Compositions and methods for preparing nucleic acid sequencing libraries using CRISPR / CAS9 immobilized on a solid support

Abstract

The present invention provides methods and compositions for generating fixed libraries of randomly fragmented double-stranded target nucleic acid fragments on a surface using immobilized CRISPR / Cas9 enzymes. The methods can be used to generate nucleic acid fragments for use in a variety of processes, including massively parallel nucleic acid sequencing.

Description

[0001] Cross-references to related patent applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 873,609, filed on July 12, 2019, which is incorporated herein by reference in its entirety for any purpose.

[0003] manual Technical Field

[0004] The present application relates to methods for generating libraries of randomly fragmented double-stranded nucleic acid fragments using CRISPR / Cas9 enzymes immobilized on solid supports, solid supports comprising immobilized CRISPR / Cas9 enzymes, and related compositions. The fixed libraries of randomly fragmented double-stranded nucleic acid fragments can be used as templates for various applications, including, for example, high-throughput, massively parallel and / or multiplexed nucleic acid sequencing. Background Art

[0005] There are a variety of methods and applications where it is desirable to generate libraries of randomly fragmented nucleic acid molecules from target double-stranded nucleic acid target molecules, such as double-stranded DNA (dsDNA) target molecules. Typically, the goal is to generate smaller nucleic acid molecules (e.g., nucleic acid fragments) from larger double-stranded nucleic acid molecules to serve as templates in nucleic acid sequencing reactions.

[0006] Many methods currently used to fragment and label double-stranded nucleic acids for use in next-generation sequencing waste nucleic acids, require expensive instruments to fragment, and the procedures for fragmenting, labeling, and recovering labeled nucleic acid fragments are difficult, cumbersome, laborious, time-consuming, inefficient, expensive, and require relatively large amounts of sample nucleic acid. In addition, the nucleic acid fragments generated by many of these methods do not fully represent the sequences contained in the sample nucleic acids that generated them. Therefore, what is needed in the art is a method that provides speed and ease of use when generating a library of randomly fragmented double-stranded nucleic acid fragments from a target double-stranded nucleic acid and can be easily applied to nucleic acid analysis methods (such as next-generation sequencing) and amplification methods. Summary of the invention

[0007] According to the present specification, the present application describes a method for preparing an immobilized library of randomly fragmented double-stranded nucleic acid fragments, the method comprising: (a) providing a solid support on which a CRISPR / Cas9 enzyme is immobilized; and (b) applying a target double-stranded nucleic acid to the solid support under conditions where the target double-stranded nucleic acid is randomly fragmented by the CRISPR / Cas9 enzyme and CRISPR / Cas9 binds to at least one strand of the double-stranded nucleic acid fragments; thereby generating an immobilized library of randomly fragmented double-stranded nucleic acid fragments.

[0008] In some embodiments, the target double-stranded nucleic acid comprises double-stranded DNA (dsDNA), double-stranded RNA (dsRNA), or double-stranded RNA / DNA hybrid. In some embodiments, the target double-stranded nucleic acid is dsDNA.

[0009] In some embodiments, the CRISPR / Cas9 enzyme is bound to a first polynucleotide that directs the CRISPR / Cas9 enzyme to bind to a target double-stranded nucleic acid in a non-sequence-specific manner. In some embodiments, the first polynucleotide is fixed to a solid support. In some embodiments, the first polynucleotide includes a 3' portion comprising a CRISPR / Cas9 terminal sequence and a 5' portion comprising a single-stranded guide RNA (sgRNA) that directs the CRISPR / Cas9 enzyme to bind to a target nucleic acid in a non-sequence-specific manner.

[0010] In some embodiments, the sgRNA comprises (GC) n or (AT) n , where n is 5-20, 10-15 or 10.

[0011] In some embodiments, the sgRNA is 10 to 40, 15 to 35, or 17 to 20 nucleotides.

[0012] In some embodiments, the first polynucleotide is biotinylated and the solid support comprises one or more biotin-binding proteins. In some embodiments, the biotin-binding protein comprises avidin, streptavidin, neutravidin, an anti-biotin antibody, a biotin receptor and / or a biotin-binding enzyme. In some embodiments, the biotin-binding enzyme comprises a biotinidase or a biotin carboxylation holoenzyme synthetase.

[0013] In some embodiments, the method includes washing the solid support having the double-stranded nucleic acid fragments immobilized thereon to remove any unbound nucleic acid.

[0014] In some embodiments, the method includes amplifying double-stranded nucleic acid fragments immobilized on a solid surface. In some embodiments, amplifying includes providing a polymerase and an amplification primer corresponding to a portion of the first polynucleotide.

[0015] In some embodiments, applying the target double-stranded nucleic acid to the solid support comprises treating the CRISPR / Cas9 enzyme with one or more agents that reduce the nucleic acid binding specificity of the CRISPR / Cas9 enzyme.

[0016] In some embodiments, the one or more agents include betaine, dimethyl sulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, and / or sulphalane.

[0017] In some embodiments, the CRISPR / Cas9 enzyme is expressed in an amount of at least 10 3 , 10 4 , 10 5 or 10 6 Enzyme / mm 2 The density is present on the solid support.

[0018] In some embodiments, the length of the double-stranded nucleic acid fragments in the immobilized library is proportional to the density of the CRISPR / Cas9 enzyme on the solid support.

[0019] In some embodiments, the solid support comprises microparticles, patterned surfaces, or wells. In some embodiments, the microparticles are beads.

[0020] In some embodiments, the method includes: (c) applying an intercalating dye to at least a portion of the immobilized library of double-stranded nucleic acid fragments to obtain a set of stained immobilized fragments; and obtaining an image of the stained immobilized fragments.

[0021] In some embodiments, applying the target double-stranded nucleic acid comprises adding a biological sample to a solid support. In some embodiments, the biological sample comprises a cell lysate. In some embodiments, the biological sample comprises whole cells. In some embodiments, the biological sample is selected from the group consisting of blood, plasma, serum, lymph, mucus, sputum, urine, semen, cerebrospinal fluid, bronchial aspirate, feces, and macerated tissue.

[0022] In some embodiments, the method comprises labeling the double-stranded nucleic acid fragments.

[0023] In some embodiments, the double-stranded nucleic acid fragment is labeled with a first tag comprising a first tag domain. In some embodiments, the first tag domain comprises a region for cluster amplification. In some embodiments, the first tag domain comprises a region for priming a sequencing reaction.

[0024] In some embodiments, the method includes releasing the fixed double-stranded nucleic acid fragment from a solid support. In some embodiments, releasing includes cleaving the CRISPR / Cas9 enzyme from a solid support. In some embodiments, releasing includes performing polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA) and nucleic acid sequence-based amplification (NASBA) or other amplification processes. In some embodiments, PCR includes suppressing PCR. In some embodiments, releasing includes applying light or heat.

[0025] The present application also describes a method for preparing an immobilized library of randomly fragmented double-stranded nucleic acid fragments, the method comprising: (a) providing a solid support having a CRISPR / Cas9 complex immobilized thereon, wherein the CRISPR / Cas9 complex comprises a CRISPR / Cas9 enzyme bound to a biotinylated first polynucleotide, the biotinylated first polynucleotide comprising a 3' portion comprising a CRISPR / Cas9 terminal sequence and a 5' portion comprising a single-stranded guide RNA (sgRNA), the single-stranded guide RNA directing the CRISPR / Cas9 enzyme to bind to a target nucleic acid in a non-sequence-specific manner, and wherein the biotinylated first polynucleotide is bound to a biotin-binding protein on the solid support; and (b) applying the target double-stranded nucleic acid to the solid support under conditions where the target double-stranded nucleic acid is randomly fragmented by the CRISPR / Cas9 complex and the CRISPR / Cas9 complex binds to at least one strand of the double-stranded nucleic acid fragments; thereby generating an immobilized library of randomly fragmented double-stranded nucleic acid fragments.

[0026] In some embodiments, the biotin binding protein comprises avidin, streptavidin, neutravidin, anti-biotin antibody, biotin receptor and / or biotin binding enzyme. In some embodiments, the biotin binding enzyme comprises biotinidase or biotin carboxylation holoenzyme synthetase.

[0027] In some embodiments, applying the target double-stranded nucleic acid to the solid support comprises treating the CRISPR / Cas9 enzyme with one or more agents that reduce the nucleic acid binding specificity of the CRISPR / Cas9 enzyme. In some embodiments, the one or more agents comprise betaine, dimethyl sulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, and / or sulfane.

[0028] In some embodiments, the method includes labeling the double-stranded nucleic acid fragments. In some embodiments, the double-stranded nucleic acid fragments are labeled with a first tag comprising a first tag domain. In some embodiments, the first tag domain comprises a region for cluster amplification. In some embodiments, the first tag domain comprises a region for initiating a sequencing reaction.

[0029] In some embodiments, the method includes releasing the fixed double-stranded nucleic acid fragment from a solid support. In some embodiments, releasing includes cleaving the CRISPR / Cas9 enzyme from the solid support. In some embodiments, releasing includes performing PCR or other amplification processes. In some embodiments, PCR includes inhibiting PCR. In some embodiments, releasing includes applying light or heat.

[0030] The present application also describes a solid support having immobilized thereon a library of double-stranded nucleic acid fragments, the library being prepared according to the method described herein.

[0031] The present application also describes a solid support having a CRISPR / Cas9 complex immobilized thereon, wherein the CRISPR / Cas9 complex comprises a CRISPR / Cas9 enzyme that randomly fragments a target double-stranded nucleic acid.

[0032] In some embodiments, the target double-stranded nucleic acid comprises double-stranded DNA (dsDNA), double-stranded RNA (dsRNA), or double-stranded RNA / DNA hybrid. In some embodiments, the target double-stranded nucleic acid is dsDNA.

[0033] In some embodiments, the CRISPR / Cas9 enzyme is bound to a first polynucleotide that directs the CRISPR / Cas9 enzyme to bind to a target double-stranded nucleic acid in a non-sequence-specific manner.

[0034] In some embodiments, the first polynucleotide is immobilized to a solid support.

[0035] In some embodiments, the first polynucleotide includes a 3' portion comprising a CRISPR / Cas9 terminal sequence and a 5' portion comprising a single-stranded guide RNA (sgRNA) that directs the CRISPR / Cas9 enzyme to bind to the target nucleic acid in a non-sequence-specific manner.

[0036] In some embodiments, the sgRNA comprises (GC) n or (AT) n , wherein n is 5-20 or 10-15. In some embodiments, n is 10.

[0037] In some embodiments, the sgRNA is 10 to 40 or 15 to 30 nucleotides. In some embodiments, the sgRNA is 17 or 20 nucleotides.

[0038] In some embodiments, the first polynucleotide is biotinylated and the solid support comprises a biotin-binding protein. In some embodiments, the biotin-binding protein comprises avidin, streptavidin, neutravidin, an anti-biotin antibody, a biotin receptor and / or a biotin-binding enzyme. In some embodiments, the biotin-binding enzyme comprises a biotinidase or a biotin carboxylation holoenzyme synthetase.

[0039] In some embodiments, the CRISPR / Cas9 enzyme is expressed in an amount of at least 10 3 , 10 4 , 10 5 or 10 6 Enzyme / mm 2 The density is present on the solid support.

[0040] In some embodiments, the solid support comprises microparticles, patterned surfaces, or wells. In some embodiments, the microparticles are beads.

[0041] The application also describes a composition comprising a solid support as described herein and one or more agents that reduce the nucleic acid binding specificity of the CRISPR / Cas9 enzyme. In some embodiments, the one or more agents include betaine, dimethyl sulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide, dimethylformamide and / or sulfane.

[0042] The present application also describes a solid support having a CRISPR / Cas9 complex immobilized thereon, wherein the CRISPR / Cas9 complex comprises a CRISPR / Cas9 enzyme bound to a biotinylated first polynucleotide, the biotinylated first polynucleotide comprising a 3' portion comprising a CRISPR / Cas9 terminal sequence and a 5' portion comprising a single-stranded guide RNA (sgRNA), the single-stranded guide RNA directing the CRISPR / Cas9 enzyme to bind to a target double-stranded nucleic acid in a non-sequence-specific manner, and wherein the biotinylated first polynucleotide is bound to a biotin-binding protein on the solid support.

[0043] In some embodiments, the biotin binding protein comprises avidin, streptavidin, neutravidin, anti-biotin antibody, biotin receptor and / or biotin binding enzyme. In some embodiments, the biotin binding enzyme comprises biotinidase or biotin carboxylation holoenzyme synthetase.

[0044] In some embodiments, the CRISPR / Cas9 complex is expressed in at least 10 3 , 10 4 , 10 5 or 10 6 Composites / mm 2 The density is present on the solid support.

[0045] In some embodiments, the solid support comprises microparticles, patterned surfaces, or wells. In some embodiments, the microparticles are beads.

[0046] The application also describes a composition comprising a solid support as described herein and one or more agents that reduce the nucleic acid binding specificity of the CRISPR / Cas9 enzyme. In some embodiments, the one or more agents include betaine, dimethyl sulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide, dimethylformamide and / or sulfane.

[0047] Other purposes and advantages will be explained in part in the following description, and in part will be obvious from the description, or can be learned through practice. These purposes and advantages will be realized and achieved by means of the elements and combinations particularly pointed out in the appended claims.

[0048] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0049] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one embodiment and, together with the description, serve to explain the principles described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 An embodiment of the present invention is shown. In this embodiment, the CRISPR / Cas9 complex is fixed on a surface. The CRISPR / Cas9 complex has a CRISPR / Cas9 (Cas9) enzyme and a biotinylated polynucleotide, which includes a 3' portion comprising a CRISPR / Cas9 terminal sequence and a 5' portion comprising a single-stranded guide RNA (sgRNA). The target double-stranded nucleic acid is applied to a solid support, randomly fragmented by the CRISPR / Cas9 complex, and the double-stranded nucleic acid fragments bind to the CRISPR / Cas9 complex fixed on the surface. The size of the nucleic acid fragment depends on the distance between the CRISPR / Cas9 enzymes fixed on the surface. DETAILED DESCRIPTION

[0051] Current protocols for sequencing nucleic acid samples typically employ sample preparation methods that convert nucleic acids (such as DNA or RNA or RNA / DNA hybrids) into template libraries. These methods can result in loss of nucleic acid samples and typically require expensive instruments for fragmentation. In addition, these sample preparation methods are typically difficult, cumbersome, and inefficient.

[0052] In standard sample preparation methods, each template contains adapter sequences at either end of the insert sequence, and many steps are typically required to modify the nucleic acid and purify the desired products of the modification reactions. These steps are performed in solution before adding the adapted fragments to a flow cell, where they are coupled to a surface via a primer extension reaction that replicates the hybridized fragments to the ends of primers covalently attached to the surface. These "breeding" templates are then passed through multiple cycles of amplification to produce monoclonal clusters of replicated templates.

[0053] By using CRISPR / Cas9 enzyme-mediated fragmentation, the number of steps required to convert a target double-stranded nucleic acid (such as DNA) into randomly fragmented double-stranded nucleic acid fragments in solution for cluster formation and sequencing can be minimized. After a purification step to remove any unbound nucleic acid, additional sequences are added to the ends of the nucleic acid fragments by PCR.

[0054] Solution-based fragmentation has disadvantages and requires several labor-intensive steps. In addition, biases may be introduced during the polymerase chain reaction (PCR) amplification step. The methods and compositions presented herein overcome these disadvantages and allow for unbiased sample preparation, cluster formation, and sequencing on a single solid support with minimal sample manipulation or transfer requirements.

[0055] The present disclosure relates to the surprising discovery that a CRISPR / Cas9 enzyme pre-coupled to the surface of a solid support (such as a flow cell) can be used under conditions that effectively randomly fragment an intact target nucleic acid and immobilize it on the solid support. In a specific embodiment, one or more strands comprising the CRISPR / Cas9 enzyme are attached to the surface of the solid support via their 5' ends. The CRISPR / Cas9 enzyme can also be contained within a complex that contains sequences that enable subsequent cluster generation and sequencing.

[0056] Method and composition proposed herein provide several advantages that are superior to solution-based fragmentation methods and less random fragmentation methods.For example, purification, partial purification or even unpurified complete target nucleic acid can be directly loaded onto solid support or flow cell to generate cluster, without the need to prepare samples in advance.In addition, the continuity of sequence information in the original complete nucleic acid can be physically retained by juxtaposing the fragment on the surface of solid support or flow cell.As another advantage, nucleic acid fragments are physically connected to the surface of solid support or flow cell, so after further operating nucleic acid fragments, reagents are purified and can be realized by, for example, the flow buffer exchange in the flow cell channel.

[0057] Furthermore, improving random fragmentation of target double-stranded nucleic acids allows libraries of randomly fragmented double-stranded nucleic acid fragments to better represent the original sample.

[0058] I. Random fragmentation on solid supports

[0059] According to the above, the present invention proposes a method for preparing a fixed library of randomly fragmented double-stranded nucleic acid fragments, and a solid support having a library of double-stranded nucleic acid fragments fixed thereon prepared according to such a method. In some embodiments, the method includes: (a) providing a solid support having a CRISPR / Cas9 enzyme fixed thereon; and (b) applying the target double-stranded nucleic acid to the solid support under conditions where the target double-stranded nucleic acid is randomly fragmented by the CRISPR / Cas9 enzyme and CRISPR / Cas9 binds to at least one strand of the double-stranded nucleic acid fragment; thereby generating a fixed library of randomly fragmented double-stranded nucleic acid fragments.

[0060] In some embodiments, the CRISPR / Cas9 enzyme is directly immobilized to a solid support.

[0061] In some embodiments, the method comprises: (a) providing a solid support having a CRISPR / Cas9 complex immobilized thereon, wherein the CRISPR / Cas9 complex comprises a CRISPR / Cas9 enzyme bound to a biotinylated first polynucleotide, the biotinylated first polynucleotide comprising a 3' portion comprising a CRISPR / Cas9 terminal sequence and a 5' portion comprising a single-stranded guide RNA (sgRNA) that directs the CRISPR / Cas9 enzyme to bind to a target double-stranded nucleic acid in a non-sequence-specific manner, and wherein the biotinylated first polynucleotide is bound to a biotin-binding protein on the solid support; and (b) applying the target double-stranded nucleic acid to the solid support under conditions where the target double-stranded nucleic acid is randomly fragmented by the CRISPR / Cas9 complex and the CRISPR / Cas9 complex binds to at least one strand of the double-stranded nucleic acid fragments; thereby generating an immobilized library of randomly fragmented double-stranded nucleic acid fragments.

[0062] As used herein, the term "random fragmentation" refers to fragmenting a target nucleic acid in a random manner to produce a series of fragments (e.g., fragments generated by fragmentation at random positions from a sequence perspective). In some embodiments, the target nucleic acid is randomly fragmented in a non-sequence-specific manner to produce a series of fragment identities. Regardless of the input amount of DNA, fragmentation can be controlled by the present invention in terms of normalized product amount and fragment size (i.e., controllable size selection). For example, Figure 1 As shown, the distance between adjacent CRISPR / Cas9 enzymes on a solid support can determine the size range of the fragments immobilized on the surface of the solid support.

[0063] In some embodiments, the target nucleic acid is fragmented in a sequence-specific manner. For example, as described below, two CRISPR / Cas9 enzyme complexes on the same solid support can include a pair of sgRNAs located on both sides of the region of interest in the target nucleic acid. In such embodiments, each pair of sgRNAs produces a single fragment. In other embodiments, a group of solid carriers (such as beads) is used, and each solid carrier in the group of solid carriers can carry different sgRNA pairs. In this way, different solid carriers will target different sequences in the target nucleic acid. In some embodiments, different sgRNAs target different target sequences in the genome. These methods can achieve multiple targeting.

[0064] As used herein, the term "CRISPR / Cas9 enzyme" generally refers to clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (Cas9). Cas9 (also known as Csn1) is a CRISPR-associated protein comprising two nuclease domains, namely the RuvC nuclease domain and the HNH nuclease domain, which is programmed by small RNA to cleave nucleic acids (e.g., DNA). CRISPR / Cas9 enzymes are generally known to those skilled in the art, as exemplified in the disclosure of U.S. Patent Application Publication No. 2019 / 0202856, the contents of which are incorporated herein by reference in their entirety. For example, the engineered CRISPR / Cas9 enzyme can be derived from Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacter jejuni Cas9 (CjCas9), Francisella novicida Cas9 (FnCas9) or Neisseria cinerea Cas9 (NcCas9). Additional variants of Cas9 known to cleave nucleic acids are known to those skilled in the art, including wild-type or naturally occurring Cas9 and mutant or modified Cas9 (e.g., Cas9D10A). It should be understood that any CRISPR / Cas9 enzyme capable of fragmenting or cleaving a target nucleic acid can be used in the present invention under conditions where the CRISPR / Cas9 enzyme is capable of randomly fragmenting or cleaving the target DNA.

[0065] As used herein, the term "CRISPR / Cas9 enzyme complex" generally refers to a CRISPR / Cas9 enzyme bound to a first polynucleotide that directs the CRISPR / Cas9 enzyme to bind to a target double-stranded nucleic acid in a non-sequence-specific or sequence-specific manner. In some embodiments, the first polynucleotide is fixed to a solid support. In some embodiments, the first polynucleotide includes a 3' portion comprising a CRISPR / Cas9 terminal sequence and a 5' portion comprising a single-stranded guide RNA (sgRNA) that directs the CRISPR / Cas9 enzyme to bind to a target nucleic acid in a non-sequence-specific or sequence-specific manner. In some embodiments, the first polynucleotide is biotinylated and the solid support includes one or more biotin-binding proteins. For example, a CRISPR / Cas9 enzyme complex may comprise a CRISPR / Cas9 enzyme bound to a biotinylated first polynucleotide comprising a 3' portion comprising a CRISPR / Cas9 terminal sequence and a 5' portion comprising a single-stranded guide RNA (sgRNA) that directs the CRISPR / Cas9 enzyme to bind to a target nucleic acid in a non-sequence-specific or sequence-specific manner, and wherein the biotinylated first polynucleotide is bound to a biotin-binding protein on a solid support.

[0066] As used herein, the term "double-stranded nucleic acid" refers to double-stranded DNA (dsDNA), double-stranded RNA (dsRNA), or double-stranded RNA / DNA hybrids. In some embodiments, the target double-stranded nucleic acid is dsDNA. Although the term "DNA" is used in conjunction with the target double-stranded nucleic acid molecule throughout this disclosure, it should be understood that any suitable nucleic acid or nucleic acid analog can be randomly fragmented.

[0067] The term "CRISPR / Cas9 end" refers to a double-stranded nucleic acid that exhibits only the nucleotide sequence necessary to form a complex with the CRISPR / Cas9 enzyme ("CRISPR / Cas9 end sequence"). The CRISPR / Cas9 end may comprise any nucleic acid or nucleic acid analog suitable for forming a functional complex with the CRISPR / Cas9 enzyme. For example, the CRISPR / Cas9 end may comprise DNA, RNA, modified bases, non-natural bases, a modified backbone, and may comprise a nick in one or both strands.

[0068] As used herein, the terms "tag" and "tag domain" refer to a portion or domain of a polynucleotide that exhibits a sequence for a desired intended purpose or application. The tag domain may include any sequence provided for any desired purpose. In some embodiments, the tag includes one or more functional sequences selected from the group consisting of the following items: a universal sequence, a primer sequence, an index sequence, a capture sequence, a barcode sequence (for example, counting or error correction), a cleavage sequence, a sequencing-related sequence, an enrichment sequence, and a combination thereof. For example, in some embodiments, the tag domain includes one or more restriction endonuclease recognition sites. In some embodiments, the tag domain includes one or more regions suitable for hybridizing with primers for cluster amplification reactions. In some embodiments, the tag domain includes one or more regions suitable for hybridizing with primers for sequencing reactions. It should be understood that any other suitable features can be incorporated into the tag domain. In some embodiments, the tag domain includes a sequence having a length between 5bp and 200bp. In some embodiments, the tag domain includes a sequence having a length between 10bp and 100bp. In some embodiments, the tag domain includes a sequence having a length between 20bp and 50bp. In some embodiments, the tag domain comprises a sequence between 5bp, 6bp, 7bp, 8bp, 9bp, 10bp, 20bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, 90bp, 100bp, 150bp, and 200bp in length.

[0069] In the methods and compositions proposed herein, the CRISPR / Cas9 enzyme is fixed to a solid support. In some embodiments, the CRISPR / Cas9 enzyme is fixed to a support via one or more polynucleotides. In some embodiments, one or more polynucleotides (such as a first polynucleotide) guide the CRISPR / Cas9 enzyme to bind to a target double-stranded nucleic acid in a non-sequence-specific manner (i.e., conditions that guide the CRISPR / Cas9 enzyme to randomly fragment or cleave a target double-stranded nucleic acid). In some embodiments, one or more polynucleotides guide the CRISPR / Cas9 enzyme to bind to a target double-stranded nucleic acid in a sequence-specific manner, and fragmentation is achieved by using a group of solid supports, wherein each solid support includes: a first CRISPR / Cas9 complex comprising a first polynucleotide, the first polynucleotide guiding the CRISPR / Cas9 enzyme to bind to the 3' of the sequence of interest in the target nucleic acid; and a second CRISPR / Cas9 complex comprising a second polynucleotide, the second polynucleotide guiding the CRISPR / Cas9 enzyme to bind to the 5' of the sequence of interest in the target nucleic acid.

[0070] In some embodiments, the first polynucleotide is fixed to a solid support. In some embodiments, the first polynucleotide includes a 3' portion comprising a CRISPR / Cas9 terminal sequence and a 5' portion comprising a single-stranded guide RNA (sgRNA), which guides the CRISPR / Cas9 complex and can be fixed directly or via a linker molecule that couples the CRISPR / Cas9 enzyme to a solid support.

[0071] As used herein, the term "single-stranded guide RNA" or "sgRNA" refers to a single-stranded RNA that is capable of hybridizing to a target sequence in a target double-stranded nucleic acid of interest. The sgRNA interacts with the CRISPR / Cas9 enzyme and the target sequence (i.e., the protospacer sequence) such that it guides the CRISPR / Cas9 enzyme to the target sequence, where the CRISPR / Cas9 enzyme cleaves the target sequence.

[0072] In some embodiments, in the case of directing the CRISPR / Cas9 enzyme in a non-sequence-specific manner, the target sequence has no sequence restrictions except that the sequence is adjacent to the protospacer sequence adjacent motif (PAM). For example, the PAM sequence of the Cas9 protein includes but is not limited to 5'-NGG, 5'-NGGNG, 5'-NNAGAAW, and 5'-ACAY. In some embodiments, in the case of directing the CRISPR / Cas9 enzyme in a sequence-specific manner, the target sequence has sequence restrictions.

[0073] In some embodiments, the sgRNA is selected to guide CRISPR / Cas9 in a non-sequence-specific manner, such that the CRISPR / Cas9 enzyme will cleave the nucleic acid in a random manner, thereby generating a range of fragment sizes.

[0074] In some embodiments, the sgRNA is 10 to 40 nucleotides, 15 to 30 nucleotides, or 17 or 20 nucleotides. Examples of sgRNAs include, but are not limited to (GC) n or (AT) n A short random oligonucleotide (randomer) or sequence, wherein n is 5 to 20, 8 to 15 or 10. Those skilled in the art are familiar with sgRNA design and construction, for example, sgRNA design tools are available on the Internet or from commercial sources. sgRNA can be chemically synthesized, enzymatically synthesized, or a combination thereof.

[0075] In some embodiments, the first polynucleotide is "biotinylated" and the solid support includes one or more biotin-binding proteins. As used herein, the term "biotinylated" refers to the process of covalently attaching biotin. Biotin-binding proteins are well known to those of ordinary skill in the art and include, but are not limited to, avidin, streptavidin, neutravidin, anti-biotin antibodies, biotin receptors, and biotin-binding enzymes, such as biotinidase, biotin carboxylation holoenzyme synthetase, and the like.

[0076] In some embodiments, one or more agents that reduce the nucleic acid binding specificity of a nucleic acid binding enzyme (such as CRISPR / Cas9) can be used alone or in combination with sgRNA (i.e., another condition that directs the CRISPR / Cas9 enzyme to randomly fragment or cleave a target double-stranded nucleic acid). In some embodiments, the CRISPR / Cas9 enzyme is treated with one or more agents to reduce the nucleic acid binding specificity of the CRISPR / Cas9 enzyme, and random fragmentation is induced when the target double-stranded nucleic acid is applied to a solid support on which the treated CRISPR / Cas9 enzyme is fixed. Binding specificity reducing agents are well known to those of ordinary skill in the art. Examples of binding specificity reducing agents include, but are not limited to, betaine, dimethyl sulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, and / or sulfane.

[0077] In some embodiments, conditions that direct the CRISPR / Cas9 enzyme to randomly fragment or cleave a target double-stranded nucleic acid may include, in addition to or as an alternative to other conditions, the use of high glycerol concentrations (e.g., >5% v / v), high concentrations of CRISPR / Cas9 enzyme / μg DNA (e.g., 100 units / μg), non-optimal buffers (e.g., with non-optimal ionic strength or pH), extended reaction times, and / or the use of Mg 2+ Other divalent cations (e.g., Mn 2+ , Cu 2+ 、Co 2+ and / or Zn 2+ ).

[0078] When referring to the immobilization of a molecule (e.g., a nucleic acid) to a solid support, the terms "immobilized" and "attached" are used interchangeably herein, and unless otherwise indicated explicitly or by context, both terms are intended to encompass direct or indirect, covalent or non-covalent attachment. In certain embodiments of the invention, covalent attachment may be preferred, but it is generally desired that the molecule (e.g., nucleic acid) remain immobilized or attached to the support under the conditions under which the support is intended to be used (e.g., in applications requiring nucleic acid amplification and / or sequencing).

[0079] Certain embodiments of the present invention can utilize solid carriers consisting of inert substrates or matrix (e.g., slides, polymer beads, etc.), which have been functionalized, for example, by applying an intermediate material layer or coating comprising reactive groups, which allow covalent attachment to biomolecules such as polynucleotides. Examples of such carriers include, but are not limited to, polyacrylamide hydrogels loaded on inert substrates (such as glass), especially polyacrylamide hydrogels as described in WO 2005 / 065814 and US2008 / 0280773, the contents of which are incorporated herein by reference in their entirety. In such embodiments, biomolecules (e.g., polynucleotides) can be covalently attached directly to intermediate materials (e.g., hydrogels), but intermediate materials themselves can be non-covalently attached to substrates or matrix (e.g., glass substrates). The term "covalently attached to a solid carrier" should be interpreted accordingly as encompassing this type of arrangement.

[0080] The terms "solid surface," "solid support," and other grammatical equivalents herein refer to any material that is suitable or can be modified to be suitable for attachment of CRISPR / Cas9 enzymes and CRISPR / Cas9 complexes. In some embodiments, the solid support comprises microparticles (such as beads), patterned surfaces, or wells. As will be appreciated by those skilled in the art, the number of possible substrates is very large. Possible substrates include, but are not limited to, glass and modified or functionalized glass, plastics (including, for example, acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon TM The solid supports and solid surfaces particularly useful for some embodiments are located within a flow cell device. Exemplary flow cells are described in further detail below.

[0081] In some embodiments, the solid support includes a patterned surface suitable for fixing CRISPR / Cas9 enzymes or complexes in an ordered pattern. "Patterned surface" refers to the arrangement of different regions in or on the exposed layer of the solid support. For example, one or more of these regions may be a feature portion where one or more CRISPR / Cas9 enzymes or complexes are present. These feature portions may be separated by gap regions where there is no CRISPR / Cas9 enzyme or complex. In some embodiments, the pattern may be an xy format of a feature portion in the form of rows and columns. In some embodiments, the pattern may be a repeated arrangement of a feature portion and / or gap regions. In some embodiments, the pattern may be a random arrangement of a feature portion and / or gap regions. In some embodiments, CRISPR / Cas9 enzymes or complexes are randomly distributed on the solid support. In some embodiments, CRISPR / Cas9 enzymes or complexes are distributed on a patterned surface. Exemplary patterned surfaces that may be used in the methods and compositions described herein are described in U.S. Patent Application Publications 2012 / 0316086A1 and 2013 / 0116153A1, each of which is incorporated herein by reference.

[0082] In some embodiments, the solid support includes an array of holes or depressions in the surface. This can be manufactured using a variety of techniques as generally known in the art, including but not limited to photolithography, imprinting techniques, molding techniques, and micro-etching techniques. Those skilled in the art will appreciate that the technique used will depend on the composition and shape of the array substrate.

[0083] The composition and geometry of the solid carrier can vary with its use. In some embodiments, the solid carrier is a planar structure, such as a slide, a chip, a microchip and / or an array. Therefore, the surface at the bottom of the substrate can be in the form of a planar layer. In some embodiments, the solid carrier includes one or more surfaces of a flow cell. As used herein, the term "flow cell" refers to a chamber comprising a solid surface, and one or more fluid reagents can flow through the solid surface. Examples of flow cells and related fluid systems and detection platforms that can be readily used in the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008); WO 04 / 018497, US 7,057,026, WO 91 / 06678, WO07 / 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.

[0084] 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 comprises microspheres or beads. The so-called "microspheres" or "beads" or "particles" or grammatical equivalents herein refer to small discrete particles. Suitable bead compositions include, but are not limited to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thoria sols, carbon graphite, titanium dioxide, latex or cross-linked dextran (such as agarose gel), cellulose, nylon, cross-linked micelles and Teflon TM , and any other material outlined herein for solid supports may be used. The "Microsphere Detection Guide" by Bangs Laboratories of Fisher, Indiana is a useful guide. In certain embodiments, the microspheres are magnetic microspheres or beads.

[0085] The beads need not be spherical; irregular particles may be used. Alternatively or in addition, the beads may be porous. Bead sizes range from nanometers (i.e., 100 nm) to millimeters (i.e., 1 mm), with beads of about 0.2 microns to about 200 microns being preferred, and beads of about 0.5 microns to about 5 microns being particularly preferred, although smaller or larger beads may be used in some embodiments.

[0086] Figure 1 A method according to one embodiment is generally shown. A CRISPR / Cas9 complex is immobilized on a surface. The CRISPR / Cas9 complex has a CRISPR / Cas9 enzyme ("Cas9") and a biotinylated polynucleotide comprising a 3' portion comprising a CRISPR / Cas9 terminal sequence and a 5' portion comprising a single-stranded guide RNA (sgRNA). A target double-stranded nucleic acid is applied to a solid support, randomly fragmented by the CRISPR / Cas9 complex, and the double-stranded nucleic acid fragments bind to the CRISPR / Cas9 complex immobilized on the surface. The size of the nucleic acid fragments depends on the distance between the immobilized CRISPR / Cas9 enzyme on the surface. For example, in some embodiments, the CRISPR / Cas9 enzyme or complex is at least 10 3 , 10 4 , 10 5 or at least 10 6 Composites / mm 2 The density is present on the solid support.

[0087] When the target double-stranded nucleic acid is applied to the solid support, the CRISPR / Cas9 enzyme will fragment the target double-stranded nucleic acid, thereby generating randomly fragmented double-stranded nucleic acid fragments that are coupled to the surface of the solid support at both ends. In some embodiments, the length of the double-stranded nucleic acid fragments can be changed by changing the density of the CRISPR / Cas9 enzyme or its complex on the surface. In some embodiments, the length of the double-stranded nucleic acid fragments in the fixed library is proportional to the density of the CRISPR / Cas9 enzyme or complex on the solid support. In certain embodiments, the length of the resulting double-stranded nucleic acid fragment is less than 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 1100 bp, 1200 bp, 1300 bp, 1400 bp, 1500 bp, 1600 bp, 1700 bp, 1800 bp, 1900 bp, 2000 bp, 2100 bp, 2200 bp, 2300 bp, 2400 bp, 2500 bp, 2600 bp, 2700 bp, 2800 bp, 2900 bp, 3000 bp, 3100 bp, 3200 bp, 3300 bp, 3400 bp, 3500 bp, 3600 bp, 3700 bp, 3800 bp, 3900 bp, 4000 bp, 4100 bp, 4200 bp, 4300 bp, 4400 bp, 4500 bp, 4600 bp, 4700 bp, 4800 bp, 4900 bp, 5000 bp, 5100 bp, 5200 bp, 5300 bp, 5400 bp, 5500 bp, 5600 bp, 5700 bp, 5800 bp, 5900 bp, 6000 bp, 6100 bp, 4700bp, 4800bp, 4900bp, 5000bp, 10000bp, 30000bp or less than 100,000bp. In such embodiments, the double-stranded nucleic acid fragments can then be amplified into clusters using standard cluster chemistry, as exemplified by the disclosures of U.S. Patents 7,985,565 and 7,115,400, each of which is incorporated herein by reference in its entirety.

[0088] In some embodiments, the length of the template is longer than the length of a template that can be properly amplified using standard cluster chemistry. For example, in some embodiments, the length of the template is longer than 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 1100 bp, 1200 bp, 1300 bp, 1400 bp, 1500 bp, 1600 bp, 1700 bp, 1800 bp, 1900 bp, 2000 bp, 2100 bp, 2200 bp, 2300 bp, 2400 bp, 2500 bp, 2600 bp, 2700 bp, 2800 bp, 2900 bp, 3000 bp, 3100 bp, 3200 bp, 3300 bp, 3400 bp, 3500 bp, 3600 bp, 3700 bp, 3800 bp, 3900 bp, 4000 bp, 4100 bp, 4200 bp, 4300 bp, 4400 bp, 4500 bp, 4600 bp, 4700 bp, 4800 bp, 4900 bp, 5000 bp, 5100 bp, 5200 bp, 5300 bp, 5400 bp, 5500 bp, 5600 bp, 5700 bp, 5800 bp, 5900 bp, 61 bp, 4800bp, 4900bp, 5000bp, 10000bp, 30000bp or longer than 100,000bp. In a specific embodiment, the length of the template can be within the range defined by the upper and lower limits selected from those exemplified above.

[0089] In certain embodiments, before cluster generation, double-stranded nucleic acid fragments immobilized on the surface of a solid support can be imaged. For example, an intercalating dye can be applied to at least a portion of a fixed library of double-stranded nucleic acid fragments to obtain a set of stained fixed fragments, which can be imaged to retain a record of the position of the backbone of the nucleic acid molecules on the surface. After cluster generation and sequencing, the coordinates of the clusters can be associated with their positions on the original backbone, thereby facilitating read alignment along molecular and genomic assemblies.

[0090] In some embodiments, the step of applying the target double-stranded nucleic acid includes adding the biological sample to the solid carrier. The biological sample can be any type that contains nucleic acid and can be deposited on a solid surface for fragmentation. For example, the sample can include nucleic acids in various purified states, including purified nucleic acids. However, the sample does not need to be completely purified, and can include nucleic acids mixed with proteins, other nucleic acid substances, other cell components, and / or any other contaminants. In some embodiments, the biological sample includes a mixture of DNA, proteins, other nucleic acid substances, other cell components, and / or any other contaminants that exist in roughly the same proportion as found in vivo. For example, in some embodiments, these components exist in the same proportion as found in intact cells. In some embodiments, the biological sample has a 260 / 280 ratio of less than 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, or less than 0.60. In some embodiments, the biological sample has a 260 / 280 ratio of at least 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7 or at least 0.60. Because the method provided herein allows nucleic acid to be combined with a solid support, any unbound nucleic acid or other contaminants can be removed only by washing the solid support after surface binding fragmentation occurs. The biological sample can include, for example, a crude cell lysate or a whole cell. For example, the crude cell lysate applied to the solid support in the method described herein does not need to undergo one or more separation steps traditionally used to separate nucleic acids from other cellular components. Exemplary separation steps are described in Maniatis et al., Molecular Cloning: A Laboratory Manual, 2d edition, 1989 and Short Protocols in Molecular Biology, edited by Ausubel et al., which are incorporated herein by reference.

[0091] Thus, in some embodiments, the biological sample may include, for example, blood, plasma, serum, lymph, mucus, sputum, urine, semen, cerebrospinal fluid, bronchial aspirate, stool, and macerated tissue or lysates thereof, or any other biological specimen containing nucleic acids. One advantage of the methods and compositions presented herein is that the biological sample can be added to the flow cell, and the subsequent lysis and purification steps can all be performed in the flow cell without further transfer or handling steps, simply by flowing the necessary reagents into the flow cell.

[0092] The present invention also provides a solid support prepared according to the above method and having a library of labeled nucleic acid fragments immobilized thereon.

[0093] II. Physical Mapping of Immobilized Polynucleotide Molecules

[0094] The method for the physical map of generating fixed polynucleotide is also proposed herein.These methods can be advantageously utilized to identify the cluster that may comprise the connection sequence (that is, from the first part and the second part of the same target polynucleotide molecule).Therefore, the relative proximity of any two clusters produced by fixing the polynucleotide provides the information that can be used for comparing the sequence information obtained from two clusters.Particularly, the distance between any two given clusters on the solid surface and the probability positive correlation of two clusters from the same target polynucleotide molecule are as described in more detail in WO 2012 / 025250, which is incorporated herein by reference in its entirety.

[0095] For example, in some embodiments, long dsDNA molecules stretched on the surface of the flow cell are fragmented in situ, thereby forming a connected dsDNA bridge on the surface of the flow cell. In addition, a physical map of the fixed DNA can then be generated. Therefore, after amplifying the fixed DNA, the physical map is related to the physical relationship of the clusters. Specifically, the physical map is used to calculate the probability that the sequence data obtained from any two clusters are connected, as described in the incorporated materials of WO 2012 / 025250.

[0096] In some embodiments, a physical map is generated by imaging nucleic acid to determine the position of fixed nucleic acid molecules on the entire solid surface. In some embodiments, fixed nucleic acid is imaged by adding an imaging agent to a solid support and detecting the signal from the imaging agent. In some embodiments, the imaging agent is a detectable label. Suitable detectable labels include but are not limited to protons, haptens, radionuclides, enzymes, fluorescent labels, chemiluminescent labels and / or developers. For example, in some embodiments, the imaging agent is an intercalating dye or a non-intercalating nucleic acid binding agent. Any suitable intercalating dye or non-intercalating nucleic acid binding agent as known in the art can be used, including but not limited to those described in US2012 / 0282617, which is incorporated herein by reference in its entirety.

[0097] In some embodiments, before cluster generation, further fragmentation fixation double-stranded nucleic acid fragments are released free ends. Cleavage bridging structures can be performed using any suitable method known in the art, as exemplified by the incorporation of materials of WO 2012 / 025250. For example, cleavage can occur as described in WO2012 / 025250 by incorporating modified nucleotides (such as uracil), by incorporating restriction endonuclease sites, or by applying solution phase CRISPR / Cas9 enzymes or complexes to bridged nucleic acid structures, as described elsewhere herein.

[0098] In certain embodiments, multiple target double-stranded nucleic acid molecules flow to a flow cell including multiple nanochannels, and the nanochannel has multiple CRISPR / Cas9 enzymes or complexes fixed thereto. As used herein, the term nanochannel refers to a narrow channel into which a long linear nucleic acid molecule flows. In some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or no more than 1000 single long chains of target nucleic acid flow into each nanochannel. In some embodiments, a single nanochannel is separated by a physical barrier that prevents a single long chain of a target nucleic acid from interacting with multiple nanochannels. In some embodiments, the solid support comprises at least 10, 50, 100, 200, 500, 1000, 3000, 5000, 10000, 30000, 50000, 80000, or at least 100000 nanochannels. In some embodiments, a CRISPR / Cas9 enzyme or complex bound to the nanochannel surface fragments nucleic acids. Continuity mapping can then be performed, for example, by following the cluster down the length of one of these channels. In some embodiments, the length of the long strand of the target nucleic acid can be at least 0.1 kb, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb, 50 kb, 55 kb, 60 kb, 65 kb, 70 kb, 75 kb, 80 kb, 85 kb, 90 kb, 95 kb, 100kb, 150kb, 200kb, 250kb, 300kb, 350kb, 400kb, 450kb, 500kb, 550kb, 600kb, 650kb, 700kb, 750kb, 800kb, 850kb, 900kb, 950kb, 1000kb, 5000kb, 10000kb, 20000kb, 30000kb or at least 50000kb.In some embodiments, the length of the long strand of the target nucleic acid is no more than 0.1 kb, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb, 50 kb, 55 kb, 60 kb, 65 kb, 70 kb, 75 kb, 80 In some embodiments, the fixed fragmentation product of nanochannel can be used for sequencing. In some embodiments, the fixed fragmentation product of nanochannel can be used for analyzing haplotype. In some embodiments, the fixed fragmentation product of nanochannel can be used for solving the phasing problem.

[0099] III. Amplification and sequencing of fixed nucleic acid fragments

[0100] Amplification The present disclosure also relates to amplifying fixed nucleic acid fragments produced according to the methods provided herein. Fixed nucleic acid fragments produced by surface-bound CRISPR / Cas9 enzyme-mediated fragmentation can be amplified according to any suitable amplification method known in the art. In some embodiments, fixed DNA fragments are amplified on a solid support. In some embodiments, the solid support is the same as the solid support on which surface-bound fragmentation occurs. In such embodiments, the methods and compositions provided herein allow sample preparation by amplification and optionally by a sequencing step on the same solid support from the initial sample introduction step.

[0101] For example, in some embodiments, a cluster amplification method is used to amplify fixed nucleic acid fragments, as exemplified by the disclosure of U.S. Patents 7,985,565 and 7,115,400, each of which is incorporated herein by reference in its entirety. U.S. Patents 7,985,565 and 7,115,400 incorporate materials describing methods for solid phase nucleic acid amplification, which allow amplification products to be fixed on a solid support to form an array consisting of a cluster or "cluster" of fixed nucleic acid molecules. Each cluster or cluster on such arrays is formed by a plurality of identical fixed polynucleotide chains and a plurality of identical fixed complementary polynucleotide chains. The array so formed is generally referred to as "clustered array" herein. The product of solid phase amplification reaction (such as those described in U.S. Patents 7,985,565 and 7,115,400) is a so-called "bridging" structure, which is formed by annealing paired fixed polynucleotide chains and fixed complementary chains (two chains are preferably fixed on a solid support via covalent attachment at 5' ends). Cluster amplification method is an example of a method in which a fixed nucleic acid template is used to produce a fixed amplicon. Other suitable methods may also be used to produce a fixed amplicon by the fixed nucleic acid fragment produced according to the method provided herein. For example, no matter whether one or both primers in each pair of amplification primers are fixed, one or more clusters or colonies can be formed via solid phase PCR.

[0102] In other embodiments, the fixed nucleic acid fragments are amplified in solution. For example, in some embodiments, the fixed nucleic acid fragments are cracked or otherwise released from a solid support, and then the amplification primers are hybridized with the released molecules in solution. In other embodiments, the amplification primers are hybridized with the fixed nucleic acid fragments to carry out one or more initial amplification steps, and then subsequent amplification steps are carried out in solution. Therefore, in some embodiments, the fixed nucleic acid template can be used to produce a solution phase amplicon.

[0103] It should be understood that any of the amplification methods described herein or generally known in the art can be used to amplify fixed nucleic acid fragments together with universal primers or target-specific primers. Suitable amplification methods include, but are not limited to, polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA), and nucleic acid sequence-based amplification (NASBA), as described in U.S. Patent 8,003,354, which is incorporated herein by reference in its entirety. The above-mentioned amplification methods can be used to amplify one or more nucleic acids of interest. For example, PCR (including multiplex PCR), SDA, TMA, NASBA, etc. can be used to amplify fixed nucleic acid fragments. In some embodiments, primers specific for nucleic acids of interest are included in the amplification reaction.

[0104] Other suitable nucleic acid amplification methods can include oligonucleotide extension and ligation, rolling circle amplification (RCA) (Lizardi et al., Nat. Genet. 19: 225-232 (1998), which is incorporated herein by reference) and oligonucleotide ligation assay (OLA) (see generally U.S. Patents 7,582,420, 5,185,243, 5,679,524 and 5,573,907; EP 0 320308B1; EP 0 336 731B1; EP0 439 182B1; WO 90 / 01069; WO 89 / 12696 and WO 89 / 09835, all of which are incorporated by reference) technology. It should be understood that these amplification methods can be designed to amplify fixed nucleic acid fragments. For example, in some embodiments, the amplification method can include a ligation probe amplification or an oligonucleotide ligation assay (OLA) reaction containing primers specific for the nucleic acid of interest. In some embodiments, the amplification method may include a primer extension-ligation reaction containing primers specific for the nucleic acid of interest. As a non-limiting example of primer extension and ligation primers that can be specifically designed to amplify the nucleic acid of interest, the amplification may include primers for GoldenGate determination (Illumina, Inc., San Diego, CA), as exemplified in U.S. Patents 7,582,420 and 7,611,869, each of which is incorporated herein by reference in its entirety.

[0105] Exemplary isothermal amplification methods that can be used in the methods of the present disclosure include, but are not limited to, multiple displacement amplification (MDA) as exemplified by, for example, Dean et al., Proc. Natl. Acad. Sci. USA 99:5261-66 (2002), or isothermal strand displacement nucleic acid amplification as exemplified by, for example, U.S. Pat. No. 6,214,587, each of which is incorporated herein by reference in its entirety. Other non-PCR-based methods that can be used in the present disclosure include, for example, Walker et al., Molecular Methods for Virus Detection, Academic Press, Inc., 1995, U.S. Patents 5,455,166 and 5,130,238 and Walker et al., Nucl. Acids Res. 20:1691-96 (1992) described in strand displacement amplification (SDA), or in, for example, Lage et al., Genome Research 13:294-307 (2003) described in hyperbranched strand displacement amplification, each of which is incorporated herein by reference in its entirety. For example, isothermal amplification methods can be used with strand displacement Phi 29 polymerase or Bst DNA polymerase large fragment 5'->3'exo for random primer amplification of genomic DNA. The use of these polymerases takes advantage of their high processivity and strand displacement activity. High processivity allows the polymerase to produce fragments of 10-20 kb in length. As described above, a polymerase with low processivity and strand displacement activity (such as Klenow polymerase) can be used to generate smaller fragments under isothermal conditions. Additional description of amplification reactions, conditions and components are detailed in the disclosure of U.S. Patent 7,670,810, which is incorporated herein by reference in its entirety.

[0106] Another nucleic acid amplification method that can be used in the present disclosure is a tag PCR, which uses a set of two-domain primers with a constant 5' region followed by a random 3' region, as described in, for example, Grothues et al., Nucleic Acids Res. 21(5): 1321-2 (1993), which is incorporated herein by reference in its entirety. A first round of amplification is performed to allow for a large number of priming of heat-denatured nucleic acids based on individual hybridization from the randomly synthesized 3' region. Due to the nature of the 3' region, it is assumed that the priming site is random throughout the genome. Unbound primers can then be removed, and further replication can be performed using primers complementary to the constant 5' region.

[0107] Sequencing The present disclosure also relates to sequencing the fixed random fragmentation double-stranded nucleic acid fragments produced according to the method provided herein. The fixed double-stranded nucleic acid fragments produced by the fragmentation mediated by the CRISPR / Cas9 enzyme of surface binding can be sequenced according to any suitable sequencing method (such as direct sequencing, including synthesis sequencing, connection sequencing, hybridization sequencing, nanopore sequencing, etc.). In some embodiments, the fixed nucleic acid fragments are sequenced on a solid support. In some embodiments, the solid support for sequencing is the same as the solid support on which surface binding fragmentation occurs. In some embodiments, the solid support for sequencing is the same as the solid support on which amplification occurs. In some embodiments, the first solid support is used for fragmentation, and the second solid support is used for amplification and sequencing.

[0108] A preferred sequencing method 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 chemistry can be polymerization (e.g., catalyzed by a polymerase). In a specific polymerase-based SBS embodiment, fluorescently labeled nucleotides are added to the primers (thereby extending the primers) in a template-dependent manner so that the detection of the order and type of nucleotides added to the primers can be used to determine the sequence of the template.

[0109] The circulation cell provides a convenient solid carrier for accommodating the amplified nucleic acid fragments produced by the method of the present disclosure. One or more amplified nucleic acid fragments of this format can be subjected to SBS or other detection techniques involving repeated delivery of reagents in a cycle. For example, in order to start the first SBS cycle, one or more labeled nucleotides, nucleic acids or DNA polymerases etc. can be made to flow into / flow through the circulation cell accommodating one or more amplified nucleic acid molecules. Those sites where primer extension causes the incorporation of labeled nucleotides can be detected. Optionally, nucleotides can also include a reversible termination property that terminates further primer extension once nucleotides are added to primers. For example, nucleotide analogs with reversible terminators can be added to primers so that subsequent extension does not occur until a deblocking agent is delivered to remove the part. Therefore, for the embodiment using reversible termination, deblocking agents can be delivered to the circulation cell (before or after detection occurs). Washing can be performed between each delivery step. Then the cycle can be repeated n times to extend the primer by n nucleotides, so as to detect a sequence with a length of n. Exemplary SBS procedures, fluidic systems, and detection platforms that can be readily adapted for use with amplicons produced by the methods of the present disclosure are described, for example, in 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.

[0110] Other sequencing procedures utilizing cyclic reactions can be used, such as pyrophosphate sequencing. Pyrophosphate sequencing detects the release of inorganic pyrophosphate (PPi) when a specific nucleotide is incorporated into a nascent nucleic acid chain (Ronaghi et al., Analytical Biochemistry 242 (1), 84-9 (1996); Ronaghi, Genome Res. 11 (1), 3-11 (2001); Ronaghi et al., Science 281 (5375), 363 (1998); US 6,210,891; US ​​6,258,568 and US. 6,274,320, each of which is incorporated herein by reference). In pyrophosphate sequencing, the released PPi can be immediately converted into adenosine triphosphate (ATP) by ATP sulfurylase for detection, and the level of ATP produced can be detected via photons generated by luciferase. Therefore, the sequencing reaction can be monitored via a luminescent detection system. The excitation radiation source for the fluorescence-based detection system is not necessary for the pyrophosphate sequencing procedure. Useful fluid systems, detectors, and procedures that may be suitable for applying pyrophosphate sequencing to amplicons produced according to the present disclosure are described in, for example, WIPO patent application serial numbers PCT / US11 / 57111, US 2005 / 0191698 A1, US 7,595,883, and US 7,244,559, each of which is incorporated herein by reference.

[0111] Some embodiments may utilize methods involving real-time monitoring of nucleic acid or DNA polymerase activity. For example, nucleotide incorporation may be detected by fluorescence resonance energy transfer (FRET) interactions between a polymerase carrying a fluorophore and a γ-phosphate labeled nucleotide or by using a zero mode waveguide (ZMW). Techniques and reagents for FRET-based sequencing are described in, for example, Levene et al., Science 299, 682–686 (2003); Lundquist et al., Opt. Lett. 33, 1026–1028 (2008); Korlach et al., Proc. Natl. Acad. Sci. USA 105, 1176–1181 (2008), the disclosures of which are incorporated herein by reference.

[0112] Some SBS embodiments include detecting the proton released when nucleotides are incorporated into extension products. For example, sequencing based on the detection of the proton released can use the commercially available electrical detector and correlation techniques from the Ion Torrent product line of Thermo Fisher Scientific, or the sequencing method and system 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 method for amplifying target nucleic acid using kinetic exclusion set forth herein can be easily applied to the substrate for detecting protons. More specifically, the method set forth herein can be used to produce an amplicon clone colony for detecting protons.

[0113] Another useful sequencing technology is nanopore sequencing (see, e.g., Deamer et al., Trends Biotechnol. 18, 147-151 (2000); Deamer et al., Acc. Chem. Res. 35:817-825 (2002); Li et al., Nat. Mater. 2:611-615 (2003), 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 pass through the nanopore. As nucleic acids or nucleotides pass through the nanopore, each nucleotide type can be identified by measuring fluctuations in the pore's conductivity (U.S. Pat. No. 7,001,792; Soni et al., Clin. Chem. 53, 1996-2001 (2007); Healy, Nanomed. 2, 459-481 (2007); Cockroft et al., J. Am. Chem. Soc. 130, 818-820 (2008), the disclosures of which are incorporated herein by reference).

[0114] Exemplary methods of array-based expression and genotyping analysis that can be applied to detection according to the present disclosure are described in the following documents: U.S. Patents 7,582,420, 6,890,741, 6,913,884 or 6,355,431, or U.S. Patent Publication Nos. 2005 / 0053980 A1, 2009 / 0186349 A1 or US 2005 / 0181440 A1, each of which is incorporated herein by reference.

[0115] The advantage of the method set forth herein is that they provide rapid and effective detection of multiple target nucleic acids in parallel. Therefore, the present disclosure provides an integrated system that can use technology known in the art (such as those illustrated above) to prepare and detect nucleic acids. Therefore, the integrated system of the present disclosure can include amplification reagents and / or sequencing reagents that can be delivered to one or more fixed DNA fragments. The system includes components such as pumps, valves, reservoirs, fluid pipelines, etc. The circulation cell can be configured for and / or for detecting target nucleic acids in the integrated system. Exemplary circulation cells are described in, for example, US2010 / 0111768A1 and U.S. Serial No. 13 / 273,666, each of which is incorporated herein by reference. As illustrated for the circulation cell, one or more fluid components of the integrated system can be used for amplification method and detection method. Taking nucleic acid sequencing embodiment as an example, one or more fluid components of the integrated system can be used for the amplification method set forth herein and for delivering sequencing reagents in sequencing method (such as those illustrated above). Alternatively, the integrated system can include a separate fluid system to perform amplification method and perform detection method. Examples of integrated sequencing systems that can generate amplified nucleic acids and also determine nucleic acid sequences include, but are not limited to, MiSeq TM The ELISA platform (Illumina, Inc., San Diego, CA) and the equipment described in U.S. Serial No. 13 / 273,666, which is incorporated herein by reference.

[0116] IV. Solid carrier with fixed CRISPR / Cas9 complex and preparation method thereof

[0117] Other embodiments presented herein include solid supports, such as beads, on which CRISPR / Cas9 enzymes are fixed. In certain embodiments, a CRISPR / Cas9 complex is fixed on a solid support, the complex comprising a CRISPR / Cas9 enzyme bound to a first polynucleotide, the first polynucleotide instructing the CRISPR / Cas9 enzyme to bind to a target nucleic acid in a non-sequence-specific manner. In certain other embodiments, a CRISPR / Cas9 complex is fixed on a solid support, the complex comprising a CRISPR / Cas9 enzyme bound to a first polynucleotide, the first polynucleotide instructing the CRISPR / Cas9 enzyme to bind to a target nucleic acid in a sequence-specific manner. In some embodiments, different solid supports instruct CRISPR / Cas9 enzymes to bind to target nucleic acids at different sequences. In some embodiments, the first CRISPR / Cas9 enzyme on a solid support binds to a first polynucleotide, the first polynucleotide instructing the first CRISPR / Cas9 enzyme to bind to a target nucleic acid 3' of a sequence of interest, and the second CRISPR / Cas9 enzyme on a solid support binds to a second polynucleotide, the second polynucleotide instructing the second CRISPR / Cas9 enzyme to bind to a target nucleic acid 5' of a sequence of interest. In some embodiments, the first polynucleotide comprises a 3' portion comprising a CRISPR / Cas9 terminal sequence and a 5' portion comprising a single-stranded guide RNA (sgRNA) that directs the CRISPR / Cas9 enzyme to bind to the target nucleic acid in a non-sequence-specific manner (or in a set of solid supports having targets to different sequences (optionally, each solid support comprises a pair of sgRNAs targeting flanking regions surrounding the sequence of interest), in a sequence-specific manner). In some embodiments, the first polynucleotide is biotinylated and the solid support comprises one or more biotin-binding proteins. The density of these surface-bound CRISPR / Cas9 enzymes or complexes can vary. For example, in some embodiments, the CRISPR / Cas9 enzymes or complexes are present in an amount of at least 10 3 , 10 4 , 10 5 or at least 10 6 Enzymes or complexes / mm 2 The density is present on the solid support.

[0118] V. Fragmentation using CRISPR / Cas9 microparticles

[0119] One embodiment proposed herein is a group of microparticles (e.g., beads) on which CRISPR / Cas9 enzymes or complexes are fixed. Using solid supports (such as beads) can provide several advantages over solution-based fragmentation. For example, in standard solution-based fragmentation, it is difficult to control the final fragment size of the fragmentation reaction. The fragment size is a function of the amount and size of CRISPR / Cas9 and nucleic acid and the ratio to the duration of the reaction. Even if these parameters are controlled, size selection classification is generally required as an additional step to remove excess small fragments shorter than the length of the combined paired reads. The method provided herein avoids these shortcomings. Specifically, the CRISPR / Cas9 enzyme or complex fixed by beads allows the final fragment size to be selected according to the spatial separation of the bound CRISPR / Cas9 enzyme or complex, regardless of the amount of CRISPR / Cas9 beads added to the fragmentation reaction. Another limitation of solution-based fragmentation is that it is usually necessary to perform some form of purification of the product of the fragmentation reaction before and after PCR amplification. This generally requires some reaction transfer between tubes. In contrast, the fragmentation products on the bead-based CRISPR / Cas9 can be washed and then released for amplification or other downstream processing, thereby avoiding the need for sample transfer. For example, in an embodiment where the CRISPR / Cas9 enzyme or complex is assembled on paramagnetic beads, purification of the fragmentation reaction product can be easily achieved by fixing the beads with a magnet and washing. Therefore, in some embodiments, fragmentation and other downstream processing (such as PCR amplification) can be performed in a single tube, vessel, droplet or other container. In some embodiments, the fragmentation and downstream processing of the sample occur on a device based on microfluidic droplets, as described in U.S. Publication No. 2013 / 0116128, which is incorporated herein by reference in its entirety. For example, in a device based on microfluidic droplets, droplets containing target nucleic acids, wash buffers or other reagents can pass through a surface containing a fixed CRISPR / Cas9 enzyme or complex. Similarly, droplets containing beads having CRISPR / Cas9 enzymes or complexes fixed thereon can contact target double-stranded nucleic acids, wash buffers or other reagents in a device based on microfluidic droplets.

[0120] When CRISPR / Cas9 beads are added to a solution of target double-stranded nucleic acid in fragmentation buffer, random fragmentation occurs, thereby attaching the target double-stranded nucleic acid to the surface of the beads. An immobilized library of randomly fragmented double-stranded nucleic acid fragments is generated.

[0121] In some embodiments, the length of the bridged fragment can be determined by the density of the CRISPR / Cas9 enzyme or complex on the bead surface. Once fragmentation is complete, any suitable method can be used to release the double-stranded nucleic acid fragments from the surface of the bead. In some embodiments, amplification methods such as suppression PCR, step-out PCR, etc. are used to release the fragmented products from the beads. In some embodiments, the fragmented products are released from the beads by cleavage. Cleavage can be, for example, chemical cleavage, enzymatic cleavage, photochemical cleavage, or a combination thereof. It should be understood that any suitable method for releasing one or more fragmented products from a solid support can be used in the methods provided herein.

[0122] Any suitable method for increasing the probability of contact can be used to effectively contact the nucleic acid with the surface-bound CRISPR / Cas9 enzyme or complex. For example, in some embodiments, the precipitation of nucleic acid on a solid surface can be used to increase the contact between the target double-stranded nucleic acid and the CRISPR / Cas9 enzyme or complex on the solid surface. Any of a variety of methods known in the art for contacting nucleic acid with a solid support can be utilized, as exemplified by the disclosure of WO 2010 / 115122, which is incorporated by reference in its entirety. As will be appreciated by those skilled in the art, nucleic acids can be precipitated onto a solid support by adding PEG, ethanol, or any of a variety of other reagents known to precipitate nucleic acids onto the surface (including, for example, any of a variety of buffers for solid phase reversible fixation (SPRI) technology).

[0123] In some embodiments, a group of beads carrying fixed CRISPR / Cas9 enzymes or complexes can be mixed with an excess of beads not carrying CRISPR / Cas9 enzymes, complexes or oligonucleotides to reduce the possibility of fragmentation on two or more different beads. Another method to reduce the possibility of fragmentation on two or more different beads includes fixing the beads to minimize contact between the beads. The fixation of beads can be achieved by any of a variety of techniques known in the art, including, for example, fixing the beads to the side of a solid surface (such as a microcentrifuge tube) via magnetic force, or any other fixing technique exemplified by the incorporation material of WO 2010 / 115122.

[0124] In some embodiments, CRISPR / Cas9 beads can be used to separate and identify nucleic acids from single cells (such as prokaryotic cells or eukaryotic cells). For example, in some embodiments, particles such as beads are coated with index CRISPR / Cas9 enzymes or complexes with the same index (all CRISPR / Cas9 enzymes or complexes present on a particular bead carry the same index, which is different from the index present on another bead). The beads can then be placed in the cell by any of a variety of methods known in the art. For example, methods for delivering beads in cells include, but are not limited to, gene guns, photothermal nanosheets (Wu et al., Anal Chem. (2011) 4: 1321-7) and peptides used in combination with cell permeabilization substances (Nitin et al., Ann Biomed Eng. (2009) 37: 2018-2027) and the like. It should be understood that any suitable method for associating nucleic acids from single cells with particles carrying index CRISPR / Cas9 enzymes or complexes can be used in the methods proposed herein.

[0125] In some embodiments, CRISPR / Cas9 enzyme or complex can be covalently attached to beads, as described in detail above. In addition or alternatively, CRISPR / Cas9 enzyme or complex can be released from beads when chemical or physical stimulation is applied. Some examples of stimulations that can trigger CRISPR / Cas9 enzyme or complex to be released from solid carriers include light and / or temperature changes (e.g., heat). In some embodiments, CRISPR / Cas9 enzyme or complex is released from solid carriers using the activity of enzymes (such as restriction endonucleases). In certain embodiments, CRISPR / Cas9 enzyme or complex can be separated from beads and move freely in cells. Once beads (or alternatively, released CRISPR / Cas9 enzyme or complex) contact chromatin or nucleic acid, fragmentation can occur. It should be understood that in eukaryotic and prokaryotic systems, not all genomic DNA is always achievable and / or can be used for fragmentation. In some embodiments, at most 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, or more than 99% of the total nucleic acid in the cell is fragmented by the CRISPR / Cas9 enzyme or complex.

[0126] In some embodiments, the double-stranded nucleic acid fragments are labeled with, for example, a first tag comprising a first tag domain. In some embodiments, the first tag domain may comprise a region for cluster amplification and / or a region for initiating a sequencing reaction. The fragments are labeled so that reads from the same cell or other biological sample can be identified by grouping reads sharing the same tag together. These reads can be considered to be derived from the same solid support or bead (and therefore from the same cell or biological sample).

[0127] In some embodiments, a method can be used to ensure that a single biological sample or target double-stranded nucleic acid is not fragmented by multiple solid supports or beads. For example, one method is to use beads of a size similar to the size of the cell. This will ensure that the cell will not be able to accommodate multiple beads. Additionally or alternatively, another method is to use a cell-to-bead ratio that is conducive to single cell targeting. For example, if the cells far outnumber the beads, the Poisson distribution of the beads within the cell means that the cells that have absorbed a single bead far exceed the cells that have absorbed two or more beads.

[0128] In some embodiments, the above-mentioned single cell method can be used to determine whether two single nucleotide polymorphisms (SNPs) or structural rearrangements are present in the same cell. For example, with respect to a heterogeneous population of cancer cells, knowing whether two SNPs are present in the same cell or in different cells can help implement the correct cancer treatment.

[0129] In some embodiments, the above-mentioned single cell method can be used to study RNA. For example, by coating beads with suitable enzymes (i.e., reverse transcriptase) and oligonucleotides for the reverse transcription step, gene expression at the single cell level can be analyzed. In one embodiment, after introducing beads coated with reverse transcriptase, oligonucleotides, and CRISPR / Cas9 enzymes, cytoplasmic RNA can be converted into cDNA and labeled and prepared in the cell.

[0130] VI. Methods for assembling long reads using immobilized CRISPR / Cas9 enzymes

[0131] The assembly of nucleic acid fragments enables the isolation of individual long nucleic acid molecules within a group of nucleic acid molecules and the conversion of each nucleic acid into a fragment library. When a library of nucleic acid fragments is labeled and sequenced, they can be assembled back into their original long molecules, for example, by reference to the tags contained by the nucleic acid fragments.

[0132] Any suitable method for increasing the probability of contact as discussed above can be used to efficiently bring the target double-stranded nucleic acid into contact with the surface-bound CRISPR / Cas9.

[0133] These methods can be performed using any of a variety of known formats, such as utilizing a combination of fragmentation reagents and bead arrays for library preparation, followed by index sequencing runs and bespoke data analysis. Any other suitable method of keeping beads statically separated from one another can be used for surface fragmentation and indexing of samples. For example, physical configurations in the substrate (such as holes or small depressions) can hold the beads so that the microspheres can rest in the holes, or other forces (magnetic or compressive forces) can be used, or chemically altered sites or active sites, such as chemically functionalized sites, electrostatically altered sites, hydrophobic and / or hydrophilic functionalized sites, or adhesive sites.

[0134] In some embodiments, the microspheres are non-covalently associated in the wells, but the wells may alternatively be chemically functionalized as generally described below, a cross-linking agent may be used, or a physical barrier such as a membrane or diaphragm over the beads may be used.

[0135] In certain embodiments, the surface of the substrate is modified to include chemically modified sites that can be used to covalently or non-covalently attach the microspheres of the present invention to discrete sites or locations on the substrate. "Chemically modified sites" in this context include, but are not limited to, adding patterns of chemical functional groups (including amino groups, carboxyl groups, oxo groups, and thiol groups) that can be used to covalently attach microspheres, which typically also contain corresponding reactive functional groups; adding patterns of adhesives that can be used to bond microspheres (either by pre-chemical functionalization to add adhesives or by directly adding adhesives); adding patterns of charged groups (similar to chemical functional groups) to electrostatically attach microspheres, such as when the microspheres contain charged groups opposite to these sites; adding patterns of chemical functional groups that give these sites different hydrophobicity or hydrophilicity, such that under appropriate experimental conditions, adding similar hydrophobic or hydrophilic microspheres will cause the microspheres to associate with these sites based on water affinity. For example, using hydrophobic sites with hydrophobic beads in an aqueous system can drive the beads to preferentially associate at these sites. As outlined above, "pattern" in this sense includes using a uniform treatment of the surface to allow beads to attach at discrete sites, to producing discrete sites for the surface treatment. As will be appreciated by those skilled in the art, this can be achieved in a variety of ways.

[0136] In some embodiments, when the target double-stranded nucleic acid is applied to a solid support, the target double-stranded nucleic acid is randomly fragmented multiple times by the CRISPR / Cas9 enzyme or complex on the solid support. Each fragment is fixed to a solid support. In some embodiments, the solid support is a bead, and the physical separation of the beads in the array chip prevents the nucleic acid molecules from reaching between the two beads. In other embodiments, the beads are in close contact, and one or more nucleic acid molecules can be stretched between two or more solid supports. In some embodiments, each solid support can fragment more than one target double-stranded nucleic acid molecule. The probability of two alleles being fragmented on the same solid support or beads is very low, and is a function of the concentration of the added nucleic acid and the number of solid support or beads. For example, in order to avoid the appearance of two alleles in the same well, it is necessary to load 0.1 times of haploid equivalents (50,000 genome equivalents) onto 1 million beads.

[0137] In some embodiments, the double-stranded nucleic acid fragments are labeled by any known labeling method. The double-stranded nucleic acid fragments can be labeled with, for example, a first tag comprising a first tag domain. In some embodiments, the first tag domain comprises a region for initiating a sequencing reaction. Once fragmentation and optional labeling are completed, the double-stranded nucleic acid fragments can be transferred from the surface of the solid support to a solution so that the single double-stranded nucleic acid fragments can be combined and prepared for the next step, such as sequencing.

[0138] Can use any suitable method known in the art to realize the release of fixed double-stranded nucleic acid fragment from solid support to solution.For example, in some embodiments, can release fragmentation molecule by being cracked from bead surface via the cracking part of the 5 ' end of oligonucleotide that for example is present in surface binding.The cracking part can be any part that is applicable to cracking nucleic acid chain from solid support.The example of the method utilizing the cracking part includes but is not limited to restriction endonuclease cracking, chemical cracking, RNase cracking, photochemical cracking etc., comprises those cracking methods and the cracking part of elaborating in WO 2006 / 064199, and the document is incorporated herein by reference in its entirety.

[0139] The label may include a "primer" region that can be used as a hybridization point to hybridize PCR step-out primers that enable the addition of additional sequences, such as amplification and sequencing primers. For example, amplification primers P5 and P7 can be added. Once added, inhibition PCR can be used, for example, to enrich for molecules that have a P5 adapter sequence at one end and P7 at the other end. In some embodiments, step-out primers that add P5 and P7 adapter sequences by inhibition PCR can be used to directly break away from a solid support (e.g., beads) for amplification.

[0140] In another embodiment, each solid support can have two types of surface grafted oligonucleotides, where the primer sequence is a P5-Read1 sequencing primer or a P7-Read 2 sequencing primer. This will produce a mixed P5-P7 CRISPR / Cas9 enzyme or complex. These can be cleaved from the solid support and then subjected to suppression PCR to enrich for P5 / P7 molecules, or directly amplified off the solid support as described above.

[0141] Equivalent content

[0142] The above written description is considered sufficient to enable those skilled in the art to practice the embodiments. The above description and examples detail certain embodiments and describe the best mode contemplated by the inventor. However, it should be understood that no matter how detailed the above appears in text, the embodiments can be practiced in many ways and should be interpreted according to the attached claims and any equivalents thereof.

[0143] The term "comprising" is intended herein to be open ended and include not only the listed elements but also any additional elements.

[0144] As used herein, the term "about" refers to numerical values, including, for example, integers, fractions, and percentages, whether or not explicitly stated. The term "about" generally refers to a numerical range (e.g., + / - 5-10% of the listed range) that one of ordinary skill in the art would consider equal to the listed value (e.g., having the same function or result). When terms such as "at least" and "about" precede a numerical value or range list, these terms modify all values ​​or ranges provided in the list. In some cases, the term "about" may include numerical values ​​rounded to the nearest significant figure.

[0145] Throughout this application, various publications, patents and / or patent applications have been cited. The disclosures of these publications are hereby incorporated by reference into this application in their entirety.

Claims

1. A method for preparing a fixed library of randomly fragmented double-stranded nucleic acid fragments, comprising: (a) providing a solid support on which a CRISPR / Cas9 enzyme is immobilized via a first polynucleotide; as well as (b) applying the target double-stranded nucleic acid to the solid support under conditions where the target double-stranded nucleic acid is randomly fragmented by the CRISPR / Cas9 enzyme and the CRISPR / Cas9 binds to at least one strand of the double-stranded nucleic acid fragments; thereby generating an immobilized library of randomly fragmented double-stranded nucleic acid fragments; in The CRISPR / Cas9 enzyme is bound to the first polynucleotide, which is immobilized via a binding protein bound to the solid support, and wherein the first polynucleotide includes a 3' portion comprising a CRISPR / Cas9 terminal sequence and a 5' portion comprising a single-stranded RNA (sgRNA), the single-stranded RNA guiding the CRISPR / Cas9 enzyme to bind to the target double-stranded nucleic acid at a protospacer adjacent motif (PAM) sequence and including a repeating subunit nucleotide sequence or a random sequence. 2 . The method of claim 1 , wherein the target double-stranded nucleic acid comprises double-stranded DNA (dsDNA), double-stranded RNA (dsRNA), or a double-stranded RNA / DNA hybrid. The method of claim 1 , wherein the target double-stranded nucleic acid is dsDNA.

4. The method according to any one of claims 1 to 3, wherein the first polynucleotide directs the CRISPR / Cas9 enzyme to bind to the target double-stranded nucleic acid in a non-sequence-specific manner.

5. The method according to claim 1, wherein the sgRNA comprises (GC) n or (AT) n , where n is 5-20. The method according to claim 5 , wherein n is 10.

7. The method of claim 6, wherein the sgRNA is 10 to 40 nucleotides.

8. The method of claim 7, wherein the sgRNA is 17 or 20 nucleotides.

9. The method of claim 3, wherein the first polynucleotide is biotinylated and the solid support comprises one or more biotin-binding proteins.

10. The method of claim 9, wherein the biotin-binding protein comprises avidin, streptavidin, neutravidin, an anti-biotin antibody, a biotin receptor and / or a biotin-binding enzyme.

11. The method of claim 10, wherein the biotin-binding enzyme comprises biotinidase or biotin carboxylation holoenzyme synthetase.

12. The method according to claim 1, comprising washing the solid support having the double-stranded nucleic acid fragment immobilized thereon to remove any unbound nucleic acid.

13. The method of claim 3, comprising amplifying the double-stranded nucleic acid fragments immobilized on the solid surface.

14. The method of claim 13, wherein the amplifying comprises providing a polymerase and an amplification primer corresponding to a portion of the first polynucleotide.

15. The method of claim 1, wherein applying the target double-stranded nucleic acid to the solid support comprises treating the CRISPR / Cas9 enzyme with one or more agents that reduce the nucleic acid binding specificity of the CRISPR / Cas9 enzyme.

16. The method of claim 15, wherein the one or more reagents comprise betaine, dimethyl sulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, and / or sulfane.

17. The method of claim 1, wherein the CRISPR / Cas9 enzyme is present in an amount of at least 10 3 , 10 4 , 10 5 or 10 6 Enzyme / mm 2 The density of is present on the solid support.

18. The method of claim 1, wherein the length of the double-stranded nucleic acid fragments in the immobilized library is proportional to the density of the CRISPR / Cas9 enzyme on the solid support.

19. The method of claim 1, wherein the solid support comprises microparticles, a patterned surface, or wells.

20. The method of claim 19, wherein the microparticles are beads.

21. The method according to claim 1, comprising: (c) applying an intercalating dye to at least a portion of the immobilized library of double-stranded nucleic acid fragments to obtain a set of stained immobilized fragments; and obtaining images of stained fixed fragments.

22. The method of claim 1, wherein applying the target double-stranded nucleic acid comprises adding a biological sample to the solid support.

23. The method of claim 22, wherein the biological sample comprises a cell lysate.

24. The method of claim 22, wherein the biological sample comprises whole cells.

25. The method of claim 22, wherein the biological sample is a blood sample.

26. The method of claim 1, comprising labeling the double-stranded nucleic acid fragments.

27. The method of claim 26, wherein the double-stranded nucleic acid fragment is labeled with a first tag comprising a first tag domain.

28. The method of claim 27, wherein the first tag domain comprises a region for cluster amplification.

29. The method of claim 27 or claim 28, wherein the first tag domain comprises a region for priming a sequencing reaction.

30. The method of claim 1, further comprising releasing the immobilized double-stranded nucleic acid fragments from the solid support.

31. The method of claim 30, wherein the releasing comprises cleaving the CRISPR / Cas9 enzyme from the solid support.

32. The method of claim 30, wherein the releasing comprises performing polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA), and nucleic acid sequence-based amplification (NASBA) or other amplification processes.

33. The method of claim 32, wherein the PCR comprises suppression PCR.

34. The method of claim 30, wherein the releasing comprises applying light or heat.

35. A method for preparing an immobilized library of randomly fragmented double-stranded nucleic acid fragments, comprising: (a) providing a solid support having a CRISPR / Cas9 complex immobilized thereon, wherein the CRISPR / Cas9 complex comprises a CRISPR / Cas9 enzyme bound to a biotinylated first polynucleotide, the biotinylated first polynucleotide comprising a 3' portion comprising a CRISPR / Cas9 terminal sequence and a 5' portion comprising a single-stranded guide RNA (sgRNA), the single-stranded guide RNA comprising a repeating subunit nucleotide sequence or a random sequence such that the CRISPR / Cas9 enzyme binds to a target nucleic acid at a protospacer adjacent motif (PAM) sequence, and wherein the biotinylated first polynucleotide is bound to a biotin-binding protein on the solid support; and (b) applying the target double-stranded nucleic acid to the solid support under conditions where the target double-stranded nucleic acid is randomly fragmented by the CRISPR / Cas9 complex and the CRISPR / Cas9 complex binds to at least one strand of the double-stranded nucleic acid fragments; thereby generating a fixed library of randomly fragmented double-stranded nucleic acid fragments.

36. The method of claim 35, wherein the biotin binding protein comprises avidin, streptavidin, neutravidin, an anti-biotin antibody, a biotin receptor and / or a biotin binding enzyme.

37. The method of claim 36, wherein the biotin binding enzyme comprises a biotinidase or a biotin carboxylating holoenzyme synthetase.

38. The method of claim 36 or claim 37, wherein applying the target double-stranded nucleic acid to the solid support comprises treating the CRISPR / Cas9 enzyme with one or more agents that reduce the nucleic acid binding specificity of the CRISPR / Cas9 enzyme.

39. The method of claim 38, wherein the one or more reagents comprise betaine, dimethyl sulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, and / or sulfane.

40. The method of claim 35, comprising labeling the double-stranded nucleic acid fragments.

41. The method of claim 40, wherein the double-stranded nucleic acid fragments are labeled with a first tag comprising a first tag domain.

42. The method of claim 41, wherein the first tag domain comprises a region for cluster amplification.

43. The method of claim 41 or claim 42, wherein the first tag domain comprises a region for priming a sequencing reaction.

44. The method of claim 35, further comprising releasing the immobilized double-stranded nucleic acid fragments from the solid support.

45. The method of claim 44, wherein the releasing comprises cleaving the CRISPR / Cas9 enzyme from the solid support.

46. ​​The method of claim 45, wherein said releasing comprises performing a PCR or other amplification process.

47. The method of claim 46, wherein the PCR comprises suppression PCR.

48. The method of claim 46, wherein the releasing comprises applying light or heat.

49. A solid support having immobilized thereon a library of double-stranded nucleic acid fragments, the library being prepared according to the method of any one of claims 1 to 48.

50. A solid support having a CRISPR / Cas9 complex immobilized thereon, wherein the CRISPR / Cas9 complex comprises a CRISPR / Cas9 enzyme bound to a first polynucleotide, the first polynucleotide comprising a single-stranded guide RNA (sgRNA), the single-stranded guide RNA comprising a repeating subunit nucleotide sequence or a random sequence such that the CRISPR / Cas9 enzyme binds to a target double-stranded nucleic acid at a protospacer adjacent motif (PAM) sequence, wherein, The CRISPR / Cas9 enzyme randomly fragments the target double-stranded nucleic acid, and wherein the first polynucleotide is immobilized to the solid support.

51. The solid support of claim 50, wherein the target double-stranded nucleic acid comprises double-stranded DNA (dsDNA), double-stranded RNA (dsRNA), or a double-stranded RNA / DNA hybrid.

52. The solid support of claim 51, wherein the target double-stranded nucleic acid is dsDNA.

53. The solid support of any one of claims 49 to 52, wherein the first polynucleotide directs the CRISPR / Cas9 enzyme to bind to the target double-stranded nucleic acid in a non-sequence-specific manner.

54. A solid support according to claim 53, wherein the first polynucleotide comprises a 3' portion comprising a CRISPR / Cas9 terminal sequence and a 5' portion comprising the single-stranded guide RNA (sgRNA), which guides the CRISPR / Cas9 enzyme to bind to the target nucleic acid in a non-sequence-specific manner.

55. The solid support of claim 54, wherein the sgRNA comprises (GC) n or (AT) n , where n is 5-20.

56. A solid support according to claim 55, wherein n is 10.

57. The solid support of claim 54, wherein the sgRNA is 10 to 40 nucleotides.

58. The solid support of claim 57, wherein the sgRNA is 17 or 20 nucleotides.

59. The solid support of claim 53, wherein the first polynucleotide is biotinylated and the solid support comprises a biotin binding protein.

60. The solid support of claim 59, wherein the biotin-binding protein comprises avidin, streptavidin, neutravidin, an anti-biotin antibody, a biotin receptor and / or a biotin-binding enzyme.

61. The solid support of claim 60, wherein the biotin-binding enzyme comprises a biotinidase or a biotin carboxylating holoenzyme synthetase.

62. The solid support of claim 50, wherein the CRISPR / Cas9 enzyme is present in an amount of at least 10 3 , 10 4 , 10 5 or 10 6 Enzyme / mm 2 The density of is present on the solid support.

63. The solid support of claim 50, wherein the solid support comprises microparticles, a patterned surface, or wells.

64. A solid support according to claim 63, wherein the microparticles are beads.

65. A composition comprising a solid support according to any one of claims 50 to 64 and one or more agents that reduce the nucleic acid binding specificity of the CRISPR / Cas9 enzyme, the one or more agents comprising betaine, dimethyl sulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide, dimethylformamide and / or sulfane.

66. A solid support having a CRISPR / Cas9 complex immobilized thereon, wherein the CRISPR / Cas9 complex comprises a CRISPR / Cas9 enzyme bound to a biotinylated first polynucleotide, the biotinylated first polynucleotide comprising a 3' portion comprising a CRISPR / Cas9 terminal sequence and a 5' portion comprising a single-stranded guide RNA (sgRNA), the single-stranded guide RNA directing the CRISPR / Cas9 enzyme to bind to a target double-stranded nucleic acid in a non-sequence-specific manner, and wherein the biotinylated first polynucleotide is bound to a biotin-binding protein on the solid support, and one or more agents that reduce the nucleic acid binding specificity of the CRISPR / Cas9 enzyme, the one or more agents comprising betaine, dimethyl sulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide, dimethylformamide and / or thioane.

67. A solid support according to claim 66, wherein the biotin binding protein comprises avidin, streptavidin, neutravidin, an anti-biotin antibody, a biotin receptor and / or a biotin binding enzyme.

68. The solid support of claim 67, wherein the biotin-binding enzyme comprises a biotinidase or a biotin carboxylating holoenzyme synthetase.

69. A solid carrier according to any one of claims 66 to 68, wherein the CRISPR / Cas9 complex is present in an amount of at least 10 3 , 10 4 , 10 5 , 10 6 Composites / mm 2 The density of is present on the solid support.

70. The solid support of claim 66, wherein the solid support comprises microparticles, a patterned surface, or wells.

71. The solid support of claim 70, wherein the microparticles are beads.

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