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

Abstract

Methods and compositions are presented for using immobilized CRISPR / Cas9 enzymes to generate immobilized libraries of randomly fragmented double-stranded target nucleic acid fragments on a surface. The methods are useful for generating nucleic acid fragments for use in a variety of processes, including massively parallel nucleic acid sequencing.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 873,609, filed July 12, 2019, which is incorporated herein by reference in its entirety for any purpose.

[0002] FIELD OF THEINVENTION The present application relates to methods for generating libraries of randomly fragmented double-stranded nucleic acid fragments using CRISPR / Cas9 enzyme immobilized on a solid support, solid supports comprising immobilized CRISPR / Cas9 enzyme, and related compositions. Immobilized libraries of randomly fragmented double-stranded nucleic acid fragments are useful as templates for a variety of applications, including, for example, high-throughput, massively parallel and / or multiplexed nucleic acid sequencing. [Background technology]

[0003] There are a variety of methods and applications in which it is desirable to generate a library of randomly fragmented nucleic acid molecules from a target double-stranded nucleic acid target molecule, such as a double-stranded DNA (dsDNA) target molecule. Often, the purpose is to generate smaller nucleic acid molecules (e.g., nucleic acid fragments) from a larger double-stranded nucleic acid molecule for use as a template in a nucleic acid sequencing reaction.

[0004] Many of the currently used methods for fragmenting and tagging double-stranded nucleic acids for use in next-generation sequencing waste nucleic acids, require expensive equipment for fragmentation, and the procedures for fragmenting, tagging, and recovering tagged nucleic acid fragments are difficult, tedious, laborious, time-consuming, inefficient, expensive, and require relatively large amounts of sample nucleic acid. In addition, many of these methods generate nucleic acid fragments that are not completely representative of the sequences contained in the sample nucleic acid from which they were generated. Therefore, what is needed in the art is a method that provides speed and ease of use in generating a library of randomly fragmented double-stranded nucleic acid fragments from a target double-stranded nucleic acid, and that can be easily applied to nucleic acid analysis methods such as next-generation sequencing and amplification methods. Summary of the Invention

[0005] According to the present specification, the present application describes a method for preparing an immobilized library of randomly fragmented, double-stranded nucleic acid fragments, comprising: (a) providing a solid support having immobilized CRISPR / Cas9 enzyme thereon; 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 where 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.

[0006] In some embodiments, the target double-stranded nucleic acid comprises double-stranded DNA (dsDNA), double-stranded RNA (dsRNA), or a double-stranded RNA / DNA hybrid. In some embodiments, the target double-stranded nucleic acid is dsDNA.

[0007] In some embodiments, CRISPR / Cas9 enzyme is bound to a first polynucleotide that induces CRISPR / Cas9 enzyme to bind to target double-stranded nucleic acid in a non-sequence specific manner.In some embodiments, the first polynucleotide is immobilized on a solid support.In some embodiments, the first polynucleotide comprises a 3' portion that comprises a CRISPR / Cas9 end sequence and a 5' portion that comprises a single-stranded guide RNA (sgRNA) that induces CRISPR / Cas9 enzyme to bind to target nucleic acid in a non-sequence specific manner.

[0008] In some embodiments, the sgRNA is n or (AT) n wherein n is 5 to 20, 10 to 15, or 10.

[0009] In some embodiments, the sgRNA is 10-40, 15-35, or 17-20 nucleotides.

[0010] 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 proteins comprise avidin, streptavidin, neutravidin, an anti-biotin antibody, a biotin receptor, and / or a biotin-binding enzyme. In some embodiments, the biotin-binding enzyme comprises biotinidase or biotin holocarboxylase synthetase.

[0011] In some embodiments, the method includes washing the solid support with the double-stranded nucleic acid fragments immobilized thereon to remove any unbound nucleic acid.

[0012] In some embodiments, the method includes amplifying the double-stranded nucleic acid fragment immobilized on the solid surface, hi some embodiments, amplifying includes providing a polymerase and an amplification primer corresponding to a portion of the first polynucleotide.

[0013] In some embodiments, applying the target double-stranded nucleic acid to the solid support comprises treating the CRISPR / Cas9 enzyme with one or more reagents that reduce the nucleic acid binding specificity of the CRISPR / Cas9 enzyme.

[0014] In some embodiments, the one or more reagents include betaine, dimethylsulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, and / or sulphalane.

[0015] In some embodiments, the CRISPR / Cas9 enzyme is 2 At least 10 per 3 , 10 4 , 10 5 , or 10 6 The enzyme is present on the solid support at a density of 100 μg / ml.

[0016] 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.

[0017] In some embodiments, the solid support comprises a microparticle, a patterned surface, or a well. In some embodiments, the microparticle is a bead.

[0018] 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.

[0019] In some embodiments, applying the target double-stranded nucleic acid comprises adding a biological sample to the 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.

[0020] In some embodiments, the method includes tagging the double-stranded nucleic acid fragments.

[0021] In some embodiments, the double-stranded nucleic acid fragment is tagged 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.

[0022] In some aspects, the method includes releasing the immobilized double-stranded nucleic acid fragments from the solid support. In some embodiments, the releasing includes cleaving the CRISPR / Cas9 enzyme from the solid support. In some embodiments, the releasing includes performing polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription-based amplification (TMA), and nucleic acid sequence-based amplification (NASBA), or other amplification process. In some embodiments, the PCR includes suppression PCR. In some embodiments, the releasing includes applying light or heat.

[0023] The present application also describes a method of preparing an immobilized library of randomly fragmented double-stranded nucleic acid fragments, the method comprising: (a) providing a solid support having immobilized CRISPR / Cas9 complexes thereon, the CRISPR / Cas9 complexes comprising a CRISPR / Cas9 enzyme bound to a biotinylated first polynucleotide, the CRISPR / Cas9 complexes comprising a 3' portion comprising a CRISPR / Cas9 end sequence and a 5' portion comprising a single-stranded guide RNA (sgRNA) that guides the CRISPR / Cas9 enzyme to bind to a target nucleic acid in a non-sequence specific manner, the biotinylated first polynucleotide being bound to a biotin-binding protein on the solid support; and (b) applying a target double-stranded nucleic acid to the solid support under conditions in which the target double-stranded nucleic acid is randomly fragmented by the CRISPR / Cas9 complexes and the CRISPR / Cas9 complexes bind 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.

[0024] 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 biotinidase or biotin holocarboxylase synthetase.

[0025] In some embodiments, applying the target double-stranded nucleic acid to the solid support comprises treating the CRISPR / Cas9 enzyme with one or more reagents that reduce the nucleic acid binding specificity of the CRISPR / Cas9 enzyme. In some embodiments, the one or more reagents comprise betaine, dimethylsulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, and / or sulfalane.

[0026] In some embodiments, the method comprises tagging the double-stranded nucleic acid fragment. In some embodiments, the double-stranded nucleic acid fragment is tagged 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.

[0027] In some aspects, the method includes releasing the immobilized double-stranded nucleic acid fragments from the solid support. In some embodiments, the releasing includes cleaving the CRISPR / Cas9 enzyme from the solid support. In some embodiments, the releasing includes performing a PCR or other amplification process. In some embodiments, the PCR includes suppression PCR. In some embodiments, the releasing includes applying light or heat.

[0028] The present application also describes a solid support having thereon immobilized libraries of double-stranded nucleic acid fragments prepared according to the methods described herein.

[0029] The present application also describes a solid support having immobilized thereon a CRISPR / Cas9 complex, the CRISPR / Cas9 complex comprising a CRISPR / Cas9 enzyme that randomly fragments a target double-stranded nucleic acid.

[0030] In some embodiments, the target double-stranded nucleic acid comprises double-stranded DNA (dsDNA), double-stranded RNA (dsRNA), or a double-stranded RNA / DNA hybrid. In some embodiments, the target double-stranded nucleic acid is dsDNA.

[0031] In some embodiments, the CRISPR / Cas9 enzyme is attached to a first polynucleotide that directs the CRISPR / Cas9 enzyme to bind the target double-stranded nucleic acid in a non-sequence specific manner.

[0032] In some embodiments, the first polynucleotide is immobilized on a solid support.

[0033] In some embodiments, the first polynucleotide comprises a 3' portion that comprises a CRISPR / Cas9 end sequence and a 5' portion that comprises a single-stranded guide RNA (sgRNA) that guides the CRISPR / Cas9 enzyme to bind to a target nucleic acid in a non-sequence specific manner.

[0034] In some embodiments, the sgRNA is n or (AT) n wherein n is 5 to 20 or 10 to 15. In some embodiments, n is 10.

[0035] In some embodiments, the sgRNA is 10-40 or 15-30 nucleotides, in some embodiments, the sgRNA is 17 or 20 nucleotides.

[0036] 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 biotinidase or biotin holocarboxylase synthetase.

[0037] In some embodiments, the CRISPR / Cas9 enzyme is 2 At least 10 per 3 , 10 4 , 10 5 , or 10 6 The enzyme is present on the solid support at a density of 100 μg / ml.

[0038] In some embodiments, the solid support comprises a microparticle, a patterned surface, or a well. In some embodiments, the microparticle is a bead.

[0039] The present application also describes compositions comprising a solid support as described herein and one or more reagents that reduce the nucleic acid binding specificity of a CRISPR / Cas9 enzyme. In some embodiments, the one or more reagents comprise betaine, dimethylsulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, and / or sulfalane.

[0040] The present application also describes a solid support having an immobilized CRISPR / Cas9 complex thereon, the CRISPR / Cas9 complex comprising a CRISPR / Cas9 enzyme bound to a biotinylated first polynucleotide comprising a 3' portion comprising a CRISPR / Cas9 end sequence and a 5' portion comprising a single-stranded guide RNA (sgRNA) that guides the CRISPR / Cas9 enzyme to bind to a target double-stranded nucleic acid in a non-sequence specific manner, the biotinylated first polynucleotide being bound to a biotin-binding protein on the solid support.

[0041] 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 biotinidase or biotin holocarboxylase synthetase.

[0042] In some embodiments, the CRISPR / Cas9 complex is 2 At least 10 per 3 , 10 4 , 10 5 , 10 6 The complexes are present on the solid support at a density of 100 .mu.m.

[0043] In some embodiments, the solid support comprises a microparticle, a patterned surface, or a well. In some embodiments, the microparticle is a bead.

[0044] The present application also describes compositions comprising a solid support as described herein and one or more reagents that reduce the nucleic acid binding specificity of a CRISPR / Cas9 enzyme. In some embodiments, the one or more reagents comprise betaine, dimethylsulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, and / or sulfalane.

[0045] Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0046] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory but are not restrictive of the scope of the appended claims.

[0047] 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]

[0048] [Figure 1] 1 shows an embodiment of the present invention. In this embodiment, a CRISPR / Cas9 complex is immobilized on a surface. The CRISPR / Cas9 complex has a CRISPR / Cas9 (Cas9) enzyme 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 and randomly fragmented by the CRISPR / Cas9 complex, and the double-stranded nucleic acid fragments are bound to the CRISPR / Cas9 complex immobilized on the surface. The size of the nucleic acid fragments depends on the distance between the CRISPR / Cas9 enzymes immobilized on the surface. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] Current protocols for sequencing nucleic acid samples routinely employ sample preparation processes that convert nucleic acids, such as DNA or RNA, or RNA / DNA hybrids, into template libraries. These methods result in loss of nucleic acid samples and often require expensive equipment for fragmentation. In addition, sample preparation methods are often difficult, laborious, and inefficient.

[0050] In standard sample preparation methods, each template contains an adaptor at either end of the insert, and many steps are often required to both modify the nucleic acid and purify the desired product of the modification reaction. These steps are carried out in solution before the matching fragments are added to the flow cell, where they are bound to the surface by a primer extension reaction that copies the fragments hybridized to the ends of primers covalently bound to the surface. These "seeding" templates then undergo several amplification cycles to generate monoclonal clusters of the copied templates.

[0051] 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 ready for clustering and sequencing can be minimized by using CRISPR / Cas9 enzyme-mediated fragmentation. Following a purification step to remove any unbound nucleic acid, additional sequences are added to the ends of the nucleic acid fragments by PCR.

[0052] Solution-based fragmentation has drawbacks and requires several laborious steps. In addition, bias can be introduced during the polymerase chain reaction (PCR) amplification step. The methods and compositions presented herein overcome these drawbacks and allow for unbiased sample preparation, clustering and sequencing on a single solid support with minimal sample manipulation or transfer requirements.

[0053] The present disclosure relates to the surprising discovery that CRISPR / Cas9 enzymes pre-bound to the surface of a solid support, such as a flow cell, can be used under conditions that effectively randomly fragment and immobilize intact target nucleic acid on the solid support. In certain embodiments, one or more strands that contain CRISPR / Cas9 enzymes are attached to the surface of the solid support via their 5' ends. CRISPR / Cas9 enzymes can also be included in a complex that contains sequences that allow for subsequent cluster generation and sequencing.

[0054] The methods and compositions presented herein provide several advantages over solution-based and less random fragmentation methods. For example, purified, partially purified, or even unpurified intact target nucleic acid can be directly loaded onto a solid support or flow cell for cluster generation without prior sample preparation. In addition, the contiguity of sequence information in the original intact nucleic acid can be physically preserved by juxtaposition of fragments on the surface of a solid support or flow cell. As an additional advantage, since the nucleic acid fragments are physically linked to the surface of a solid support or flow cell, purification of reagents after further manipulation of the nucleic acid fragments can be achieved, for example, by flow-through buffer exchange in the flow cell channel.

[0055] Furthermore, improved random fragmentation of target double-stranded nucleic acids allows the library of randomly fragmented double-stranded nucleic acid fragments to better represent the original sample. I. Random Fragmentation on Solid Supports

[0056] In accordance with the above, provided herein is a method for preparing an immobilized library of randomly fragmented double-stranded nucleic acid fragments, and a solid support having a library of immobilized double-stranded nucleic acid fragments thereon, prepared according to such method. In some embodiments, the method includes: (a) providing a solid support having an immobilized CRISPR / Cas9 enzyme thereon; and (b) applying a target double-stranded nucleic acid to the solid support under conditions in which 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.

[0057] In some embodiments, the CRISPR / Cas9 enzyme is directly immobilized on a solid support.

[0058] In some embodiments, a method is described that includes: (a) providing a solid support having immobilized CRISPR / Cas9 complexes thereon, the CRISPR / Cas9 complexes comprising a CRISPR / Cas9 enzyme bound to a biotinylated first polynucleotide, the CRISPR / Cas9 complexes comprising a 3' portion comprising a CRISPR / Cas9 end sequence and a 5' portion comprising a single-stranded guide RNA (sgRNA) that guides the CRISPR / Cas9 enzyme to bind to a target double-stranded nucleic acid in a non-sequence specific manner, the biotinylated first polynucleotide being bound to a biotin-binding protein on the solid support; and (b) applying a target double-stranded nucleic acid to the solid support under conditions in which the target double-stranded nucleic acid is randomly fragmented by the CRISPR / Cas9 complexes and the CRISPR / Cas9 complexes bind 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.

[0059] As used herein, the term "randomly fragmented" refers to fragmentation of a target nucleic acid in a random manner, generating a variety of fragments (e.g., fragments generated by fragmentation at random positions in terms of sequence). In some embodiments, the target nucleic acid is randomly fragmented in a non-sequence specific manner to generate a variety of fragment identities. Fragmentation is controllable in terms of both normalized product amount and fragment size, regardless of DNA input (i.e., controllable size selection). For example, as shown in Figure 1, the distance between adjacent CRISPR / Cas9 enzymes on a solid support can determine the size range of fragments immobilized on the surface of the solid support.

[0060] In some embodiments, the target nucleic acid is fragmented in a sequence-specific manner. For example, as discussed below, two CRISPR / Cas9 enzyme complexes on the same solid support can contain a pair of sgRNAs that flank the region of interest of the target nucleic acid. In such embodiments, each pair of sgRNAs generates a single fragment. In other embodiments, a population of solid supports (e.g., beads) is used, and each solid support in the population of solid supports can carry a different pair of sgRNAs. In this way, different solid supports target different sequences in the target nucleic acid. In some embodiments, different sgRNA pairs target different target sequences in the genome. These methods allow multiplex targeting.

[0061] 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) enzyme. Cas9, also known as Csn1, is a CRISPR-associated protein that contains two nuclease domains, RuvC nuclease domain and HNH nuclease domain, which are programmed by small RNA to cleave nucleic acids (e.g., DNA). CRISPR / Cas9 enzymes are well known to those skilled in the art and are exemplified, for example, by 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 mutants of Cas9 known to cleave nucleic acids are known to those of skill in the art and include wild-type or naturally occurring Cas9 and mutant or modified Cas9 (e.g., Cas9D10A). It will 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 that allow the CRISPR / Cas9 enzyme to randomly fragment or cleave the target DNA.

[0062] As used herein, the term "CRISPR / Cas9 enzyme complex" generally refers to a CRISPR / Cas9 enzyme bound to a first polynucleotide that induces 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 immobilized on a solid support. In some embodiments, the first polynucleotide comprises a 3' portion that comprises a CRISPR / Cas9 end sequence and a 5' portion that comprises a single-stranded guide RNA (sgRNA) that induces 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 comprises one or more biotin-binding proteins. For example, a CRISPR / Cas9 enzyme complex can include a CRISPR / Cas9 enzyme bound to a biotinylated first polynucleotide, the first polynucleotide comprising a 3' portion comprising a CRISPR / Cas9 end 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, wherein the biotinylated first polynucleotide is bound to a biotin-binding protein on a solid support.

[0063] 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 throughout this disclosure in connection with a target double-stranded nucleic acid molecule, it should be understood that any suitable nucleic acid or nucleic acid analog can be randomly fragmented.

[0064] The term "CRISPR / Cas9 end" refers to a double-stranded nucleic acid that exhibits only the nucleotide sequence required to form a complex with a CRISPR / Cas9 enzyme (the "CRISPR / Cas9 end sequence"). A CRISPR / Cas9 end can include any nucleic acid or nucleic acid analog suitable for forming a functional complex with a CRISPR / Cas9 enzyme. For example, a CRISPR / Cas9 end can include DNA, RNA, modified bases, unnatural bases, modified backbones, and can include nicks in one or both strands.

[0065] 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 use. A tag domain may include any sequence provided for any desired purpose. In some embodiments, a tag includes one or more functional sequences selected from the group consisting of a universal sequence, a primer sequence, an index sequence, a capture sequence, a barcode sequence (e.g., used for counting or error correction), a cleavage sequence, a sequencing-related sequence, a sequence for enrichment, and combinations thereof. For example, in some embodiments, a tag domain includes one or more restriction endonuclease recognition sites. In some embodiments, a tag domain includes one or more regions suitable for hybridization with a primer for a cluster amplification reaction. In some embodiments, a tag domain includes one or more regions suitable for hybridization with a primer for a sequencing reaction. It will be understood that any other suitable features may be incorporated into the tag domain. In some embodiments, a tag domain includes a sequence having a length between 5 and 200 bp. In some embodiments, a tag domain includes a sequence having a length between 10 and 100 bp. In some embodiments, the tag domain comprises a sequence having a length between 20 and 50 bp, in some embodiments, the tag domain comprises a sequence having a length between 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, and 200 bp.

[0066] In the methods and compositions presented herein, CRISPR / Cas9 enzyme is immobilized on a solid support. In some embodiments, CRISPR / Cas9 enzyme is immobilized on a support via one or more polynucleotides. In some embodiments, one or more polynucleotides, such as the first polynucleotide, induce CRISPR / Cas9 enzyme to bind to target double-stranded nucleic acid in a non-sequence specific manner (i.e., the condition induces CRISPR / Cas9 enzyme to randomly fragment or cleave target double-stranded nucleic acid). In some embodiments, one or more polynucleotides direct the CRISPR / Cas9 enzyme to bind to the target double stranded nucleic acid in a sequence specific manner, and fragmentation is accomplished using a population of solid supports, each solid support comprising a first CRISPR / Cas9 complex comprising a first polynucleotide that directs the CRISPR / Cas9 enzyme to bind 3' of a sequence of interest in the target nucleic acid, and a second CRISPR / Cas9 complex comprising a second polynucleotide that directs the CRISPR / Cas9 enzyme to bind 5' of the sequence of interest in the target nucleic acid.

[0067] In some embodiments, the first polynucleotide is immobilized on a solid support. In some embodiments, the first polynucleotide comprises a 3' portion comprising a CRISPR / Cas9 end sequence and a 5' portion comprising a single-stranded guide RNA that guides the CRISPR / Cas9 complex so that it can be immobilized on a solid support directly or via a linker molecule that binds the CRISPR / Cas9 enzyme.

[0068] As used herein, the term "single-stranded guide RNA" or "sgRNA" refers to a single-stranded RNA that can hybridize 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), thereby guiding the CRISPR / Cas9 enzyme to the target sequence, where the CRISPR / Cas9 enzyme cleaves the target sequence.

[0069] In some embodiments, when the CRISPR / Cas9 enzyme is induced in a non-sequence-specific manner, the target sequence does not have sequence restrictions, except that the sequence is adjacent to a protospacer adjacent motif (PAM). For example, PAM sequences of Cas9 protein include, but are not limited to, 5'-NGG, 5'-NGGNG, 5'-NNAGAAW, and 5'-ACAY. In some embodiments, when the CRISPR / Cas9 enzyme is induced in a sequence-specific manner, the target sequence has sequence restrictions.

[0070] In some embodiments, sgRNAs are selected that direct CRISPR / Cas9 in a non-sequence specific manner, such that the CRISPR / Cas9 enzyme cleaves the nucleic acid in a random manner to generate a range of fragment sizes.

[0071] In some embodiments, the sgRNA is 10-40 nucleotides, 15-30 nucleotides, or 17 or 20 nucleotides. n or (AT) n Examples of suitable sgRNAs include, but are not limited to, short random sequences or sequences of 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. sgRNAs may be chemically synthesized, enzymatically synthesized, or a combination thereof.

[0072] In some embodiments, the first polynucleotide is "biotinylated" and the solid support comprises one or more biotin-binding proteins. As used herein, the term "biotinylation" refers to the process of covalently binding biotin. Biotin-binding proteins are well known to those skilled 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 holocarboxylase synthetase, and the like.

[0073] In some embodiments, one or more reagents that reduce the nucleic acid binding specificity of a nucleic acid binding enzyme such as CRISPR / Cas9 may be used alone or in combination with an sgRNA (i.e., another condition that induces 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 reagents to reduce the nucleic acid binding specificity of the CRISPR / Cas9 enzyme and induce random fragments when the target double-stranded nucleic acid is applied to a solid support having the treated immobilized CRISPR / Cas9 enzyme thereon. Reagents that reduce binding specificity are well known to those of skill in the art. Examples of binding specificity reducers include, but are not limited to, betaine, dimethyl sulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, and / or sulphalane.

[0074] In some embodiments, conditions that induce the CRISPR / Cas9 enzyme to randomly fragment or cleave the target double-stranded nucleic acid include, in addition to or instead of other conditions, the use of high glycerol concentrations (e.g., >5% v / v), high concentrations of CRISPR / Cas9 enzyme / μg of DNA (e.g., 100 units / μg), non-optimal buffers (e.g., having non-optimal ionic strength or pH), long reaction times, and / or low Mg concentrations. 2+ Use of divalent cations other than Mn 2+ , Cu 2+ , Co 2+ and / or Zn 2+ ).

[0075] 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 both terms are intended to encompass direct or indirect, covalent or non-covalent attachment, unless otherwise indicated explicitly or by context. In certain embodiments of the present disclosure, covalent attachment may be preferred, but generally, what is required is that the molecule (e.g., a nucleic acid) remains immobilized or attached to the support under conditions in which the support is intended to be used, for example in applications requiring nucleic acid amplification and / or sequencing.

[0076] Certain embodiments of the present disclosure may utilize solid supports comprised of an inert substrate or matrix (e.g., glass slides, polymeric beads, etc.) that have been "functionalized" by application of a layer or coating of an intermediate material that contains reactive groups that allow for covalent attachment to biomolecules, such as polynucleotides. Examples of such supports include, but are not limited to, polyacrylamide hydrogels supported on an inert substrate such as glass, particularly those described in WO 2005 / 065814 and US 2008 / 0280773, the contents of which are incorporated herein by reference in their entirety. In such embodiments, the biomolecule (e.g., polynucleotide) may be covalently attached directly to the intermediate material (e.g., hydrogel), although the intermediate material may itself be non-covalently attached to the substrate or matrix (e.g., glass substrate). The term "covalent attachment to a solid support" should be interpreted accordingly to encompass this type of array.

[0077] The terms "solid surface", "solid support" and other grammatical equivalents 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 understood 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 (e.g., acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon™, etc.), polysaccharides, nylon or nitrocellulose, ceramics, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glass, plastics, fiber optic bundles, and various other polymers. In some embodiments, particularly useful solid supports and solid surfaces are placed in a flow cell device. Exemplary flow cells are shown in more detail below.

[0078] In some embodiments, the solid support comprises a patterned surface suitable for immobilization of CRISPR / Cas9 enzymes or complexes in a regular pattern. A "patterned surface" refers to an arrangement of different regions in or on an exposed layer of a solid support. For example, one or more regions can be features in which one or more CRISPR / Cas9 enzymes or complexes are present. The features can be separated by interstitial regions in which the CRISPR / Cas9 enzymes or complexes are absent. In some embodiments, the pattern can be an xy format of features in rows and columns. In some embodiments, the pattern can be a repeating sequence of features and / or interstitial regions. In some implementations, the pattern can be a random sequence of features and / or interstitial regions. In some embodiments, the CRISPR / Cas9 enzymes or complexes are randomly distributed on the solid support. In some embodiments, the CRISPR / Cas9 enzymes or complexes are distributed on a patterned surface. Exemplary patterned surfaces that can be used in the methods and compositions described herein are described in U.S. Patent Application Publication Nos. 2012 / 0316086(A1) and 2013 / 0116153(A1), each of which is incorporated herein by reference.

[0079] In some embodiments, the solid support comprises an array of wells or depressions on its surface, which can be manufactured as commonly known in the art using a variety of techniques, including but not limited to photolithography, stamping techniques, molding techniques, and microetching techniques. As understood in the art, the technique used will depend on the composition and shape of the array substrate.

[0080] The composition and geometry of the solid support may vary depending on its use. In some embodiments, the solid support is a planar structure, such as a slide, a chip, a microchip, and / or an array. Thus, the surface of the substrate may be in the form of a planar layer. In some embodiments, the solid support comprises one or more surfaces of a flow cell. As used herein, the term "flow cell" refers to a chamber that comprises a solid surface through which one or more fluidic reagents can flow. Examples of flow cells and associated fluidic systems and detection platforms that can readily be used in the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497, U.S. Pat. No. 7,057,026, WO 91 / 06678, WO 07 / 123744, U.S. Pat. No. 7,329,492, U.S. Pat. No. 7,211,414, U.S. Pat. No. 7,315,019, U.S. Pat. No. 7,405,281, and U.S. Patent Application No. 2008 / 0108082, each of which is incorporated herein by reference.

[0081] In some embodiments, the solid support or its surface is non-planar, such as the inside or outside surface of a tube or vessel. In some embodiments, the solid support comprises a microsphere or bead. By "microsphere" or "bead" or "particle" or grammatical equivalents herein is meant a small discrete particle. Suitable bead compositions include, but are not limited to, plastic, ceramic, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thoria sol, carbon graphite, titanium dioxide, latex, or cross-linked dextran such as Sepharose, cellulose, nylon, cross-linked micelles, and Teflon™, as well as any other material outlined herein for solid supports can all be used. The "Microsphere Detection Guide" by Bangs Laboratories, Fishers, Ind. is a useful guide. In certain embodiments, the microsphere is a magnetic microsphere or bead.

[0082] The beads need not be spherical. Irregular particles may be used. Alternatively or additionally, 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 about 0.5 to about 5 microns being particularly preferred, although smaller or larger beads may be used in some embodiments.

[0083] FIG. 1 generally illustrates a method according to one embodiment. A CRISPR / Cas9 complex is immobilized on a surface. The CRISPR / Cas9 complex has a biotinylated polynucleotide comprising a CRISPR / Cas9 enzyme ("Cas9"), 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 and randomly fragmented by the CRISPR / Cas9 complex, and the double-stranded nucleic acid fragments are bound to the CRISPR / Cas9 complex immobilized on the surface. The size of the nucleic acid fragments depends on the distance between the CRISPR / Cas9 enzymes immobilized on the surface. For example, in some embodiments, the CRISPR / Cas9 complex is 1 mm 2 At least 10 per 3 , 10 4 , 10 5 complexes, or at least 10 6 The complexes are immobilized on the solid support at a density of 100 .mu.m.

[0084] When the target double-stranded nucleic acid is applied to the solid support, the CRISPR / Cas9 enzyme fragments the target double-stranded nucleic acid, thus generating randomly fragmented double-stranded nucleic acid fragments that are attached at both ends to the surface of the solid support. In some embodiments, the length of the double-stranded nucleic acid fragments can be varied by varying the density of the CRISPR / Cas9 enzyme or complexes thereof on the surface. 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 or complexes on the solid support. In certain embodiments, the length of the resulting double stranded nucleic acid fragments is 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 1100bp, 1200bp, 1300bp, 1400bp, 1500bp, 1600bp, 1700bp, 1800bp, 1900bp, 2000bp, 2100bp, 2200bp, 2300bp, 2400bp, 2500bp, 2600bp, 2700bp, 2800bp, 2900bp, 3000bp, 3100bp, 3200bp, 3300bp, 3400bp, 3500bp, 3600bp, 3700bp, 3800bp, 3900bp, 4000bp, 4100bp, 4200bp, 4300bp, 4400bp, 4500bp, 4600bp, 4700bp, 4800bp, 4900bp, 5000bp, 5100bp, 5200bp, 5300bp, 5400bp, 5500bp, 5600bp, 5700bp, 5800bp, 5900bp, 6000bp, 6100bp, 6200bp, 6300bp, 6400bp, 6500bp, 6600bp, 6700bp, 6800bp, 6900bp, 7000bp, 7100bp, 7200bp In such embodiments, the double-stranded nucleic acid fragments may then be amplified into clusters using standard cluster chemistry, as exemplified by the disclosures in U.S. Patent Nos. 7,985,565 and 7,115,400, the contents of each of which are incorporated herein by reference in their entirety.

[0085] In some embodiments, the template length is longer than can be adequately amplified using standard cluster chemistry. For example, in some embodiments, the template length is 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 1100bp, 1200bp, 1300bp, 1400bp, 1500bp, 1600bp, 1700bp, 1800bp, 1900bp, 2000bp, 2100bp, 2200bp, 2300bp, 2400bp, 2500bp, 2600bp, 2700bp, 2800bp, 2900bp, 3000bp, 3100bp, 3200bp, 3300bp, 3400bp, 3500bp, 3600bp, 3700bp, 3800bp, 3900bp, 4000bp, 4100bp, 4200bp, 4300bp, 4400bp, 4500bp, 4600bp, 4700bp, 4800bp, 4900bp, 5000bp, 5100bp, 5200bp, 5300bp, 5400bp, 5500bp, 5600bp, 5700bp, 5800bp, 5900bp, 6000bp, 6100bp, 6200bp, 6300bp, 6400bp, 6500bp, 6600bp, 6700bp, 6800bp, 6900bp, 700 In certain embodiments, the length of the template may be greater than 100 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, 10000 bp, 30000 bp, or 100,000 bp. In certain embodiments, the length of the template may be within a range defined by upper and lower limits selected from those exemplified above.

[0086] In certain embodiments, the double-stranded nucleic acid fragments immobilized on the surface of the solid support can be imaged prior to cluster generation. For example, an intercalating dye can be applied to at least a portion of the immobilized library of double-stranded nucleic acid fragments to obtain a set of stained immobilized fragments, which can be imaged to preserve a record of the location of the backbone of the nucleic acid molecules on the surface. Following cluster generation and sequencing, the coordinates of the clusters can be associated with their location on the original backbone, thus aiding in the alignment of the molecules and reads along the genome assembly.

[0087] In some embodiments, applying the target double-stranded nucleic acid comprises adding a biological sample to the solid support. 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 contain nucleic acid in various states of purification, including purified nucleic acid. However, the sample need not be completely purified and can contain, for example, nucleic acid mixed with protein, other nucleic acid species, other cellular components, and / or any other contaminants. In some embodiments, the biological sample contains a mixture of DNA, protein, other nucleic acid species, other cellular components, and / or any other contaminants that are present in approximately the same proportions as found in vivo. For example, in some embodiments, the components are found in the same proportions 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, less than 0.7, or at least 0.60. To allow the nucleic acid to be bound to the solid support by the methods provided herein, any unbound nucleic acid or other contaminants can be removed by simply washing the solid support after surface-bound fragmentation has occurred. The biological sample can include, for example, a crude cell lysate or whole cells. For example, the crude cell lysate applied to the solid support in the methods described herein does not need to be subjected to one or more of the separation steps traditionally used to isolate nucleic acid 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, ed. Ausubel, et al., which are incorporated herein by reference.

[0088] Thus, in some embodiments, the biological sample may include, for example, blood, plasma, serum, lymph, mucus, sputum, urine, semen, cerebrospinal fluid, bronchial aspirate, feces, and macerated tissues thereof, or lysates thereof, or any other biological specimen containing nucleic acid.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 occur within the flow cell without further movement or handling steps, simply by flowing the necessary reagents through the flow cell.

[0089] Also provided herein is a solid support having thereon a library of immobilized tagged nucleic acid fragments prepared according to the above methods. II. Physical Maps of Immobilized Polynucleotide Molecules

[0090] Also provided herein is a method for generating a physical map of immobilized polynucleotide.The method can advantageously be utilized to identify clusters that are likely to contain linked sequences (i.e., first and second parts from the same target polynucleotide molecule).Therefore, the relative proximity of any two clusters obtained from immobilized polynucleotide provides information useful for aligning the sequence information obtained from the two clusters.Specifically, the distance between any two given clusters on a solid surface positively correlates with the probability that the two clusters are derived from the same target polynucleotide molecule, as described in more detail in International Publication WO2012 / 025250, the entirety of which is incorporated herein by reference.

[0091] As an example, in some embodiments, long dsDNA molecules extending on the surface of a flow cell are fragmented in situ, resulting in a line of connected dsDNA bridges across the surface of the flow cell.Furthermore, a physical map of the immobilized DNA can be generated.The physical map thus correlates the physical relationship of the clusters after the immobilized DNA is amplified.Specifically, the physical map calculates the probability that the sequence data obtained from any two clusters are linked, as described in the incorporated materials of WO2012 / 025250.

[0092] In some embodiments, the physical map is generated by imaging the nucleic acid to establish the position of the immobilized nucleic acid molecule across the solid surface. In some embodiments, the immobilized nucleic acid is imaged by adding an imaging agent to the solid support and detecting a signal from the imaging agent. In some embodiments, the imaging agent may be a detectable label. Suitable detectable labels include, but are not limited to, protons, haptens, radionuclides, enzymes, fluorescent labels, chemiluminescent labels, and / or colorants. 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 known in the art may be used, including, but not limited to, those described in U.S. Patent Application Publication No. 2012 / 0282617, which is incorporated herein by reference in its entirety.

[0093] In some embodiments, the immobilized double-stranded nucleic acid fragments are further fragmented to release free ends prior to cluster generation. Cleavage of the bridge structure can be performed using any suitable methodology known in the art, as exemplified by the incorporated material in WO 2012 / 025250. For example, cleavage can be performed by incorporation of modified nucleotides such as uracil, as described in WO 2012 / 025250, by incorporation of restriction endonuclease sites, or by incorporation of modified nucleotides such as uracil, as described in WO 2012 / 025250. Alternatively, this can occur by applying a solution-phase CRISPR / Cas9 enzyme or complex to the bridge nucleic acid structure, as described elsewhere herein.

[0094] In certain embodiments, multiple target double-stranded nucleic acid molecules are flowed over a flow cell that includes multiple nanochannels, the nanochannels having multiple CRISPR / Cas9 enzymes or complexes immobilized thereon. As used herein, the term "nanochannel" refers to a narrow channel through which long linear nucleic acid molecules flow. In some embodiments, 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 1000 individual long strands of target nucleic acid flow into each nanochannel. In some embodiments, the individual nanochannels are separated by physical barriers that prevent individual long strands of 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, CRISPR / Cas9 enzymes or complexes bound to the nanochannel surface fragment the nucleic acid. Continuous mapping can then be performed, for example, by tracking clusters along the length of one of these channels. In some embodiments, the length of the target nucleic acid is 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, 100 kb, 1 It may be 50kb, 200kb, 250kb, 300kb, 350kb, 400kb, 450kb, 500kb, 550kb, 600kb, 650kb, 700kb, 750kb, 800kb, 850kb, 900kb, 950kb, 1000kb, 5000kb, 10000kb, 20000kb, 30000kb in length, or at least 50000kb in length.In some embodiments, the length of the target nucleic acid is 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, 100 kb, 150 ... kb, 90 kb, 95 kb, 100 kb, 150 kb, 200 kb, 250 kb, 300 kb, 350 kb, 400 kb, 450 kb, 500 kb, 550 kb, 600 kb, 650 kb, 700 kb, 750 kb, 800 kb, 850 kb, 900 kb, 950 kb, or 1000 kb or less in length. As an example, a flow cell with 1000 or more nanochannels with immobilized fragmentation products mapped into the nanochannels can be used to sequence the genome of an organism with short "positioned" reads. In some embodiments, immobilized fragmentation products mapped into the nanochannels can be used to analyze haplotypes. In some embodiments, immobilized fragmentation products mapped into the nanochannels can be used to analyze phasing problems. III. Amplification and Sequencing of Immobilized Nucleic Acid Fragments

[0095] Amplification. The present disclosure further relates to the amplification of immobilized nucleic acid fragments generated according to the methods provided herein. The immobilized nucleic acid fragments generated by surface-bound CRISPR / Cas9 enzyme-mediated fragments can be amplified according to any suitable amplification method known in the art. In some embodiments, the immobilized DNA fragments are amplified on a solid support. In some embodiments, the solid support is the same solid support on which surface-bound fragmentation occurs. In such embodiments, the methods and compositions provided herein allow sample preparation to proceed on the same solid support from the initial sample introduction step, through amplification, and optionally through sequencing steps.

[0096] For example, in some embodiments, the immobilized nucleic acid fragments are amplified using cluster amplification methodology, as exemplified by the disclosures of U.S. Patent Nos. 7,985,565 and 7,115,400, the contents of each of which are incorporated herein by reference in their entirety. The incorporated materials of U.S. Patent Nos. 7,985,565 and 7,115,400 describe a method of solid-phase nucleic acid amplification that allows the amplification products to be immobilized on a solid support to form an array consisting of clusters or "colonies" of immobilized nucleic acid molecules. Each cluster or colony on such an array is formed from a plurality of identical immobilized polynucleotide strands and a plurality of identical immobilized complementary polynucleotide strands. Arrays so formed are generally referred to herein as "clustered arrays." The product of a solid-phase amplification reaction as described in US Patent Nos. 7,985,565 and 7,115,400 is a so-called "bridged" structure formed by annealing a pair of immobilized polynucleotide strands and immobilized complementary strands, both strands being immobilized on a solid support at their 5' ends, preferably via covalent bonds. Cluster amplification is an example of a method for generating immobilized amplicons using immobilized nucleic acid templates. Other suitable methodologies can also be used to generate immobilized amplicons from immobilized nucleic acid fragments generated according to the methods provided herein. For example, one or more clusters or colonies can be formed by solid-phase PCR, regardless of whether one or both primers of each pair of amplification primers are immobilized.

[0097] In other embodiments, the immobilized nucleic acid fragments are amplified in solution. For example, in some embodiments, the immobilized nucleic acid fragments are cleaved or otherwise released from the solid support, and then amplification primers are hybridized to the released molecules in solution. In other embodiments, amplification primers are hybridized to the immobilized nucleic acid fragments for one or more initial amplification steps, followed by subsequent amplification steps in solution. Thus, in some embodiments, immobilized nucleic acid templates can be used to generate liquid phase amplicons.

[0098] It will be understood that any of the amplification methodologies described herein or generally known in the art can be used with universal or target-specific primers to amplify nucleic acids. Suitable methods for amplification include, but are not limited to, polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription-based amplification (TMA) and nucleic acid sequence-based amplification (NASBA), as described in U.S. Pat. No. 8,003,354, the entirety of which is incorporated herein by reference. The above 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 immobilized nucleic acid fragments. In some embodiments, primers specifically directed to the nucleic acid of interest are included in the amplification reaction.

[0099] Other methods suitable for amplifying polynucleotides include oligonucleotide extension and ligation, rolling circle amplification (RCA) (Lizardi et al., Nat. Genet. 19:225-232 (1998), incorporated herein by reference), and oligonucleotide ligation assay (OLA) (generally described in U.S. Pat. Nos. 7,582,420, 5,185,243, 5,679,524, and 5,573,907, EP 0 320 308(B1), EP 0 336 731(B1), EP 0 439 182(B1), WO 90 / 01069, WO 89 / 12696, and WO 89 / 09835, all of which are incorporated by reference) techniques. It will be understood that these amplification methods can be designed to amplify nucleic acids. For example, in some embodiments, the amplification method can include a ligation probe amplification or oligonucleotide ligation assay (OLA) reaction that includes a primer specifically directed to the nucleic acid of interest. In some embodiments, the amplification method can include a primer extension ligation reaction that includes a primer specifically directed to 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 can include primers used in the GoldenGate assay (Illumina, Inc., San Diego, CA) as exemplified in U.S. Patent Nos. 7,582,420 and 7,611,869, each of which is incorporated herein by reference in its entirety.

[0100] 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, for example, by Dean et al., Proc. Natl. Acad. Sci. USA 99:5261-66 (2002), or isothermal strand displacement nucleic acid amplification, as exemplified, for example, by 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 strand displacement amplification (SDA), as described, for example, in Walker et al., Molecular Methods for Virus Detection, Academic Press, Inc., 1995, U.S. Patent Nos. 5,455,166 and 5,130,238, and Walker et al., Nucl.Acids Res.20:1691-96 (1992), or hyperbranched strand displacement amplification, as described, for example, in Lage et al., Genome Research 13:294-307 (2003), each of which is incorporated herein by reference in its entirety. For example, isothermal amplification methods can be used with strand-displacing Phi 29 polymerase or large fragment, 5'→3' exo- of Bst DNA polymerase for random primer amplification of genomic DNA. The use of these polymerases takes advantage of their high processivity and strand displacement activity. Due to their high processivity, polymerases can produce fragments of 10-20 kb in length. As noted above, polymerases with low processivity and strand displacement activity, such as Klenow polymerase, can be used to generate smaller fragments under isothermal conditions. Further description of the amplification reaction, conditions and components is described in detail in the disclosure of U.S. Patent No. 7,670,810, which is incorporated herein by reference in its entirety.

[0101] Another nucleic acid amplification method useful in the present disclosure is tagged PCR, which uses a population of two-domain primers with a constant 5' region followed by a random 3' region, as described, for example, in Grothues, et al. Nucleic Acids Res. 21(5):1321-2 (1993), which is incorporated herein by reference in its entirety. The first round of amplification is carried out on the heat-denatured nucleic acid to allow multiple initiations based on individual hybridization from the randomly synthesized 3' region. Due to the nature of the 3' region, the initiation sites are considered to be random throughout the genome. Unbound primers can then be removed, and further replication can be carried out using primers complementary to the constant 5' region.

[0102] Sequencing. The present disclosure further relates to sequencing of the immobilized randomly fragmented double-stranded nucleic acid fragments generated according to the methods provided herein. The immobilized double-stranded nucleic acid fragments generated by surface-bound CRISPR / Cas9 enzyme-mediated fragmentation can be sequenced according to any suitable sequencing method, such as direct sequencing, such as sequencing by synthesis, sequencing by ligation, sequencing by hybridization, nanopore sequencing, etc. In some embodiments, the immobilized nucleic acid fragments are sequenced on a solid support. In some embodiments, the solid support for sequencing is the same solid support on which the surface-bound fragmentation occurs. In some embodiments, the solid support for sequencing is the same solid support on which the amplification occurs. In some embodiments, a first solid support is used for fragmentation and a second solid support is used for amplification and sequencing.

[0103] One sequencing methodology 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 an amplicon thereof) is monitored to determine the sequence of nucleotides in the template. The underlying chemical process can be polymerization (e.g., when catalyzed by a polymerase enzyme). In certain polymer-based SBS embodiments, fluorescently labeled nucleotides are added to the primer (thereby extending the primer) in a template-dependent manner such that detection of the order and type of nucleotides added to the primer can be used to determine the sequence of the template.

[0104] The flow cell provides a convenient solid support for housing the amplified nucleic acid fragments produced by the disclosed method. One or more amplified nucleic acid fragments in such a format can be subjected to SBS or other detection techniques that involve repeated delivery of reagents in cycles. For example, to initiate a first SBS cycle, one or more labeled nucleotides, nucleic acids, DNA polymerase, etc. can be flowed / passed through a flow cell housing one or more amplified nucleic acid molecules. The site at which the labeled nucleotide is incorporated by primer extension can be detected. Optionally, the nucleotide can further include a reversible termination property that terminates further primer extension once the nucleotide is added to the primer. For example, a nucleotide analog with a reversible terminator moiety can be added to the primer to prevent further extension from occurring until a deblocking agent is delivered to remove the moiety. Thus, in embodiments using reversible termination, a deblocking reagent can be delivered to the flow cell (before or after detection occurs). Washing can be performed between the various delivery steps. The cycle is then repeated n times to extend the primer by n nucleotides, thereby allowing detection of sequences of length n. Exemplary SBS procedures, fluidic systems, and detection platforms that can be readily adapted for use with amplicons generated by the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497, U.S. Patent No. 7,057,026, WO 91 / 06678, WO 07 / 123744, U.S. Patent No. 7,329,492, U.S. Patent No. 7,211,414, U.S. Patent No. 7,315,019, U.S. Patent No. 7,405,281, and U.S. Patent No. 2008 / 0108082, each of which is incorporated herein by reference.

[0105] Other sequencing procedures using cyclic reactions can be used, such as pyrosequencing. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) when a specific nucleotide is incorporated into a nascent nucleic acid chain (described in 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); U.S. Patent No. 6,210,891; U.S. Patent No. 6,258,568; and U.S. Patent No. 6,274,320, each of which is incorporated herein by reference). In pyrosequencing, the released PPi can be detected by its immediate conversion to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP produced can be detected via luciferase-generated photons. Therefore, the sequencing reaction can be monitored through a luminescence detection system. The excitation radiation source used in the fluorescence-based detection system is not necessary for the pyrosequencing procedure. Useful fluid systems, detectors and procedures that can be adapted to the application of pyrosequencing to the amplicons generated according to the present disclosure are described, for example, in International Application No. PCT / US11 / 57111, US Patent Application Publication No. 2005 / 0191698(A1), US Patent No. 7,595,883 and US Patent No. 7,244,559, each of which is incorporated herein by reference.

[0106] Some embodiments can utilize methods that include real-time monitoring of nucleic acid or DNA polymerase activity. For example, nucleotide incorporation can be detected via fluorescence resonance energy transfer (FRET) interactions between fluorophore-bearing polymerase and γ-phosphate-labeled nucleotides, or using zero-mode waveguides (ZMW). Techniques and reagents for FRET-based sequencing are described, for example, in 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.

[0107] Some SBS embodiments include detection of protons released upon incorporation of a nucleotide into an extension product. For example, sequencing based on detection of released protons can use electrical detectors and related technologies commercially available in Thermo Fisher Scientific's IonTorrent product line, or the sequencing methods and systems described in U.S. Patent Application Publication Nos. 2009 / 0026082 (A1), 2009 / 0127589 (A1), 2010 / 0137143 (A1), or 2010 / 0282617 (A1), each of which is incorporated herein by reference. The methods described herein for amplifying target nucleic acids using binding equilibrium exclusion can be easily adapted to substrates used to detect protons. More specifically, the methods described herein can be used to produce clonal populations of amplicons used to detect protons.

[0108] Another useful sequencing technique 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 a nanopore, each nucleotide type can be identified by measuring the fluctuations in the electrical conductance of the pore (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).

[0109] Exemplary methods for array-based expression and genotyping that can be applied for detection according to the present disclosure are described in U.S. Pat. Nos. 7,582,420, 6,890,741, 6,913,884, or 6,355,431, or U.S. Patent Application Publication Nos. 2005 / 0053980(A1), 2009 / 0186349(A1), or 2005 / 0181440(A1), each of which is incorporated herein by reference.

[0110] An advantage of the methods described herein is that they provide rapid and efficient detection of multiple target nucleic acids in parallel. Thus, the present disclosure provides an integrated system that can prepare and detect nucleic acids using techniques known in the art, such as those exemplified above. Thus, the integrated system of the present disclosure can include fluidic components that can deliver amplification and / or sequencing reagents to one or more immobilized DNA fragments, and the system includes components such as pumps, valves, reservoirs, fluid lines, etc. A flow cell can be configured and / or used in the integrated system for detecting target nucleic acids. Exemplary flow cells are described, for example, in U.S. Patent No. 2010 / 0111768(A1) and U.S. Patent Application No. 13 / 273,666, each of which is incorporated herein by reference. As exemplified for the flow cell, one or more of the fluidic components of the integrated system can be used for amplification and detection methods. Taking the nucleic acid sequencing embodiment as an example, one or more of the fluidic components of the integrated system can be used for delivery of sequencing reagents in the amplification methods described herein and sequencing methods as exemplified above. Alternatively, an integrated system may include separate fluidic systems for performing the amplification method and for performing the detection method. Examples of integrated sequencing systems that can generate amplified nucleic acids and sequence the nucleic acids include, but are not limited to, the MiSeq™ platform (Illumina Inc., San Diego, Calif.) and the devices described in U.S. Patent Application No. 13 / 273,666, which is incorporated herein by reference. IV. Solid Supports with Immobilized CRISPR / Cas9 Complexes and Methods of Preparation

[0111] Other embodiments presented herein include solid supports such as beads having immobilized CRISPR / Cas9 enzymes thereon. In certain embodiments, the solid supports have CRISPR / Cas9 complexes thereon, including CRISPR / Cas9 enzymes bound to a first polynucleotide that induces the CRISPR / Cas9 enzyme to bind to a target nucleic acid in a non-sequence specific manner. In certain other embodiments, the solid supports have CRISPR / Cas9 enzymes bound to a first polynucleotide that induces the CRISPR / Cas9 enzyme to bind to a target nucleic acid in a sequence specific manner. They have immobilized CRISPR / Cas9 complexes thereon. In some embodiments, different solid supports induce the CRISPR / Cas9 enzyme to bind to a target nucleic acid with different sequences. In some embodiments, the first CRISPR / Cas9 enzyme on the solid support binds to a first polynucleotide that induces the first CRISPR / Cas9 enzyme to bind to the 3' of the target nucleic acid of the sequence of interest, and the second CRISPR / Cas9 enzyme on the solid support binds to a second polynucleotide that induces the second CRISPR / Cas9 enzyme to bind to the 5' of the target nucleic acid of interest. In some embodiments, the first polynucleotide comprises a 3' portion that comprises a CRISPR / Cas9 terminal sequence, and a 5' portion that comprises a single-stranded guide RNA (sgRNA) that induces the CRISPR / Cas9 enzyme to bind to the target nucleic acid in a non-sequence-specific manner (or, optionally, in a sequence-specific manner for a population of solid supports that target different sequences, where each solid support comprises a pair of sgRNAs that target adjacent regions around the sequence of interest). 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 CRISPR / Cas9 complexes are distributed over a 1 mm 2 At least 10 per 3 , 10 4 , 10 5 or at least 10 6 The enzyme or complex is present on the solid support at a density of 100 μg / ml. V. Fragmentation using CRISPR / Cas9 microparticles

[0112] One embodiment presented herein is a population of microparticles (e.g., beads) with CRISPR / Cas9 enzymes or complexes immobilized thereon. The use of 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 size of the fragments is a function of the ratio of CRISPR / Cas9s to the amount and size of the nucleic acid, as well as to the reaction time. Even when these parameters are controlled, a size selection fractionation is generally required as an additional step to remove excess small fragments that are shorter than the combined length of the paired reads. The methods provided herein avoid these drawbacks. Specifically, bead-immobilized CRISPR / Cas9 enzymes or complexes allow the selection of the final fragment size as a function of the spatial separation of the bound CRISPR / Cas9 enzymes or complexes, regardless of the amount of CRISPR / Cas9 beads added to the fragmentation reaction. A further limitation of solution-based fragmentation is the need to perform some form of purification of the products of the fragmentation reaction both before and after PCR amplification. This typically requires some transfer of the reaction from tube to tube. In contrast, the fragmentation products on bead-based CRISPR / Cas9s can be washed and later released for amplification or other downstream processing, thus avoiding the need for sample transfer. For example, in embodiments where the CRISPR / Cas9s enzyme or complex is assembled on a paramagnetic bead, purification of the fragmentation reaction products can be easily achieved by immobilizing and washing the beads with a magnet. Thus, in some embodiments, fragmentation and other downstream processing, such as PCR amplification, can all be performed in a single tube, vessel, droplet, or other container. In some embodiments, the fragmentation and downstream processing of the sample is performed in a microfluidic droplet-based device, as described in U.S. Patent Application Publication No. 2013 / 0116128, which is incorporated herein by reference in its entirety. For example, in a microfluidic droplet-based device, droplets containing target nucleic acid, wash buffer, or other reagents can be passed over a surface containing immobilized CRISPR / Cas9 enzyme or complex.Similarly, droplets containing beads having immobilized CRISPR / Cas9s enzymes or complexes thereon may be contacted with target double-stranded nucleic acids, wash buffers, or other reagents in a microfluidic droplet-based device.

[0113] Adding CRISPR / Cas9 beads to a solution of target double-stranded nucleic acid in fragment buffer results in random fragmentation and ligation of the target double-stranded nucleic acid to the surface of the beads, generating an immobilized library of randomly fragmented double-stranded nucleic acid fragments.

[0114] In some embodiments, the length of the bridge fragment may be determined by the density of the CRISPR / Cas9 enzyme or complex on the surface of the bead. Once fragmentation is complete, the double-stranded nucleic acid fragments can be released from the surface of the bead using any suitable method. In some embodiments, the fragmentation products are released from the bead using an amplification method such as suppression PCR, step-out PCR, etc. In some embodiments, the fragmentation products are released from the bead by cleavage. The cleavage can be, for example, chemical, enzymatic, photochemical, or a combination thereof. It will be understood that any suitable method for releasing one or more fragmentation products from a solid support can be utilized in the methods provided herein.

[0115] The nucleic acid can be efficiently contacted with the surface-bound CRISPR / Cas9 enzyme or complex using any suitable method for increasing the probability of contact. For example, in some embodiments, precipitation of the nucleic acid onto a solid surface can be utilized to increase the contact between the target double-stranded nucleic acid and the CRISPR / Cas9 enzyme or complex on the solid surface. Any one of a number of methods known in the art can be utilized to contact the nucleic acid with a solid support, 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, the nucleic acid can be precipitated onto the solid support by adding PEG, ethanol, or any one of a variety of other agents known to precipitate nucleic acids onto a surface, including, for example, any one of a number of buffers used in solid phase reversible immobilization (SPRI) technology.

[0116] In some embodiments, a population of beads with immobilized CRISPR / Cas9 enzymes or complexes can be mixed with excess beads that do not have CRISPR / Cas9s enzymes, complexes, or oligonucleotides, thereby reducing the possibility of fragmentation across two or more different beads. Another method for reducing the possibility of fragmentation across two or more different beads includes immobilizing the beads so that contact between the beads is minimized. Immobilization of the beads can be achieved by any of a number of techniques known in the art, including, for example, immobilization of the beads via magnetism to the side of a solid surface such as a microcentrifuge tube, or any other immobilization technique exemplified by the incorporated materials of WO2010 / 115122.

[0117] In some embodiments, CRISPR / Cas9 beads can be used to isolate and identify nucleic acids from single cells, such as prokaryotic or eukaryotic cells. For example, in some embodiments, particles, such as beads, are coated with indexed CRISPR / Cas9 enzymes or complexes that share the same index (all of the CRISPR / Cas9 enzymes or complexes present on a particular bead carry the same index, which differs from the indexes present on another bead). The beads can then be placed inside a cell via any one of a variety of methods known in the art. For example, methods for delivering beads inside a cell include, but are not limited to, gene guns, photothermal nanoblades (Wu et al. Anal Chem. (2011) 4:1321-7), and peptides used with cell-permeable agents (Nitin et al Ann Biomed Eng. (2009) 37:2018-2027), and the like. It will be understood that any suitable method for associating nucleic acids from a single cell with particles bearing indexed CRISPR / Cas9 enzymes or complexes can be used in the methods presented herein.

[0118] In some embodiments, the CRISPR / Cas9 enzyme or complex can be covalently attached to the beads, as detailed herein above. Additionally or alternatively, the CRISPR / Cas9 enzyme or complex can be released from the beads upon application of a chemical or physical stimulus. Some examples of stimuli that can trigger the release of the CRISPR / Cas9 enzyme or complex from the solid support include light and / or temperature change (e.g., heat). In some embodiments, the CRISPR / Cas9 enzyme or complex is released from the solid support using the activity of an enzyme, such as a restriction endonuclease. In certain embodiments, the CRISPR / Cas9 enzyme or complex is separated from the beads and can move freely within the cell. When the beads (or alternatively, the released CRISPR / Cas9 enzyme or complex) come into contact with chromatin or nucleic acid, fragmentation can occur. It will be understood that in eukaryotic and prokaryotic systems, not all genomic DNA is always accessible and / or available for fragmentation. In some embodiments, up to 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 of the total nucleic acid in 99% of the cells is fragmented by the CRISPR / Cas9 enzyme or complex.

[0119] In some embodiments, the double-stranded nucleic acid fragments are tagged with a first tag, for example comprising a first tag domain. In some embodiments, the first tag domain may comprise a region for cluster amplification and / or a region for priming a sequencing reaction. The tagging of the fragments allows for identification of reads from the same cell or other biological sample by grouping together reads that share the same tag. These reads can be considered as originating from the same solid support or bead (and therefore from the same cell or biological sample).

[0120] In some embodiments, approaches can be used to ensure that individual biological samples or target double-stranded nucleic acids are not fragmented by multiple solid supports or beads. For example, one approach is to use beads of similar size to cells. This ensures that cells cannot accommodate multiple beads. Additionally or alternatively, another approach is to use a cell-to-bead ratio that favors single cell targeting. For example, if there are many more cells than beads, the Poisson distribution of beads in cells means that cells that have taken up one bead will far outnumber cells that have taken up two or more beads.

[0121] In some embodiments, the above single cell approach can be used to determine whether two single nucleotide polymorphisms (SNPs) or structural rearrangements are present in the same cell.For example, in the case of a heterogeneous population of cancer cells, knowing whether two SNPs are present in the same cell or in different cells can help to implement appropriate cancer treatment.

[0122] In some embodiments, the above single cell approach can be used to study RNA. For example, gene expression at the single cell level can be analyzed by coating beads with suitable enzymes (i.e., reverse transcriptase) and oligonucleotides for the reverse transcription step. In one embodiment, after introducing beads coated with reverse transcriptase, oligonucleotides, and CRISPR / Cas9 enzymes, cytoplasmic RNA can be converted to cDNA, tagged, and prepared in the cell. VI. How to Assemble Long Reads Using Immobilized CRISPR / Cas9 Enzymes

[0123] Assembly of nucleic acid fragments allows for the isolation of individual long nucleic acid molecules within a population of nucleic acid molecules and the conversion of each nucleic acid into a fragment library. When a library of nucleic acid fragments is tagged and sequenced, the nucleic acid fragments can be reassembled back into the original long molecules, for example, by reference to the tags they contain.

[0124] The target double-stranded nucleic acid can be efficiently contacted with the surface-bound CRISPR / Cas9s using any suitable method for increasing the probability of contact, as described herein above.

[0125] This method can be carried out using any one of a variety of known formats, for example, using a combination of fragmentation reagents and bead arrays for library preparation, followed by indexed sequencing and custom data analysis. Any other suitable method that keeps the beads statically separated from each other can be used for surface fragmentation and indexing of the sample. For example, physical configurations such as wells or small depressions in the substrate that can hold the beads so that the microspheres can remain in the wells, or the use of other forces (magnetic or compressive), or chemically altered or activated sites such as chemically functionalized sites, electrostatically altered sites, hydrophobically and / or hydrophilically functionalized sites, or adhesive spots.

[0126] In some embodiments, the microspheres are non-covalently associated with the wells, although the wells may be further chemically functionalized, cross-linking agents may be used, or a physical barrier, such as a film or membrane over the beads, may be used.

[0127] In certain embodiments, the surface of the substrate is modified to contain chemically modified sites that can be used to attach, either covalently or non-covalently, the microspheres of the present invention to distinct sites or locations on the substrate. "Chemically modified sites" in this context include, but are not limited to, the addition of a pattern of chemical functional groups that can be used to covalently attach microspheres, including amino, carboxy, oxo, and thiol groups, which generally also contain corresponding reactive functional groups; the addition of a pattern of adhesives that can be used to attach microspheres (either by prior chemical functionalization for the addition of adhesives or by direct addition of adhesives); the addition of a pattern of charged groups (similar to chemical functionality) for electrostatic attachment of microspheres; for example, the addition of a pattern of chemical functional groups that render sites of different hydrophobicity or hydrophilicity such that, if the microspheres contain oppositely charged groups to the sites, the addition of similarly hydrophobic or hydrophilic microspheres under suitable experimental conditions will cause the microspheres to associate with the sites based on water affinity. For example, the use of hydrophobic beads and hydrophobic sites in an aqueous system preferentially encourages the association of beads to the sites. As outlined above, "pattern" in this sense includes the use of uniform treatments of a surface that allow for attachment of beads at discrete sites, as well as the use of treatments of a surface that result in discrete sites. As will be appreciated by those skilled in the art, this can be achieved in a variety of ways.

[0128] In some embodiments, when the target double-stranded nucleic acid is applied to the 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 immobilized on 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 molecule from reaching between two beads. In other embodiments, the beads are in close contact, and one or more nucleic acid molecules can extend between two or more solid supports. In some embodiments, two or more target double-stranded nucleic acid molecules can be fragmented per solid support. The probability that two alleles are fragmented on the same solid support or bead is low and is a function of the concentration of the nucleic acid added and the number of solid supports or beads. For example, 0.1x haplome equivalent (50,000 genome equivalent) needs to be loaded on one million beads to avoid two alleles in the same well.

[0129] In some embodiments, the double-stranded nucleic acid fragments are tagged by any of the known methods for tagging. The double-stranded nucleic acid fragments can be tagged with a first tag, for example, comprising a first tag domain. In some embodiments, the first tag domain comprises a region for priming a sequencing reaction. Once fragmentation and optionally tagging are completed, the double-stranded nucleic acid fragments can be transferred from the surface of the solid support to a solution, and the individual double-stranded nucleic acid fragments can be pooled and prepared for the next step, such as sequencing.

[0130] The release or liberation of the immobilized double-stranded nucleic acid fragments from the solid support into solution can be accomplished using any suitable method known in the art. For example, in some embodiments, the fragmented molecules can be liberated by cleavage from the surface of the bead, for example, via a cleavage moiety present at the 5' end of the oligonucleotide bound to the surface. The cleavage moiety can be any moiety suitable for cleaving a nucleic acid strand from a solid support. Examples of methods that utilize a cleavage moiety include, but are not limited to, restriction endonuclease cleavage, chemical cleavage, RNase cleavage, photochemical cleavage, and the like, including the cleavage methods and cleavage moieties described in WO2006 / 064199, the entirety of which is incorporated herein by reference.

[0131] The tag may include a "primer" region that can be used as a hybridization point to hybridize a PCR step-out primer to which additional sequences, such as amplification and sequencing primers, can be added. For example, amplification primers P5 and P7 can be added. Once added, suppression PCR can be used, for example, to enrich for molecules with a P5 adaptor at one end and P7 at the other end. In some embodiments, amplification can be performed directly from the solid support (e.g., beads) using step-out primers that add the P5 and P7 adaptor sequences by suppression PCR.

[0132] In another embodiment, each solid support can have two surface-grafted oligonucleotides, where the primer sequence is either P5-lead 1 sequencing primer or P7-lead 2 sequencing primer. This results in a mixed P5-P7 CRISPR / Cas9 enzyme or complex, which can be cleaved from the solid support and then enriched for P5 / P7 molecules by suppression PCR or directly amplified from the solid support. Equivalent

[0133] The foregoing specification is believed to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and examples set forth certain embodiments in detail and explain the best mode contemplated by the inventors. However, no matter how detailed the foregoing appears in the text, it will be understood that the embodiments can be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0134] The term "comprising," as used herein, is intended to be open ended, encompassing not only the recited elements, but any additional elements as well.

[0135] As used herein, the term "about" refers to numerical values, including, for example, integers, fractions, and percentages, whether or not expressly indicated. The term generally refers to a range of numerical values ​​(e.g., ±5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When a term such as at least and about precedes a list of numerical values ​​or ranges, the term modifies all of the values ​​or ranges provided in the list. In some cases, the term about may include numerical values ​​that are rounded to the nearest significant figure.

[0136] Throughout this application, various publications, patents, or patent applications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application. In certain embodiments, for example, the following are provided: (Item 1) 1. A method for preparing an immobilized library of randomly fragmented, double-stranded nucleic acid fragments, comprising: (a) providing a solid support having an immobilized CRISPR / Cas9 enzyme thereon; (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 where 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. (Item 2) 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. (Item 3) 2. The method of claim 1, wherein the target double-stranded nucleic acid is dsDNA. (Item 4) 4. The method of any one of items 1 to 3, wherein the CRISPR / Cas9 enzyme is bound to a first polynucleotide that induces the CRISPR / Cas9 enzyme to bind to the target double-stranded nucleic acid in a non-sequence specific manner. (Item 5) 5. The method of claim 4, wherein the first polynucleotide is immobilized on a solid support. (Item 6) 6. The method of claim 4 or 5, wherein the first polynucleotide comprises a 3' portion comprising a CRISPR / Cas9 end sequence and a 5' portion comprising a single-stranded guide RNA (sgRNA) that guides the CRISPR / Cas9 enzyme to bind to the target nucleic acid in a non-sequence specific manner. (Item 7) The sgRNA is n or (AT) n and n is 5 to 20. (Item 8) 8. The method according to item 7, wherein n is 10. (Item 9) 7. The method according to item 6, wherein the sgRNA is 10 to 40 nucleotides. (Item 10) 10. The method of claim 9, wherein the sgRNA is 17 or 20 nucleotides. (Item 11) 11. The method according to any one of items 4 to 10, wherein the first polynucleotide is biotinylated and the solid support comprises one or more biotin-binding proteins. (Item 12) 12. The method of claim 11, wherein the biotin-binding protein comprises avidin, streptavidin, neutravidin, an anti-biotin antibody, a biotin receptor, and / or a biotin-binding enzyme. (Item 13) Item 13. The method according to item 12, wherein the biotin-binding enzyme comprises biotinidase or biotin holocarboxylase synthetase. (Item 14) 14. The method according to any one of items 1 to 13, comprising washing the solid support with the double-stranded nucleic acid fragment immobilized thereon to remove any unbound nucleic acid. (Item 15) 15. The method according to any one of items 4 to 14, comprising amplifying the double-stranded nucleic acid fragment immobilized on the solid surface. (Item 16) 16. The method of claim 15, wherein the amplifying comprises providing a polymerase and an amplification primer that corresponds to a portion of the first polynucleotide. (Item 17) 17. The method of any one of items 1 to 16, wherein applying the target double-stranded nucleic acid to the solid support comprises treating the CRISPR / Cas9 enzyme with one or more reagents that reduce the nucleic acid binding specificity of the CRISPR / Cas9 enzyme. (Item 18) 19. The method of claim 18, wherein the one or more reagents comprise betaine, dimethylsulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, and / or sulphalane. (Item 19) The CRISPR / Cas9 enzyme is 2 At least 10 per 3 、10 4 、10 5 , or 10 6 19. The method according to any one of items 1 to 18, wherein the enzyme is present on the solid support at a density of about 1000 μg / ml. (Item 20) 20. The method of any one of items 1 to 19, wherein the length of the double-stranded nucleic acid fragments in the immobilized library is proportional to the density of CRISPR / Cas9 enzyme on the solid support. (Item 21) 21. The method of any one of items 1 to 20, wherein the solid support comprises a microparticle, a patterned surface, or a well. (Item 22) 22. The method of claim 21, wherein the microparticles are beads. (Item 23) (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. (Item 24) 24. The method according to any one of items 1 to 23, wherein applying the target double-stranded nucleic acid comprises adding a biological sample to the solid support. (Item 25) 25. The method of claim 24, wherein the biological sample comprises a cell lysate. (Item 26) 25. The method of claim 24, wherein the biological sample comprises whole cells. (Item 27) 25. The method of claim 24, wherein 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. (Item 28) 28. The method according to any one of items 1 to 27, comprising tagging the double-stranded nucleic acid fragment. (Item 29) 30. The method of claim 28, wherein the double-stranded nucleic acid fragments are tagged with a first tag comprising a first tag domain. (Item 30) 30. The method of claim 29, wherein the first tag domain comprises a region for cluster amplification. (Item 31) 31. The method of claim 29 or 30, wherein the first tag domain comprises a region for priming a sequencing reaction. (Item 32) 32. The method according to any one of items 1 to 31, comprising liberating the immobilized double-stranded nucleic acid fragment from the solid support. (Item 33) 33. The method of claim 32, wherein the releasing comprises cleavage of the CRISPR / Cas9 enzyme from the solid support. (Item 34) 33. The method of claim 32, wherein the releasing comprises performing a polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription-based amplification (TMA), and nucleic acid sequence-based amplification (NASBA), or other amplification process. (Item 35) 35. The method of claim 34, wherein the PCR comprises suppression PCR. (Item 36) 33. The method of claim 32, wherein said releasing comprises applying light or heat. (Item 37) 1. A method for preparing an immobilized library of randomly fragmented, double-stranded nucleic acid fragments, comprising: (a) providing a solid support having an immobilized CRISPR / Cas9 complex thereon, the CRISPR / Cas9 complex comprising a CRISPR / Cas9 enzyme bound to a biotinylated first polynucleotide comprising a 3' portion comprising a CRISPR / Cas9 end 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, the biotinylated first polynucleotide being bound to a biotin-binding protein on the solid support; (b) applying a target double-stranded nucleic acid to the solid support under conditions in which 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. (Item 38) 38. The method of claim 37, wherein the biotin-binding protein comprises avidin, streptavidin, neutravidin, an anti-biotin antibody, a biotin receptor, and / or a biotin-binding enzyme. (Item 39) Item 39. The method according to item 38, wherein the biotin-binding enzyme comprises biotinidase or biotin holocarboxylase synthetase. (Item 40) 40. The method of claim 38 or 39, wherein applying the target double-stranded nucleic acid to the solid support comprises treating the CRISPR / Cas9 enzyme with one or more reagents that reduce the nucleic acid binding specificity of the CRISPR / Cas9 enzyme. (Item 41) 41. The method of claim 40, wherein the one or more reagents comprise betaine, dimethylsulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, and / or sulfalane. (Item 42) 42. The method according to any one of items 37 to 41, comprising tagging the double-stranded nucleic acid fragment. (Item 43) 43. The method of claim 42, wherein the double-stranded nucleic acid fragment is tagged with a first tag comprising a first tag domain. (Item 44) Item 44. The method of item 43, wherein the first tag domain comprises a region for cluster amplification. (Item 45) 45. The method of claim 43 or 44, wherein the first tag domain comprises a region for priming a sequencing reaction. (Item 46) 46. ​​The method according to any one of items 37 to 45, comprising liberating the immobilized double-stranded nucleic acid fragment from the solid support. (Item 47) 47. The method of claim 46, wherein the releasing comprises cleavage of the CRISPR / Cas9 enzyme from the solid support. (Item 48) 48. The method of claim 47, wherein the releasing comprises performing a PCR or other amplification process. (Item 49) 49. The method of claim 48, wherein the PCR comprises suppression PCR. (Item 50) 49. The method of claim 48, wherein said releasing comprises applying light or heat. (Item 51) 46. ​​A solid support having a library of immobilized double-stranded nucleic acid fragments thereon, prepared according to the method according to any one of items 1 to 45. (Item 52) 1. A solid support having an immobilized CRISPR / Cas9 complex thereon, the CRISPR / Cas9 complex comprising a CRISPR / Cas9 enzyme that randomly fragments a target double-stranded nucleic acid. (Item 53) 53. The solid support according to item 52, wherein the target double-stranded nucleic acid comprises double-stranded DNA (dsDNA), double-stranded RNA (dsRNA), or a double-stranded RNA / DNA hybrid. (Item 54) 54. The solid support according to item 53, wherein the target double-stranded nucleic acid is dsDNA. (Item 55) 55. The solid support according to any one of items 51 to 54, wherein the CRISPR / Cas9 enzyme is bound to a first polynucleotide that induces the CRISPR / Cas9 enzyme to bind the target double-stranded nucleic acid in a non-sequence specific manner. (Item 56) 56. The solid support according to item 55, wherein the first polynucleotide is immobilized on the solid support. (Item 57) 57. The solid support according to item 55 or 56, wherein the first polynucleotide comprises a 3' portion comprising a CRISPR / Cas9 end sequence and a 5' portion comprising a single-stranded guide RNA (sgRNA) that guides the CRISPR / Cas9 enzyme to bind to the target nucleic acid in a non-sequence specific manner. (Item 58) The sgRNA is n or (AT) n Item 58. The solid support according to item 57, comprising: (Item 59) 59. The method of claim 58, wherein n is 10. (Item 60) 58. The method of item 57, wherein the sgRNA is 10 to 40 nucleotides. (Item 61) 61. The method of claim 60, wherein the sgRNA is 17 or 20 nucleotides. (Item 62) 52. The solid support according to any one of items 55 to 51, wherein the first polynucleotide is biotinylated and the solid support comprises a biotin-binding protein. (Item 63) 63. The solid support according to item 62, wherein the biotin-binding protein comprises avidin, streptavidin, neutravidin, an anti-biotin antibody, a biotin receptor, and / or a biotin-binding enzyme. (Item 64) Item 64. The solid support according to item 63, wherein the biotin-binding enzyme comprises biotinidase or biotin holocarboxylase synthetase. (Item 65) The CRISPR / Cas9 enzyme is 2 At least 10 per 3 、10 4 、10 5 , or 10 6 65. The solid support according to any one of items 52 to 64, wherein the enzyme is present on the solid support at a density of about 1000 μg / ml. (Item 66) 66. The solid support according to any one of items 52 to 65, wherein the solid support comprises a microparticle, a patterned surface, or a well. (Item 67) Item 67. The solid support according to item 66, wherein the microparticles are beads. (Item 68) 68. A composition comprising the solid support according to any one of items 52 to 67 and one or more reagents that reduce the nucleic acid binding specificity of the CRISPR / Cas9 enzyme. (Item 69) 70. The composition of claim 68, wherein the one or more reagents comprise betaine, dimethylsulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, and / or sulfalane. (Item 70) 1. A solid support having an immobilized CRISPR / Cas9 complex thereon, the CRISPR / Cas9 complex comprising a CRISPR / Cas9 enzyme bound to a biotinylated first polynucleotide comprising a 3' portion comprising a CRISPR / Cas9 end 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, the biotinylated first polynucleotide being bound to a biotin-binding protein on the solid support. (Item 71) 71. The solid support according to item 70, wherein the biotin-binding protein comprises avidin, streptavidin, neutravidin, an anti-biotin antibody, a biotin receptor, and / or a biotin-binding enzyme. (Item 72) 72. The solid support according to item 71, wherein the biotin-binding enzyme comprises biotinidase or biotin holocarboxylase synthetase. (Item 73) The CRISPR / Cas9 complex is 2 At least 10 per 3 、10 4 、10 5 、10 6 73. The solid support according to any one of items 70 to 72, wherein the complex is present on the solid support at a density of about 1000 .mu.m. (Item 74) 74. The solid support according to any one of items 70 to 73, wherein the solid support comprises a microparticle, a patterned surface, or a well. (Item 75) 75. The solid support according to claim 74, wherein the microparticles are beads. (Item 76) 76. A composition comprising the solid support according to any one of items 70 to 75 and one or more reagents that reduce the nucleic acid binding specificity of the CRISPR / Cas9 enzyme. (Item 77) 77. The composition of claim 76, wherein the one or more reagents comprise betaine, dimethylsulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, and / or sulfalane.

Claims

1. 1. A method for preparing an immobilized library of randomly fragmented, double-stranded nucleic acid fragments, comprising: (a) providing a solid support having thereon an immobilized polynucleotide of a CRISPR / Cas9 complex, the CRISPR / Cas9 complex comprising a CRISPR / Cas9 enzyme associated with 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 double-stranded nucleic acid in a non-sequence specific manner, the biotinylated first polynucleotide being immobilized on the solid support via binding to a biotin-binding protein on the solid support; (b) applying a target double-stranded nucleic acid to the solid support under conditions in which 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, and the double-stranded nucleic acid fragments remain bound to the CRISPR / Cas9 complex immobilized on the solid support; thereby generating an immobilized library of randomly fragmented, double-stranded nucleic acid fragments.

2. The method of claim 1 , wherein the biotin-binding protein comprises avidin, streptavidin, neutravidin, an anti-biotin antibody, a biotin receptor, and / or a biotin-binding enzyme.

3. The method of claim 2 , wherein the biotin-binding enzyme comprises biotinidase or biotin holocarboxylase synthetase.

4. 4. The method of claim 2 or 3, wherein applying the target double-stranded nucleic acid to the solid support comprises treating the CRISPR / Cas9 enzyme with one or more reagents.

5. 5. The method of claim 4, wherein the one or more reagents comprise betaine, dimethylsulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, and / or sulfalane.

6. The method of any one of claims 1 to 5, comprising tagging the double-stranded nucleic acid fragments.

7. 7. The method of claim 6, wherein the double-stranded nucleic acid fragments are tagged with a first tag comprising a first tag domain.

8. 8. The method of claim 7, wherein the first tag domain comprises a region for cluster amplification.

9. 9. The method of claim 7 or 8, wherein the first tag domain comprises a region for priming a sequencing reaction.

10. The method of any one of claims 1 to 9, further comprising liberating the immobilized double-stranded nucleic acid fragments from the solid support.

11. 11. The method of claim 10, wherein the releasing comprises cleavage of the CRISPR / Cas9 enzyme from the solid support.

12. The method of claim 11 , wherein the releasing comprises performing a PCR or other amplification process.

13. The method of claim 12, wherein the PCR comprises suppression PCR.

14. The method of claim 12 , wherein the liberating comprises applying light or heat.

15. (a) a solid support having thereon an immobilized polynucleotide of a CRISPR / Cas9 complex, the CRISPR / Cas9 complex comprising a CRISPR / Cas9 enzyme associated with 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 double-stranded nucleic acid in a non-sequence specific manner, the target double-stranded nucleic acid remaining bound to the CRISPR / Cas9 complex immobilized on the solid support, and the biotinylated first polynucleotide being immobilized on the solid support via binding to a biotin-binding protein on the solid support; (b) one or more reagents; 13. A composition comprising: wherein the one or more reagents comprise betaine, dimethylsulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, and / or sulfalane.

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