.S. Patent No. 6,410,278 by Eiken Kagaku Kabushiki Kaishi, Tokyo, Japan, and PCT Application No. WO 00 / 28082; U.S. Patent Nos. 5,591,609; 5,614,389; 5,773,733; 5,834,202; and 6,448,017 by Auerbach; and U.S. Patent Nos. 6,124,120; and 6,280,949 by Lizardi. In some embodiments, the Cas-mediated amplification provided herein is used to amplify a library of target nucleic acid fragments.

[0129] In some embodiments, when the target nucleic acid has a universal primer sequence, such as that produced using the methods provided above, a guide RNA, such as a crRNA, can target the universal primer sequence of the nucleic acid fragments in the library to isothermally amplify the library of nucleic acid fragments.

[0130] Figures 1A-1CDisclosed is a method of amplifying nucleic acid fragments generated from a Nextera library product (purchased from Illumina, Inc., San Diego, CA) according to the present disclosure. In some embodiments, the Cas proteins provided herein require a PAM sequence to recognize the target. For example, the Cas9 protein requires a sequence of the motif NGG adjacent to the target sequence. Sequences complementary to the PAM sequence can be incorporated into the flowcell primers (e.g., P5 and P7) added to the library inserts to generate PAM-modified P5 and P7 primers capable of targeting sequences containing the PAM. The sequences of the standard universal primers P5 and P7 are shown in Figure 1A (SEQ ID No.1 and SEQ ID No.2). The sequences of the PAM-modified primers P5 and P7 are also shown in Figure 1A (SEQ ID No.3 and SEQ ID No.4). Thus, in some embodiments, the PAM-modified primers P5 and P7 are added to the nucleic acid fragments in the library. In certain embodiments, the PAM-modified primers P5 and P7 are added to the nucleic acid fragments using limited cycle PCR.

[0131] As Figures 1B-1C shown, DNA fragments (e.g., Nextera amplicons in solution) contain P5 and P7 primer sequences (SEQ ID No.1 and SEQ ID No.2) at the ends of the amplicons. A CRISPR-Cas9 system with a guide RNA of SEQ ID No.7 that contains a region targeting the P5 primer sequence (see Figure 1B ) and a CRISPR-Cas9 system with a guide RNA of SEQ ID No.8 that contains a region targeting the P7 primer sequence (see Figure 1C ) are added. The CRISPR-Cas9 system opens some regions of the double-stranded DNA, creating R-loop structures near both ends of the DNA fragment by binding the crRNA to the primer sequence. Then, truncated PAM-modified P5 and P7 primers (SEQ ID No.5 and SEQ ID No.6) can be used to amplify the DNA fragment.

[0132] Thus, in some embodiments, the first strand of the target double-stranded nucleic acid contains a universal sequence, and wherein the crRNA or its derivative contains a sequence complementary to the region of the universal sequence. In some embodiments, the primer contains a sequence of the region of the universal sequence.

[0133] In some embodiments, the universal primer has the sequence of SEQ ID No.3. In some embodiments, the crRNA contains the sequence of SEQ ID No.7. In some embodiments, the primer contains the sequence of SEQ ID No.5.

[0134] In some embodiments, the universal primer sequence has the sequence of SEQ ID No. 4. In some embodiments, the crRNA comprises the sequence of SEQ ID No. 8. In some embodiments, the primer comprises the sequence of SEQ ID No. 6.

[0135] In some embodiments, the first strand of the target double-stranded nucleic acid comprises a first universal sequence, and wherein the crRNA or its derivative of the first system comprises a sequence complementary to a region of the first universal sequence, and the second strand of the target double-stranded nucleic acid comprises a second universal sequence, and wherein the crRNA or its derivative of the second system comprises a sequence complementary to a region of the second universal sequence. In some embodiments, the first primer comprises a sequence of a region of the first universal sequence, and the second primer comprises a sequence of a region of the second universal sequence. In certain embodiments, the first universal sequence has the sequence of SEQ ID No. 3, the crRNA or its derivative of the first system comprises the sequence of SEQ ID No. 7, and the first primer comprises the sequence of SEQ ID No. 5, and the second universal sequence has the sequence of SEQ ID No. 4, the crRNA or its derivative of the second system comprises the sequence of SEQ ID No. 8, and the second primer comprises the sequence of SEQ ID No. 6.

[0136] The methods provided herein can be used for isothermal amplification for, e.g., sequencing in cluster amplification developed by Illumina, Inc. (San Diego, CA). In some embodiments, the target nucleic acid of the present method can be immobilized on a surface for amplification. For example, in some embodiments, the immobilized nucleic acid fragments are amplified using a cluster amplification method, as exemplified by the disclosures of U.S. Patent Nos. 7,985,565 and 7,115,400, the contents of each of which are hereby incorporated by reference in their entirety. The incorporated materials of U.S. Patent Nos. 7,985,565 and 7,115,400 describe methods of solid-phase nucleic acid amplification that allow amplification products to be immobilized on a solid support to form an array comprising clusters or "colonies" of immobilized nucleic acid molecules. Each cluster or colony on such an array is formed by multiple identical immobilized polynucleotide chains and multiple identical immobilized complementary polynucleotide chains. The arrays so formed are generally referred to herein as "clustered arrays". The products of solid-phase amplification reactions such as those described in U.S. Patent Nos. 7,985,565 and 7,115,400 are the so-called "bridged" structures formed by annealing of paired immobilized polynucleotide chains and immobilized complementary chains, with the two chains preferably covalently attached at the 5' end to the solid support. The cluster amplification method is an example of a method in which immobilized nucleic acid templates are used to generate immobilized amplicons. Other suitable methods can also be used to generate immobilized amplicons from the immobilized nucleic acid fragments produced according to the methods provided herein. For example, one or more clusters or colonies can be formed via solid-phase PCR, whether one or both primers in each pair of amplification primers are immobilized.

[0137] As used herein, the terms "solid surface", "solid support" and other grammatical equivalents herein refer to any material suitable for or that can be modified to be suitable for attaching polynucleotides. Possible substrates include, but are not limited to: glass and modified or functionalized glass, plastics (including acrylics, polystyrene, and copolymers of styrene and other materials, polypropylene, polyethylene, polybutene, polyurethane, Teflon TMetc.), polysaccharides, nylon or nitrocellulose, ceramics, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glass, plastics, optical fiber bundles, and many other polymers. In some embodiments, the solid support and the solid surface are located within a flow cell device. In some embodiments, the solid support includes a patterned surface adapted to immobilize molecules in an ordered pattern. A "patterned surface" refers to the arrangement of different regions in or on the exposed layer of the solid support. In some embodiments, the solid support includes an array of pores or recesses in the surface. The composition and geometry of the solid support can vary depending on its use. In some embodiments, the solid support is a planar structure, such as a slide, chip, microchip, and / or array. Thus, the surface of the substrate can be in the form of a planar layer. In some embodiments, the solid support includes one or more surfaces of a flow cell. As used herein, the term "flow cell" refers to a chamber containing a solid surface through which one or more fluid reagents can flow. Examples of flow cells and associated fluid systems and detection platforms that can be readily used in the methods of the present disclosure are described in the following: e.g., Bentley et al., Nature 456:53-59 (2008); WO 04 / 018497; US 7,057,026; WO 91 / 06678; WO 07 / 123744; US 7,329,492; US 7,211,414; US 7,315,019; US 7,405,281 and US 2008 / 0108082, each of which is incorporated herein by reference. In some embodiments, the solid support or its surface is non-planar, such as the inner or outer surface of a tube or container. In some embodiments, the solid support includes microspheres or beads. As used herein, the terms "microspheres", "beads", "particles", or grammatical equivalents are intended to mean small, discrete particles made of a variety of materials including but not limited to plastics, ceramics, glass, and polystyrene. In certain embodiments, the microspheres are magnetic microspheres or beads. Optionally or additionally, the beads can be porous. The size of the beads ranges from nanometers, e.g., 100 nm, to millimeters, e.g., 1 mm.

[0138] In other embodiments, the immobilized nucleic acid fragments are amplified in solution. For example, in some embodiments, the immobilized nucleic acid fragments are cleaved or otherwise released from the solid support, and then amplification primers hybridize to the released molecules in solution. In other embodiments, for one or more initial amplification steps, the amplification primers are hybridized to the immobilized fragments, followed by subsequent amplification steps in solution. Thus, in some embodiments, the immobilized nucleic acid template can be used to generate solution-phase amplicons. It will be appreciated that any of the amplification methods described herein can be utilized with universal or target-specific primers to amplify the immobilized nucleic acid fragments.

[0139] The nucleic acids amplified according to the methods provided herein can be sequenced according to any suitable sequencing method, such as direct sequencing, including sequencing by synthesis, sequencing by ligation, sequencing by hybridization, nanopore sequencing, and the like. In some embodiments, immobilized DNA fragments are sequenced on a solid support. In some embodiments, the solid support used for sequencing is the same solid support on which amplification occurs.

[0140] In some embodiments, the sequencing method used in the methods provided herein is sequencing by synthesis (SBS). In SBS, the extension of a nucleic acid primer along a nucleic acid template (e.g., a target nucleic acid or its amplicon) is monitored to determine the sequence of nucleotides in the template. The underlying chemical process can be polymerization (e.g., as catalyzed by a polymerase). In certain polymerase-based SBS embodiments, fluorescently labeled nucleotides are added to the primer in a template-dependent manner (thereby extending the primer) such that the order and type of nucleotides added to the primer can be used to determine the sequence of the template.

[0141] Other sequencing procedures that use cyclic reactions can be utilized, such as pyrosequencing. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as a particular nucleotide is incorporated into a nascent nucleic acid strand (Ronaghi, et al., 1996, Analytical Biochemistry 242(1), 84-9; Ronaghi, 2001, Genome Res. 11(1), 3-11; Ronaghi et al., 1998, Science 281(5375), 363; US 6,210,891; US 6,258,568 and US 6,274,320, each of which is incorporated herein by reference). In pyrosequencing, the released PPi can be detected by adenosine triphosphate (ATP) that is immediately converted by ATP sulfurylase, and the level of ATP generated can be detected via protons generated by luciferase. Thus, the sequencing reaction can be monitored via a luminescence detection system. An excitation radiation source for a fluorescence-based detection system is not required for the pyrosequencing procedure. Useful fluid systems, detectors, and procedures that can be adapted for use in pyrosequencing amplicons generated according to the present disclosure are described in, for example, WIPO Patent Application Serial No. PCT / US11 / 57111, US2005 / 0191698A1, US 7,595,883, and US 7,244,559, each of which is incorporated herein by reference.

[0142] Some embodiments can utilize methods involving real-time monitoring of DNA polymerase activity. For example, nucleotide incorporation can be detected by fluorescence resonance energy transfer (FRET) interactions between a polymerase carrying a fluorophore and γ-phosphate-labeled nucleotides, or using zero-mode waveguides (ZMWs). Techniques and reagents for FRET-based sequencing are described, for example, in Levene et al., 2003, Science 299, 682–686; Lundquist et al., 2008, Opt. Lett. 33, 1026–1028; Korlach et al., 2008, Proc. Natl. Acad. Sci. USA 105, 1176–1181, the disclosures of which are incorporated herein by reference.

[0143] Some SBS embodiments include detecting protons released upon incorporation of a nucleotide into an extension product. For example, sequencing based on detection of released protons can use an electronic detector and related techniques commercially available from Ion Torrent (Guilford, CT, a Life Technologies subsidiary), or the sequencing methods and systems described in US 2009 / 0026082 A1, US 2009 / 0127589 A1, US 2010 / 0137143 A1, or US 2010 / 0282617 A1, each of which is incorporated herein by reference. The methods listed herein for amplifying a target nucleic acid using kinetic exclusion can be readily applied to a substrate used to detect protons. More specifically, the methods listed herein can be used to generate a clonal population of amplicons for detecting protons.

[0144] Another useful sequencing technique is nanopore sequencing (see, e.g., Deamer et al., 2000, Trends Biotechnol. 18, 147–151; Deamer et al., 2002, Acc. Chem. Res. 35:817-825; Li et al., 2003, Nat. Mater. 2:611–615), the disclosures of which are incorporated herein by reference. In some nanopore embodiments, the target nucleic acid or individual nucleotides removed from the target nucleic acid are passed through a nanopore. As the nucleic acid or nucleotide passes through the nanopore, each nucleotide type can be identified by measuring fluctuations in the electrical conductivity of the pore. (U.S. Patent No. 7,001,792; Soni et al., 2007, Clin. Chem., 53, 1996–200; Healy, 2007, Nanomed. 2, 459–481; Cockroft et al., 2008, J. Am. Chem. Soc., 130, 818–820, the disclosures of which are incorporated herein by reference. In other nanopore sequencing embodiments, DNA fragments to be sequenced create unique nanopore current signatures using γ-phosphate modified nucleotides.

[0145] It will be apparent from the foregoing description that changes and modifications may be made to the invention described herein to adapt it to various uses and conditions. Such embodiments are also within the scope of the following claims.

[0146] In any variable definition herein, the recitation of a column of elements includes that variable as a definition of any single element or combination (or sub-combination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any separate embodiment and any combination with any other embodiment or portion thereof.

[0147] All patents and publications mentioned in this specification are incorporated herein by reference to the same extent as if each individual patent and publication were specifically and individually indicated to be incorporated by reference.

Claims

1. A method for amplifying a target double-stranded nucleic acid, the method comprising: (a) providing a system having: clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA), trans-activating crRNA (tracrRNA), and CRISPR-associated (Cas) 9 protein, wherein the crRNA comprises a target-specific nucleotide region complementary to a region of the first strand of the target double-stranded nucleic acid; (b) contacting the target double-stranded nucleic acid with the system to form a complex; (c) hybridizing a primer to the second strand of the target double-stranded nucleic acid, wherein the primer is immobilized on a substrate, and wherein the primer comprises a sequence complementary to a region of the second strand of the target double-stranded nucleic acid, and (d) using a polymerase to extend a nucleic acid complementary to the second strand of the target double-stranded nucleic acid from the primer.

2. A method for amplifying a target double-stranded nucleic acid, the method comprising: (a) providing a system having: clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA), trans-activating crRNA (tracrRNA), and CRISPR-associated (Cas) 9 protein, wherein the crRNA comprises a target-specific nucleotide region complementary to a region of the first strand of the target double-stranded nucleic acid; (b) contacting the target double-stranded nucleic acid with the system to form a complex; (c) hybridizing a primer to the second strand of the target double-stranded nucleic acid, the primer comprising a sequence complementary to a region of the second strand of the target double-stranded nucleic acid, and (d) using a polymerase to extend a nucleic acid complementary to the second strand of the target double-stranded nucleic acid from the primer; wherein steps (b) to (d) are carried out under isothermal conditions.

3. The method according to claim 1 or 2, the method further comprising repeating steps (a) to (d) one or more times.

4. The method according to claim 1 or 2, wherein the target double-stranded nucleic acid is linearly amplified.

5. The method according to claim 1 or 2, wherein the target double-stranded nucleic acid is exponentially amplified.

6. The method according to claim 1 or 2, wherein, The system comprises a polynucleotide containing the crRNA and the tracrRNA.

7. The method according to claim 1 or 2, wherein the first strand of the target double-stranded nucleic acid comprises a sequence complementary to a 5'-NGG protospacer adjacent motif (PAM).

8. The method according to claim 1 or 2, wherein the first strand of the target double-stranded nucleic acid comprises a universal sequence, and wherein the crRNA comprises a sequence complementary to a region of the universal sequence.

9. The method according to claim 8, wherein the primer comprises a sequence of a region of the universal sequence.

10. The method according to claim 8, wherein the universal sequence comprises the nucleotide sequence of SEQ ID No.

3.

11. The method according to claim 10, wherein the crRNA comprises the nucleotide sequence of SEQ ID No.

7.

12. The method according to claim 11, wherein the primer comprises the nucleotide sequence of SEQ ID No.

5.

13. The method according to claim 8, wherein the universal sequence has the nucleotide sequence of SEQ ID No.

4.

14. The method according to claim 13, wherein the crRNA comprises the nucleotide sequence of SEQ ID No.

8.

15. The method according to claim 14, wherein the primer comprises the nucleotide sequence of SEQ ID No.

6.

16. The method according to claim 1 or 2, wherein the Cas9 protein comprises two inactivated nuclease domains.

17. The method according to claim 16, wherein the two inactivated nuclease domains comprise a first mutation in the domain that cleaves the strand complementary to the clustered regularly interspaced short palindromic repeat RNA and a second mutation in the domain that cleaves the strand non-complementary to the clustered regularly interspaced short palindromic repeat RNA.

18. The method according to claim 17, wherein the first mutation is D10A and the second mutation is H840A.

19. The method according to claim 1 or 2, wherein the polymerase is a strand displacement polymerase.

20. The method according to claim 19, wherein the polymerase is selected from the group consisting of: Bst, Bsu, and Phi29.

21. The method according to claim 1 or 2, the method further comprising: applying at least one transposase and the transposon end composition to a sample comprising the target double-stranded nucleic acid under conditions for a transposition reaction between the target double-stranded nucleic acid and at least one transposon end composition comprising a transfer strand to produce a mixture, wherein the target double-stranded nucleic acid is fragmented to produce a plurality of target nucleic acid fragments, and incorporating a universal primer sequence into each of the plurality of target nucleic acid fragments, wherein the crRNA comprises a target-specific nucleotide region complementary to a region of the universal primer sequence.

22. The method according to claim 21, wherein the universal primer sequence is incorporated into the plurality of target nucleic acid fragments by a PCR reaction.

23. The method according to claim 22, wherein the universal primer sequence has a sequence complementary to the 5'-NGG protospacer adjacent motif (PAM).

24. The method according to claim 22, wherein the universal primer sequence comprises the nucleotide sequence of SEQ ID No.

3.

25. The method according to claim 24, wherein the crRNA comprises the nucleotide sequence of SEQ ID No.

7.

26. The method according to claim 25, wherein the primer comprises the nucleotide sequence of SEQ ID No.

5.

27. The method according to claim 22, wherein the universal primer sequence comprises the nucleotide sequence of SEQ ID No.

4.

28. The method according to claim 27, wherein the crRNA comprises the nucleotide sequence of SEQ ID No.

8.

29. The method according to claim 28, wherein the primer comprises the nucleotide sequence of SEQ ID No.

6.

30. The method according to claim 21, wherein two universal primer sequences are incorporated into both ends of each of the plurality of target nucleic acid fragments.

31. The method according to claim 30, wherein each of the two universal primer sequences comprises the nucleotide sequences of SEQ ID No. 3 and SEQ ID No.

4.

32. A method for amplifying a target double-stranded nucleic acid, the method comprising: (a) providing a first system having: a first clustered regularly interspaced short palindromic repeat (CRISPR) RNA (crRNA), and a first CRISPR-associated (Cas) 9 protein, wherein the first crRNA comprises a target-specific nucleotide region complementary to a region of the first strand of the target double-stranded nucleic acid; (b) providing a second system having: a second crRNA, and a second Cas9 protein, wherein the second crRNA comprises a target-specific nucleotide region complementary to a region of the second strand of the target double-stranded nucleic acid; (c) contacting the target double-stranded nucleic acid with the first system and the second system; (d) hybridizing a first primer to the second strand of the target double-stranded nucleic acid, wherein the first primer comprises a sequence complementary to a region of the second strand of the target double-stranded nucleic acid, and hybridizing a second primer to the first strand of the target double-stranded nucleic acid, wherein the second primer comprises a sequence complementary to a region of the first strand of the target double-stranded nucleic acid, wherein the first primer or the second primer is immobilized on a substrate, and (e) extending the 3'-ends of the first primer and the second primer with a polymerase to produce first and second target double-stranded nucleic acids, wherein the first system further comprises a first trans-activating crRNA (tracrRNA), or the second system further comprises a second trans-activating crRNA (tracrRNA).

33. A method for amplifying a target double-stranded nucleic acid, the method comprising: (a) providing a first system having: a first clustered regularly interspaced short palindromic repeat (CRISPR) RNA (crRNA), and a first CRISPR-associated (Cas) 9 protein, wherein the first crRNA comprises a target-specific nucleotide region complementary to a region of the first strand of the target double-stranded nucleic acid; (b) providing a second system having: a second crRNA, and a second Cas9 protein, wherein the second crRNA comprises a target-specific nucleotide region complementary to a region of the second strand of the target double-stranded nucleic acid; (c) contacting the target double-stranded nucleic acid with the first system and the second system; (d) hybridizing a first primer to the second strand of the target double-stranded nucleic acid, wherein the first primer comprises a sequence complementary to a region of the second strand of the target double-stranded nucleic acid, and hybridizing a second primer to the first strand of the target double-stranded nucleic acid, wherein the second primer comprises a sequence complementary to a region of the first strand of the target double-stranded nucleic acid, and (e) extending the 3'-ends of the first primer and the second primer with a polymerase to produce first and second target double-stranded nucleic acids, wherein the first system further comprises a first trans-activating crRNA (tracrRNA), or the second system further comprises a second trans-activating crRNA (tracrRNA); wherein steps (c) to (e) are carried out under isothermal conditions.

34. The method according to claim 32 or 33, further comprising repeating steps (a) to (e) one or more times.

35. The method according to claim 32 or 33, wherein (i) the first system comprises a polynucleotide containing the first crRNA and the first tracrRNA, or (ii) the second system comprises a polynucleotide containing the second crRNA and the second tracrRNA.

36. The method according to claim 32 or 33, wherein the first strand and the second strand of the target double-stranded nucleic acid each comprise a sequence complementary to a 5'-NGG protospacer adjacent motif (PAM).

37. The method according to claim 32 or 33, wherein: the first strand of the target double-stranded nucleic acid comprises a first universal sequence, and wherein the crRNA of the first system comprises a sequence complementary to a region of the first universal sequence, and the second strand of the target double-stranded nucleic acid comprises a second universal sequence, and wherein the crRNA of the second system comprises a sequence complementary to a region of the second universal sequence.

38. The method according to claim 37, wherein the first primer comprises a sequence of a region of the first universal sequence, and the second primer comprises a sequence of a region of the second universal sequence.

39. The method according to claim 38, wherein: the first universal sequence comprises the nucleotide sequence of SEQ ID No. 3, the crRNA of the first system comprises the nucleotide sequence of SEQ ID No. 7, and the first primer comprises the nucleotide sequence of SEQ ID No. 5, and the second universal sequence comprises the nucleotide sequence of SEQ ID No. 4, the crRNA of the second system comprises the nucleotide sequence of SEQ ID No. 8, and the second primer comprises the nucleotide sequence of SEQ ID No.

6.

40. The method according to claim 32 or 33, wherein the Cas9 protein comprises two inactivated nuclease domains.

41. The method according to claim 40, wherein the two inactivated nuclease domains comprise a first mutation in the domain that cleaves the strand complementary to the first crRNA or the second crRNA and a second mutation in the domain that cleaves the strand non-complementary to the first crRNA or the second crRNA.

42. The method according to claim 41, wherein the first mutation is D10A and the second mutation is H840A.

43. The method according to claim 32 or 33, wherein the polymerase is a strand displacement polymerase.

44. The method according to claim 43, wherein the polymerase is selected from the group consisting of: Bst, Bsu, and Phi29.

45. The method according to any one of claims 1, 2, 32, or 33, wherein the target double-stranded nucleic acid is DNA.

46. The method according to any one of claims 1, 2, 32 or 33, wherein the target double-stranded nucleic acid is RNA.

47. The method according to any one of claims 1, 2, 32 or 33, wherein the target double-stranded nucleic acid is genomic DNA.

48. The method according to any one of claims 1, 2, 32 or 33, wherein the target double-stranded nucleic acid comprises chromosomal DNA or a fragment thereof.

49. The method according to any one of claims 1, 2, 32 or 33, wherein the target double-stranded nucleic acid comprises a genome or a portion of a genome.

50. The method according to any one of claims 1, 2, 32 or 33, the method further comprising sequencing the amplified target double-stranded nucleic acid.

51. The method according to claim 50, wherein the sequencing comprises using one or more of sequencing by synthesis, bridge PCR, chain termination sequencing, hybridization sequencing, nanopore sequencing, and ligation sequencing.

52. A method for amplifying a target double-stranded nucleic acid having a first strand and a second strand, the method comprising: (a) providing a system comprising: a polynucleotide comprising clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) and trans-activating crRNA (tracrRNA), and CRISPR-associated (Cas) 9 protein, wherein the crRNA comprises a target-specific nucleotide region complementary to a region of the first strand of the target double-stranded nucleic acid; (b) contacting the target double-stranded nucleic acid with the system to form a complex; (c) hybridizing a primer to the second strand of the target double-stranded nucleic acid, wherein the primer is immobilized on a substrate, and wherein the primer comprises a sequence complementary to a region of the second strand of the target double-stranded nucleic acid; (d) extending a nucleic acid complementary to the second strand of the target double-stranded nucleic acid from the primer using a polymerase; and (e) repeating steps (a) to (d).

53. The method according to claim 52, wherein the target double-stranded nucleic acid is immobilized on the substrate.

54. The method according to claim 52, wherein the substrate comprises a flow cell.

55. The method according to claim 52, wherein the substrate comprises beads.

56. The method according to claim 52, wherein the target double-stranded nucleic acid is amplified exponentially.

57. The method according to claim 52, wherein the Cas9 protein comprises two inactivated nuclease domains.

58. The method according to claim 57, wherein the two inactivated nuclease domains comprise a first mutation in the domain that cleaves the strand complementary to the crRNA and a second mutation in the domain that cleaves the strand non-complementary to the crRNA.

59. The method according to claim 58, wherein the first mutation is D10A and the second mutation is H840A.

60. The method according to claim 52, wherein the first strand of the target double-stranded nucleic acid comprises a sequence complementary to a 5'-NGG protospacer adjacent motif (PAM).

61. The method according to claim 52, wherein the first strand of the target double-stranded nucleic acid comprises a universal sequence, and the primer comprises a sequence of a region of the universal sequence.

62. The method according to claim 61, wherein the universal sequence comprises the sequence of SEQ ID NO:03 or SEQ ID NO:

04.

63. The method according to claim 52, wherein the primer comprises a P5 or P7 nucleotide sequence.

64. The method according to claim 63, wherein the primer comprises the sequence of SEQ ID NO:05 or SEQ ID NO:

06.

65. The method according to claim 52, wherein the polymerase is a strand displacement polymerase.

66. The method according to claim 65, wherein the strand displacement polymerase is selected from the group consisting of: Bst, Bsu, and Phi29.

67. A system for amplifying a target double-stranded nucleic acid having a first strand and a second strand, the system comprising: (a) A complex, the complex comprising: (i) A polynucleotide comprising a clustered regularly interspaced short palindromic repeat (CRISPR) RNA (crRNA) and a trans-activating crRNA (tracrRNA), and a CRISPR-associated (Cas) 9 protein, wherein the crRNA comprises a target-specific nucleotide region complementary to a region of the first strand of the target double-stranded nucleic acid; and (ii) the target double-stranded nucleic acid in contact with the polynucleotide and the Cas protein; (b) A primer immobilized on a substrate, wherein the primer hybridizes to the second strand of the target double-stranded nucleic acid, and wherein the primer comprises a sequence complementary to a region of the second strand of the target double-stranded nucleic acid; and (c) A polymerase adapted to extend a nucleic acid complementary to the second strand of the target double-stranded nucleic acid from the primer.

68. The system according to claim 67, wherein the target double-stranded nucleic acid is immobilized on the substrate.

69. The system according to claim 67, wherein the substrate comprises a flow cell.

70. The system according to claim 67, wherein the substrate comprises beads.

Images